Variant FC domains and uses thereof

Abstract

The present disclosure relates to variant Fc domain monomers, fusion proteins, conjugates, compositions, and related methods for treating or preventing disease. In particular, the present invention features variant Fc domain monomers comprising mutations at positions (220), (252, 254), and / or (256) or (309, 311), and / or (434) according to the Kabat index numbering. The present invention also features variant Fc domain monomers comprising mutations at position (220) according to the Kabat index numbering, wherein the variant Fc domain monomer is between 200 and 300 amino acid residues in length and / or has a mass between about 20 kDa and about 40 kDa.

Description

[Technical Field]

[0001] The present invention relates to variant FC domains and uses thereof. [Background technology]

[0002] The usefulness of many therapeutic agents, such as small molecule therapeutics and biologics, e.g., peptides, polypeptides, and polynucleotides, is challenged by insufficient serum half-lives. This necessitates the administration of such therapeutic agents more frequently and / or at higher doses, or the use of sustained-release formulations to maintain serum levels necessary for therapeutic efficacy. Frequent systemic drug administration is associated with significant adverse side effects. For example, frequent systemic injections can cause significant discomfort to the subject, carry a high risk of administration-related infections, and may require hospitalization or frequent visits to the hospital, especially when the therapeutic agent is administered intravenously. Furthermore, in long-term treatments, daily intravenous injections can also lead to significant side effects such as tissue scarring and vascular lesions caused by repeated vascular perforation. Similar problems are known for all frequent systemic administration of therapeutic agents. All of these factors lead to reduced patient compliance and increased costs to the health care system.

[0003] There is a need for new and more effective methods of increasing the half-life and efficacy of therapeutic agents. Summary of the Invention

[0004] The present disclosure provides Fc domain monomers, conjugates comprising Fc domain monomers, and fusion proteins comprising Fc domain monomers, where the Fc domain monomers are mutant variants of a parent Fc polypeptide (e.g., an IgG1 or IgG2 polypeptide). The Fc domain monomers may contain one or more mutations that contribute to increased half-life and / or efficacy. The one or more mutations may also minimize aggregation during manufacturing, thereby increasing productivity and reducing costs. The Fc domain monomers may also be optimized for size (e.g., measured by kDa or amino acid residues) to maximize tissue distribution to tissues of interest and / or minimize renal clearance.

[0005] In one aspect, the disclosure provides a variant Fc domain monomer (e.g., a variant of a parent Fc polypeptide). The variant Fc domain monomer may comprise a substitution at amino acid position 220. The variant Fc domain monomer may comprise amino acid substitutions at position 220 and positions 252, 254, and 256. The variant Fc domain monomer may comprise amino acid substitutions at positions 309, 311, and 434. In some embodiments, the substitution at position 220 is serine, the substitution at position 252 is tyrosine, the substitution at position 254 is threonine, the substitution at position 256 is glutamic acid, the substitution at position 309 is aspartic acid, the substitution at position 311 is histidine, and / or the substitution at position 434 is serine. In some embodiments, the variant Fc domain monomer comprises substitutions at positions 220, 252, 254, and 256, where numbering is according to the EU index as Kabat, where the substitution at position 220 is serine, the substitution at position 252 is tyrosine, the substitution at position 254 is threonine, and the substitution at position 256 is glutamic acid. In some embodiments, the substitution at position 220 is cysteine ​​to serine (C220S). In some embodiments, the substitution at position 252 is methionine to tyrosine (M252Y). In some embodiments, the substitution at position 254 is serine to threonine (S254T). In some embodiments, the substitution at position 252 is threonine to glutamate (T256E). In some embodiments, the substitution at position 309 is valine to aspartic acid (V309D). In some embodiments, the substitution at position 311 is glutamine to histidine (Q311H). In some embodiments, the substitution at position 434 is asparagine to serine (N434S). Amino acid numbering of the variant Fc monomers shown above and throughout this disclosure is according to the EU index as per Kabat. The amino acid substitutions are relative to the wild-type Fc monomer amino acid sequence, e.g., wild-type human IgG1 or IgG2.

[0006] In some embodiments, the variant Fc domain monomer comprises fewer than about 300 amino acid residues (e.g., fewer than about 300, fewer than about 295, fewer than about 290, fewer than about 285, fewer than about 280, fewer than about 275, fewer than about 270, fewer than about 265, fewer than about 260, fewer than about 255, fewer than about 250, fewer than about 245, fewer than about 240, fewer than about 235, fewer than about 230, fewer than about 225, or fewer than about 220 amino acid residues). In some embodiments, the variant Fc domain monomer is less than about 40 kDa (e.g., less than about 35 kDa, less than about 30 kDa, less than about 25 kDa).

[0007] In some embodiments, the variant Fc domain monomer comprises at least 200 amino acid residues (e.g., at least 210, at least 220, at least 230, at least 240, at least 250, at least 260, at least 270, at least 280, at least 290, or at least 300 amino acid residues). In some embodiments, the variant Fc domain monomer is at least 20 kDa (e.g., at least 25 kDa, at least 30 kDa, or at least 35 kDa).

[0008] In some embodiments, the variant Fc domain monomer comprises 200-400 amino acid residues (e.g., 200-250, 250-300, 300-350, 350-400, 200-300, 250-350, or 300-400 amino acid residues). In some embodiments, the variant Fc domain monomer is between 200 and 300 amino acid residues in length (e.g., between 210-300, 230-300, 250-300, 270-300, 290-300, 210-290, 220-280, 230-270, 240-260, or 245-255 amino acid residues). In specific embodiments, the variant Fc domain monomer is between 240 and 255 amino acid residues (e.g., 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, or 254 amino acid residues). In even more specific embodiments, the variant Fc domain monomer is 246 amino acid residues in length. In some embodiments, the variant Fc domain monomer is 20 to 40 kDa (e.g., 20 to 25 kDa, 25 to 30 kDa, 35 to 40 kDa, 20 to 30 kDa, 25 to 35 kDa, or 30 to 40 kDa). In some embodiments, the variant Fc domain monomer has a mass between about 20 kDa and about 40 kDa (eg, 20 kDa to 25 kDa, 25 kDa to 30 kDa, 30 kDa to 35 kDa, 35 kDa to 40 kDa).

[0009] In some embodiments, a variant Fc domain monomer comprises an amino acid sequence, or a region thereof, that is at least 90% (e.g., at least 95%, at least 98%) identical to any one of SEQ ID NOs: 1-52 or 56-58. In some embodiments, a variant Fc domain monomer comprises an amino acid sequence, or a region thereof, that is at least 90% (e.g., at least 95%, at least 98%) identical to any one of SEQ ID NOs: 1-19. In some embodiments, a variant Fc domain monomer comprises an amino acid sequence, or a region thereof, that is at least 90% (e.g., at least 95%, at least 98%) identical to any one of SEQ ID NOs: 1-19.

[0010] In some embodiments, the variant Fc domain monomer comprises a region of any one of SEQ ID NOs: 1-19, 23-29, or 31, wherein the region comprises positions 220, 252, 254, and 256. In some embodiments, the region comprises at least 40 amino acid residues, at least 50 amino acid residues, at least 60 amino acid residues, at least 70 amino acid residues, at least 80 amino acid residues, at least 90 amino acid residues, at least 100 amino acid residues, at least 110 amino acid residues, at least 120 amino acid residues, at least 130 amino acid residues, at least 140 amino acid residues, at least 150 amino acid residues, at least 160 amino acid residues, at least 170 amino acid residues, at least 180 amino acid residues, at least 190 amino acid residues, or at least 200 amino acid residues.

[0011] In some embodiments, the variant Fc domain monomer comprises a region of any one of SEQ ID NOs: 31-52, wherein the region includes positions 220, 309, 311, and 434. In some embodiments, the region includes at least 215 amino acid residues, at least 220 amino acid residues, at least 225 amino acid residues, at least 230 amino acid residues, at least 235 amino acid residues, at least 240 amino acid residues, or at least 245 amino acid residues.

[0012] In another aspect, the disclosure provides a variant Fc domain monomer comprising a serine at amino acid position 220, where amino acid numbering is according to the EU index as Kabat, and the variant Fc domain monomer is between 200 and 300 amino acid residues in length (e.g., between 210 and 300, between 230 and 300, between 250 and 300, between 270 and 300, between 290 and 300, between 210 and 290, between 220 and 280, between 230 and 270, between 240 and 260, or between 245 and 255 amino acid residues). In some embodiments, the variant Fc domain monomer comprises a serine at amino acid position 220, a tyrosine at position 252, a threonine at position 254, and / or a glutamic acid at position 256. In some embodiments, the variant Fc domain monomer comprises a serine at amino acid position 220, an aspartic acid at position 309, a histidine at position 311, and / or a serine at position 434. In some embodiments, the variant Fc domain monomer further comprises one or more (1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) additional mutations (e.g., amino acid deletions, additions, and / or substitutions) relative to the corresponding human wild-type Fc sequence.

[0013] In another aspect, the disclosure provides a variant Fc domain monomer comprising a serine at amino acid position 220, where the amino acid numbering is according to the EU index as Kabat, and the variant Fc domain monomer has a mass of between about 20 kDa and about 40 kDa (e.g., 20 kDa to 25 kDa, 25 kDa to 30 kDa, 30 kDa to 35 kDa, 35 kDa to 40 kDa). In some embodiments, the variant Fc domain monomer comprises a serine at amino acid position 220, a tyrosine at position 252, a threonine at position 254, and / or a glutamic acid at position 256. In some embodiments, the variant Fc domain monomer comprises a serine at amino acid position 220, an aspartic acid at position 309, a histidine at position 311, and / or a serine at position 434.

[0014] In some embodiments, the variant Fc domain monomer is a variant of human IgG1 or human IgG2. In some embodiments, the variant Fc domain monomer is a variant of human IgG1.

[0015] In some embodiments, the N-terminus of the variant Fc domain monomer comprises between 10 and 20 residues of the Fab domain (e.g., residues 11, 12, 13, 14, 15, 16, 17, 18, or 19). In certain embodiments, the N-terminus of the variant Fc domain monomer is any one of amino acid residues 198-205. In some embodiments, the N-terminus of the variant Fc domain monomer is amino acid residue 201 (e.g., Asn201). In certain embodiments, the N-terminus of the variant Fc domain monomer is amino acid residue 202 (e.g., Val202). In other embodiments, the C-terminus of the variant Fc domain monomer is any one of amino acid residues 437-447. In another embodiment, the C-terminus of the variant Fc domain monomer is amino acid residue 446 (e.g., Gly446). In some embodiments, the C-terminus of the variant Fc domain monomer is amino acid residue 447 (eg, Lys447).

[0016] In some embodiments, the variant Fc domain monomer comprises an amino acid sequence, or a region thereof, that is at least 90% identical (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% identical) to a sequence of SEQ ID NOs: 1-29, 31-52, and 56-58 (e.g., SEQ ID NOs: 1-19, SEQ ID NOs: 20-29, SEQ ID NOs: 31-52, and 56-58).

[0017] In another aspect, the invention provides a variant Fc domain comprising a dimer of variant Fc domain monomers, each independently selected from any one of the variant Fc domain monomers described herein, wherein the variant Fc domain has a mass between about 50 kDa and about 70 kDa (e.g., about 51 kDa, about 52 kDa, about 53 kDa, about 54 kDa, about 55 kDa, about 56 kDa, about 57 kDa, about 58 kDa, about 59 kDa, about 60 kDa, about 61 kDa, about 62 kDa, about 63 kDa, about 64 kDa, about 65 kDa, about 66 kDa, about 67 kDa, about 68 kDa, or about 69 kDa). In some embodiments, the variant Fc domain monomers dimerize (e.g., homodimers or heterodimers) to form a variant Fc domain. In some embodiments, the variant Fc domain is at least 40 kDa (e.g., at least 45 kDa, at least 50 kDa, at least 55 kDa, at least 60 kDa, at least 65 kDa, at least 70 kDa, at least 75 kDa, or at least 80 kDa). In some embodiments, the variant Fc domain has a mass between 40 kDa and 80 kDa (e.g., between about 42 kDa and about 50 kDa, about 48 kDa and about 55 kDa, about 53 kDa and about 60 kDa, about 58 kDa and about 65 kDa, about 62 kDa and about 70 kDa, about 68 kDa and about 75 kDa, or about 72 kDa and about 80 kDa). In certain embodiments, the variant Fc domain is between 55 kDa and 62 kDa (e.g., about 56 kDa, about 57 kDa, about 58 kDa, about 59 kDa, about 60 kDa, or about 61 kDa). In preferred embodiments, the variant Fc domain is a homodimer comprising two variant Fc domain monomers (e.g., a homodimer in which each variant Fc domain monomer comprises any one of SEQ ID NOS: 1-52 or 56-58).

[0018] In another aspect, the disclosure provides a conjugate comprising a variant Fc domain described herein and at least one therapeutic agent, wherein the variant Fc domain monomer is covalently conjugated to the at least one therapeutic agent by a linker. In some embodiments, the conjugate has a structure represented by formula (1): [ka] wherein A is independently a therapeutic agent; each E comprises a variant Fc domain monomer or a polypeptide comprising a variant Fc domain monomer; L is a linker; n is 1 or 2; T is an integer from 1 to 20; The curved line connected to E indicates that each LA is covalently attached to E (e.g., by a linker or bond). or a pharmaceutically acceptable salt thereof.

[0019] In some embodiments, the therapeutic agent (A) is a small molecule therapeutic agent. In certain embodiments, the therapeutic agent (A) is a monomeric (e.g., single) small molecule therapeutic agent. In some embodiments, the therapeutic agent (A) is a multimer (e.g., 2 or more, 3 or more, 4 or more, or 5 or more) of small molecule therapeutic agents. In some embodiments, when (A) is a multimer (e.g., 2 or more, 3 or more, 4 or more, or 5 or more) of small molecule therapeutic agents, each of the (As) can be the same or a different small molecule agent. In certain embodiments, when the therapeutic agent (A) is a multimer (e.g., 2 or more, 3 or more, 4 or more, or 5 or more) of small molecule agents, each of the small molecule agents is linked by any linker described herein. In some embodiments, the linker has a trivalent structure (e.g., a trivalent linker). A trivalent linker has three arms, each arm covalently attached to a component of the conjugate (e.g., a first arm conjugated to a first therapeutic agent, a second arm conjugated to a second therapeutic agent, and a third arm conjugated to a fusion protein or variant Fc domain monomer).

[0020] In some embodiments, each linker comprises a polyethylene glycol (PEG) linker comprising between about 2 and 10 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) PEG units. In some embodiments, at least one arm of the trivalent linker comprises a polyethylene glycol (PEG) linker comprising between about 2 and 10 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) PEG units.

[0021] In some embodiments, the therapeutic agent (A) is an antiviral agent, an antifungal agent, or an antibacterial agent. In some embodiments, the therapeutic agent is an antiviral agent. In some embodiments, the therapeutic agent is an antifungal agent. In further embodiments, the therapeutic agent is an antibacterial agent.

[0022] In some embodiments, the conjugate is at least 40 kDa (e.g., at least 45 kDa, at least 50 kDa, at least 55 kDa, at least 60 kDa, at least 65 kDa, at least 70 kDa, at least 75 kDa, or at least 80 kDa). In some embodiments, the conjugate has a mass between about 40 kDa and about 80 kDa (e.g., 40 kDa to 50 kDa, 45 kDa to 55 kDa, 50 kDa to 60 kDa, 55 kDa to 65 kDa, 60 kDa to 70 kDa, 65 kDa to 75 kDa, or 70 kDa to 80 kDa). In certain embodiments, the conjugate has a mass between 58 kDa and 70 kDa (e.g., about 59 kDa, about 60 kDa, or about 61 kDa, 62 kDa, 63 kDa, 64 kDa, 65 kDa, 66 kDa, 67 kDa, 68 kDa, or 69 kDa).

[0023] In another aspect, the disclosure provides a fusion protein comprising a variant Fc domain monomer and at least one polypeptide therapeutic, wherein the variant Fc domain monomer is covalently conjugated to the polypeptide therapeutic by a linker. In some embodiments, the fusion protein has the structure: (P2-L2) n2 -B-(L1-P1) n1 wherein B is a variant Fc domain monomer, a polypeptide comprising a variant Fc domain monomer, or a conjugate (e.g., any conjugate described herein); P1 and P2 are each independently a polypeptide therapeutic; L1 and L2 are each independently a linker; n1 and n2 are each independently 0 or 1, and at least one of n1 and n2 is 1. Includes.

[0024] In some embodiments, the fusion protein comprises fewer than about 500 amino acid residues (e.g., less than about 495, less than about 490, less than about 485, less than about 480, less than about 475, less than about 470, less than about 465, less than about 460, less than about 455, less than about 450, less than about 445, less than about 440, less than about 435, less than about 430, less than about 425, less than about 420, less than about 415, less than about 410, less than about 405, less than about 400, less than about 395, less than about 390, less than about In some embodiments, the variant Fc domain monomer comprises less than about 50 kDa (e.g., less than about 45 kDa, less than about 40 kDa, less than about 35 kDa, less than about 350 kDa, less than about 345 kDa, less than about 340 kDa, less than about 335 kDa, less than about 330 kDa, less than about 325 kDa, less than about 320 kDa, less than about 315 kDa, less than about 310 kDa, less than about 305 kDa, less than about 300 kDa, less than about 295 kDa, less than about 290 kDa, less than about 285 kDa, less than about 280 kDa, less than about 275 kDa, less than about 270 kDa, less than about 265 kDa, less than about 260 kDa, or less than about 255 kDa).

[0025] In some embodiments, the fusion protein comprises at least 250 amino acid residues (e.g., at least about 250, at least about 260, at least about 270, at least about 280, at least about 290, at least about 300 amino acid residues, at least about 310, at least about 320, at least about 330, at least about 340, at least about 350, at least about 360, at least about 370, at least about 380, at least about 390, at least about 400, at least about 410, at least about 420, at least about 430, at least about 440, at least about 450, at least about 460, at least about 470, at least about 480, or at least about 490). In some embodiments, the fusion protein is at least about 30 kDa (e.g., at least about 35 kDa, at least about 40 kDa, or at least about 45 kDa).

[0026] In some embodiments, the fusion protein comprises 250-500 amino acid residues (e.g., 250-300, 300-350, 350-400, 200-300, 250-350, 300-400, 350-450, or 400-500 amino acid residues). In some embodiments, the variant Fc domain monomer is 30-50 kDa (e.g., 30-35 kDa, 30-40 kDa, 35-45 kDa, or 40-50 kDa).

[0027] In some embodiments, the therapeutic polypeptides each independently comprise fewer than about 200 amino acid residues (e.g., fewer than about 195, fewer than about 190, fewer than about 185, fewer than about 180, fewer than about 175, fewer than about 170, fewer than about 165, fewer than about 160, fewer than about 155, fewer than about 150, fewer than about 145, fewer than about 140, fewer than about 135, fewer than about 130, fewer than about 125, fewer than about 120, fewer than about 115, fewer than about 110, fewer than about 105, fewer than about 100, fewer than about 95, fewer than about 90, fewer than about 85, fewer than about 80, fewer than about 75, fewer than about 70, fewer than about 65, fewer than about 60, fewer than about 55, fewer than about 50, fewer than about 45, fewer than about 40, fewer than about 35, fewer than about 30, fewer than about 25, fewer than about 20, or fewer than about 15 amino acid residues).

[0028] In some embodiments, the therapeutic polypeptides each independently comprise at least about 10 amino acid residues (e.g., at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95 amino acid residues, at least about 100, at least about 105, at least about 110, at least about 115, at least about 120, at least about 125, at least about 130, at least about 135, at least about 140, at least about 145, at least about 150, at least about 155, at least about 160, at least about 165, at least about 170, at least about 175, at least about 180, at least about 185, at least about 190, or at least about 195 amino acid residues).

[0029] In some embodiments, n1 is 1 and n2 is 0, and the fusion protein has the structure: B-L1-P1 Includes.

[0030] In some embodiments, a linker (L1) is conjugated to the C-terminus of the Fc domain monomer (B) and the N-terminus of the polypeptide therapeutic agent (P1). In some embodiments, a linker (L1) is conjugated to the N-terminus of the Fc domain monomer (B) and the C-terminus of the polypeptide therapeutic agent (P1). In some embodiments, L1 is a peptide linker comprising between 2 and 200 amino acids. In some embodiments, L1 is a peptide linker comprising between 5 and 25 amino acids. In some embodiments, L1 is a peptide linker comprising (GS) x , (GGS) x , (GGGGS) x , (GGSG) x , (SGGG) x(x is an integer from 1 to 10). In some embodiments, B, L1, and P1 are expressed as a single polypeptide chain. In some embodiments, the linker (L1) is conjugated to the N-terminus of the Fc domain monomer (B) and the N-terminus of the polypeptide therapeutic agent (P1). In some embodiments, the linker (L1) is conjugated to the C-terminus of the Fc domain monomer (B) and the C-terminus of the polypeptide therapeutic agent (P1). In some embodiments, L1 comprises a chemical linker covalently conjugated to each of B and P1. In some embodiments, B and P1 are expressed as separate polypeptide chains and then each is covalently conjugated to L1.

[0031] In some embodiments, n1 is 1, n2 is 1, and the fusion protein has the structure: P2-L2-B-L1-P1 Includes.

[0032] In some embodiments, linker (L2) is conjugated to the C-terminus of the polypeptide therapeutic (P2) and the N-terminus of the Fc domain monomer (B), and linker (L1) is conjugated to the C-terminus of the Fc domain monomer (B) and the N-terminus of the polypeptide therapeutic (P1). In some embodiments, L1 and L2 are each independently selected peptide linkers comprising between 2 and 200 amino acids. In some embodiments, L1 and L2 are each independently selected peptide linkers comprising between 5 and 25 amino acids. In some embodiments, L1 and L2 are each independently selected peptide linkers comprising between 5 and 25 amino acids. In some embodiments, L1 and L2 are each independently selected peptide linkers comprising between 5 and 25 amino acids. x , (GGS) x , (GGGGS) x , (GGSG) x , (SGGG) x(x is an integer from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10)). In some embodiments, P2, L2, B, L1, and P1 are expressed together as a single polypeptide chain. In some embodiments, linker (L2) is conjugated to the N-terminus of the polypeptide therapeutic agent (P2) and the N-terminus of the Fc domain monomer (B), and linker (L1) is conjugated to the N-terminus of the polypeptide therapeutic agent (P1) and the C-terminus of the Fc domain monomer (B). In some embodiments, linker (L2) is conjugated to the C-terminus of the polypeptide therapeutic agent (P2) and the N-terminus of the Fc domain monomer (B), and linker (L1) is conjugated to the C-terminus of the polypeptide therapeutic agent (P1) and the C-terminus of the Fc domain monomer (B). In some embodiments, L2 comprises a chemical linker covalently conjugated to each of B and P2, and L1 comprises a chemical linker covalently conjugated to each of B and P1. In some embodiments, P2, B, and P1 are expressed as separate polypeptide chains, and P2 and B are then each covalently conjugated to L2, and P1 and B are each then covalently conjugated to L1.

[0033] In some embodiments of any aspect described herein, the variant Fc domain monomers dimerize to form an Fc domain, hi some embodiments, each of the variant Fc domain monomers in the Fc domain have the same amino acid sequence, thereby forming a homodimeric Fc domain.

[0034] In another aspect, the present disclosure provides a pharmaceutical composition comprising any variant Fc domain monomer described herein, any conjugate described herein, any fusion protein described herein, or any Fc domain, and a pharmaceutically acceptable carrier.

[0035] In another aspect, the disclosure provides a method of treating or preventing a respiratory disorder in a subject, the method comprising administering any of the compositions described herein to the subject. In some embodiments, the respiratory disorder is an infectious disease. In some embodiments, the infectious disease is a viral infection. In some embodiments, the viral infection is selected from the group including RSV, influenza, dengue, betacoronavirus (e.g., COVID-19), and Zika virus. In some embodiments, the infectious disease is a bacterial infection. In some embodiments, the respiratory disorder is selected from the group including chronic obstructive pulmonary disease (COPD), chronic bronchitis, cystic fibrosis, bronchiectasis, and pneumonia.

[0036] In some embodiments, the ratio of the concentration of the Fc domain monomer, conjugate, fusion protein, or Fc domain in the epithelial lining fluid is at least 30% of the concentration of the Fc domain monomer, conjugate, fusion protein, or Fc domain in the plasma within 2 hours after administration. In some embodiments, the ratio of the concentrations is at least 45% within 2 hours after administration. In some embodiments, the ratio of the concentrations is at least 55% within 2 hours after administration. In some embodiments, the ratio of the concentrations is at least 60% within 2 hours after administration. In certain embodiments of the above, the route of administration is by injection, e.g., intramuscular, subcutaneous, intraperitoneal, or intravenous injection. In certain embodiments of the above, the route of administration is oral.

[0037] In another aspect, the present disclosure provides a method for treating or preventing liver damage in a subject, the method comprising administering any of the compositions described herein to the subject. In some embodiments, the liver damage is an infection (e.g., a viral infection such as hepatitis A, hepatitis B, or hepatitis C), a fungal infection, or a bacterial infection. In some embodiments, the liver damage is selected from the group consisting of primary cholangitis, primary sclerosing cholangitis, hepatocellular carcinoma, cholangiocarcinoma, hepatocellular adenoma, nonalcoholic fatty liver disease (NAFLD), acute liver failure, and cirrhosis.

[0038] In another aspect, the present disclosure provides a method for treating or preventing a central nervous system (CNS) disorder in a subject, the method comprising administering any of the compositions described herein to the subject. In some embodiments, the CNS disorder is an infectious disease. In some embodiments, the infectious disease is a viral infection, a bacterial infection, or a fungal infection. In some embodiments, the viral infection is selected from the group including herpes simplex virus (HSV) 1, HSV2, Epstein-Barr virus, varicella-zoster virus, poliovirus, coxsackievirus, West Nile virus, La Crosse virus, western equine encephalitis, eastern equine encephalitis, Powassan virus, or rabies virus. In some embodiments, the CNS disorder is selected from the group including cancer, Alzheimer's disease, Parkinson's disease, epilepsy, multiple sclerosis, schizophrenia, and meningitis.

[0039] In another aspect, the present disclosure provides a method of treating or preventing a muscle disorder in a subject, the method comprising administering any of the compositions described herein to the subject. In some embodiments, the muscle disorder is myositis or cancer. In some embodiments, the myositis is caused by injury, infection, or an immune disorder.

[0040] In another aspect, the disclosure provides a method of treating or preventing a skin disorder in a subject, the method comprising administering any of the compositions described herein to the subject. In some embodiments, the skin disorder is an infection (e.g., a viral infection (HSV1, HSV2, or varicella-zoster virus), a fungal infection, or a bacterial infection. In some embodiments, the skin disorder is selected from the group including eczema, psoriasis, acne, rosacea, herpes, cellulitis, basal cell carcinoma, squamous cell carcinoma, and melanoma.

[0041] In another aspect, the disclosure provides a method of treating or preventing an ocular disorder in a subject, the method comprising administering any of the compositions described herein to the subject. In some embodiments, the ocular disorder is an infection (e.g., a viral infection (HSV1 or HSV2), a fungal infection, or a bacterial infection. In some embodiments, the ocular disorder is selected from age-related macular degeneration, cataracts, and glaucoma.

[0042] In another aspect, the present disclosure provides a method of treating or preventing a vascular disorder in a subject, the method comprising administering to the subject any of the compositions described herein. In some embodiments, the vascular disorder is an infection (e.g., a viral infection, a fungal infection, or a bacterial infection).

[0043] In some embodiments of any of the aspects described herein, the variant Fc domain monomer (e.g., each variant Fc domain monomer of an Fc domain) comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the variant Fc domain monomer comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 1.

[0044] In some embodiments of any of the aspects described herein, the variant Fc domain monomer (e.g., each variant Fc domain monomer of an Fc domain) comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the variant Fc domain monomer comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 2.

[0045] In some embodiments of any of the aspects described herein, the variant Fc domain monomer (e.g., each variant Fc domain monomer of an Fc domain) comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments, the variant Fc domain monomer comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 3.

[0046] In some embodiments of any of the aspects described herein, the variant Fc domain monomer (e.g., each variant Fc domain monomer of an Fc domain) comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, the variant Fc domain monomer comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 4.

[0047] In some embodiments of any of the aspects described herein, the variant Fc domain monomer (e.g., each variant Fc domain monomer of an Fc domain) comprises the amino acid sequence of SEQ ID NO: 5. In some embodiments, the variant Fc domain monomer comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 5.

[0048] In some embodiments of any of the aspects described herein, the variant Fc domain monomer (e.g., each variant Fc domain monomer of an Fc domain) comprises the amino acid sequence of SEQ ID NO: 6. In some embodiments, the variant Fc domain monomer comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 6.

[0049] In some embodiments of any of the aspects described herein, the variant Fc domain monomer (e.g., each variant Fc domain monomer of an Fc domain) comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the variant Fc domain monomer comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:8.

[0050] In some embodiments of any of the aspects described herein, the variant Fc domain monomer (e.g., each variant Fc domain monomer of an Fc domain) comprises the amino acid sequence of SEQ ID NO: 9. In some embodiments, the variant Fc domain monomer comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 9.

[0051] In some embodiments of any of the aspects described herein, the variant Fc domain monomer (e.g., each variant Fc domain monomer of an Fc domain) comprises the amino acid sequence of SEQ ID NO: 10. In some embodiments, the variant Fc domain monomer comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 10.

[0052] In some embodiments of any of the aspects described herein, the variant Fc domain monomer (e.g., each variant Fc domain monomer of an Fc domain) comprises the amino acid sequence of SEQ ID NO: 11. In some embodiments, the variant Fc domain monomer comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 11.

[0053] In some embodiments of any of the aspects described herein, the variant Fc domain monomer (e.g., each variant Fc domain monomer of an Fc domain) comprises the amino acid sequence of SEQ ID NO: 12. In some embodiments, the variant Fc domain monomer comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 12.

[0054] In some embodiments of any of the aspects described herein, the variant Fc domain monomer (e.g., each variant Fc domain monomer of an Fc domain) comprises the amino acid sequence of SEQ ID NO: 13. In some embodiments, the variant Fc domain monomer comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 13.

[0055] In some embodiments of any of the aspects described herein, the variant Fc domain monomer (e.g., each variant Fc domain monomer of an Fc domain) comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments, the variant Fc domain monomer comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 14.

[0056] In some embodiments of any of the aspects described herein, the variant Fc domain monomer (e.g., each variant Fc domain monomer of an Fc domain) comprises the amino acid sequence of SEQ ID NO: 15. In some embodiments, the variant Fc domain monomer comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 15.

[0057] In some embodiments of any of the aspects described herein, the variant Fc domain monomer (e.g., each variant Fc domain monomer of an Fc domain) comprises the amino acid sequence of SEQ ID NO: 16. In some embodiments, the variant Fc domain monomer comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 16.

[0058] In some embodiments of any of the aspects described herein, the variant Fc domain monomer (e.g., each variant Fc domain monomer of an Fc domain) comprises the amino acid sequence of SEQ ID NO: 17. In some embodiments, the variant Fc domain monomer comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 17.

[0059] In some embodiments of any of the aspects described herein, the variant Fc domain monomer (e.g., each variant Fc domain monomer of an Fc domain) comprises the amino acid sequence of SEQ ID NO: 18. In some embodiments, the variant Fc domain monomer comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 18.

[0060] In some embodiments of any of the aspects described herein, the variant Fc domain monomer (e.g., each variant Fc domain monomer of an Fc domain) comprises the amino acid sequence of SEQ ID NO: 19. In some embodiments, the variant Fc domain monomer comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 19.

[0061] In some embodiments of any of the aspects described herein, the variant Fc domain monomer (e.g., each variant Fc domain monomer of an Fc domain) comprises the amino acid sequence of SEQ ID NO: 20. In some embodiments, the variant Fc domain monomer comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 20.

[0062] In some embodiments of any of the aspects described herein, the variant Fc domain monomer (e.g., each variant Fc domain monomer of an Fc domain) comprises the amino acid sequence of SEQ ID NO: 21. In some embodiments, the variant Fc domain monomer comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 21.

[0063] In some embodiments of any of the aspects described herein, the variant Fc domain monomer (e.g., each variant Fc domain monomer of an Fc domain) comprises the amino acid sequence of SEQ ID NO: 22. In some embodiments, the variant Fc domain monomer comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 22.

[0064] In some embodiments of any of the aspects described herein, the variant Fc domain monomer (e.g., each variant Fc domain monomer of an Fc domain) comprises the amino acid sequence of SEQ ID NO: 23. In some embodiments, the variant Fc domain monomer comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 23.

[0065] In some embodiments of any of the aspects described herein, the variant Fc domain monomer (e.g., each variant Fc domain monomer of an Fc domain) comprises the amino acid sequence of SEQ ID NO: 24. In some embodiments, the variant Fc domain monomer comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 24.

[0066] In some embodiments of any of the aspects described herein, the variant Fc domain monomer (e.g., each variant Fc domain monomer of an Fc domain) comprises the amino acid sequence of SEQ ID NO: 25. In some embodiments, the variant Fc domain monomer comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 25.

[0067] In some embodiments of any of the aspects described herein, the variant Fc domain monomer (e.g., each variant Fc domain monomer of an Fc domain) comprises the amino acid sequence of SEQ ID NO: 26. In some embodiments, the variant Fc domain monomer comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 26.

[0068] In some embodiments of any of the aspects described herein, the variant Fc domain monomer (e.g., each variant Fc domain monomer of an Fc domain) comprises the amino acid sequence of SEQ ID NO: 27. In some embodiments, the variant Fc domain monomer comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 27.

[0069] In some embodiments of any of the aspects described herein, the variant Fc domain monomer (e.g., each variant Fc domain monomer of an Fc domain) comprises the amino acid sequence of SEQ ID NO: 28. In some embodiments, the variant Fc domain monomer comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 28.

[0070] In some embodiments of any of the aspects described herein, the variant Fc domain monomer (e.g., each variant Fc domain monomer of an Fc domain) comprises the amino acid sequence of SEQ ID NO: 29. In some embodiments, the variant Fc domain monomer comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 29.

[0071] In some embodiments of any of the aspects described herein, the variant Fc domain monomer (e.g., each variant Fc domain monomer of an Fc domain) comprises the amino acid sequence of SEQ ID NO: 30. In some embodiments, the variant Fc domain monomer comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 30.

[0072] In some embodiments of any of the aspects described herein, the variant Fc domain monomer (e.g., each variant Fc domain monomer of an Fc domain) comprises the amino acid sequence of SEQ ID NO: 31. In some embodiments, the variant Fc domain monomer comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 31.

[0073] In some embodiments of any of the aspects described herein, the variant Fc domain monomer (e.g., each variant Fc domain monomer of an Fc domain) comprises the amino acid sequence of SEQ ID NO: 32. In some embodiments, the variant Fc domain monomer comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 32.

[0074] In some embodiments of any of the aspects described herein, the variant Fc domain monomer (e.g., each variant Fc domain monomer of an Fc domain) comprises the amino acid sequence of SEQ ID NO: 33. In some embodiments, the variant Fc domain monomer comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 33.

[0075] In some embodiments of any of the aspects described herein, the variant Fc domain monomer (e.g., each variant Fc domain monomer of an Fc domain) comprises the amino acid sequence of SEQ ID NO: 34. In some embodiments, the variant Fc domain monomer comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 34.

[0076] In some embodiments of any of the aspects described herein, the variant Fc domain monomer (e.g., each variant Fc domain monomer of an Fc domain) comprises the amino acid sequence of SEQ ID NO: 35. In some embodiments, the variant Fc domain monomer comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 35.

[0077] In some embodiments of any of the aspects described herein, the variant Fc domain monomer (e.g., each variant Fc domain monomer of an Fc domain) comprises the amino acid sequence of SEQ ID NO: 36. In some embodiments, the variant Fc domain monomer comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 36.

[0078] In some embodiments of any of the aspects described herein, the variant Fc domain monomer (e.g., each variant Fc domain monomer of an Fc domain) comprises the amino acid sequence of SEQ ID NO: 37. In some embodiments, the variant Fc domain monomer comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 37.

[0079] In some embodiments of any of the aspects described herein, the variant Fc domain monomer (e.g., each variant Fc domain monomer of an Fc domain) comprises the amino acid sequence of SEQ ID NO: 38. In some embodiments, the variant Fc domain monomer comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 38.

[0080] In some embodiments of any of the aspects described herein, the variant Fc domain monomer (e.g., each variant Fc domain monomer of an Fc domain) comprises the amino acid sequence of SEQ ID NO: 39. In some embodiments, the variant Fc domain monomer comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 39.

[0081] In some embodiments of any of the aspects described herein, the variant Fc domain monomer (e.g., each variant Fc domain monomer of an Fc domain) comprises the amino acid sequence of SEQ ID NO: 40. In some embodiments, the variant Fc domain monomer comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 40.

[0082] In some embodiments of any of the aspects described herein, the variant Fc domain monomer (e.g., each variant Fc domain monomer of an Fc domain) comprises the amino acid sequence of SEQ ID NO: 41. In some embodiments, the variant Fc domain monomer comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 41.

[0083] In some embodiments of any of the aspects described herein, the variant Fc domain monomer (e.g., each variant Fc domain monomer of an Fc domain) comprises the amino acid sequence of SEQ ID NO: 42. In some embodiments, the variant Fc domain monomer comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 42.

[0084] In some embodiments of any of the aspects described herein, the variant Fc domain monomer (e.g., each variant Fc domain monomer of an Fc domain) comprises the amino acid sequence of SEQ ID NO: 43. In some embodiments, the variant Fc domain monomer comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 43.

[0085] In some embodiments of any of the aspects described herein, the variant Fc domain monomer (e.g., each variant Fc domain monomer of an Fc domain) comprises the amino acid sequence of SEQ ID NO: 44. In some embodiments, the variant Fc domain monomer comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 44.

[0086] In some embodiments of any of the aspects described herein, the variant Fc domain monomer (e.g., each variant Fc domain monomer of an Fc domain) comprises the amino acid sequence of SEQ ID NO: 45. In some embodiments, the variant Fc domain monomer comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 45.

[0087] In some embodiments of any of the aspects described herein, the variant Fc domain monomer (e.g., each variant Fc domain monomer of an Fc domain) comprises the amino acid sequence of SEQ ID NO: 46. In some embodiments, the variant Fc domain monomer comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 46.

[0088] In some embodiments of any of the aspects described herein, the variant Fc domain monomer (e.g., each variant Fc domain monomer of an Fc domain) comprises the amino acid sequence of SEQ ID NO: 47. In some embodiments, the variant Fc domain monomer comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 47.

[0089] In some embodiments of any of the aspects described herein, the variant Fc domain monomer (e.g., each variant Fc domain monomer of an Fc domain) comprises the amino acid sequence of SEQ ID NO: 48. In some embodiments, the variant Fc domain monomer comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 48.

[0090] In some embodiments of any of the aspects described herein, the variant Fc domain monomer (e.g., each variant Fc domain monomer of an Fc domain) comprises the amino acid sequence of SEQ ID NO: 49. In some embodiments, the variant Fc domain monomer comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 49.

[0091] In some embodiments of any of the aspects described herein, the variant Fc domain monomer (e.g., each variant Fc domain monomer of an Fc domain) comprises the amino acid sequence of SEQ ID NO: 50. In some embodiments, the variant Fc domain monomer comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 50.

[0092] In some embodiments of any of the aspects described herein, the variant Fc domain monomer (e.g., each variant Fc domain monomer of an Fc domain) comprises the amino acid sequence of SEQ ID NO: 51. In some embodiments, the variant Fc domain monomer comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 51.

[0093] In some embodiments of any of the aspects described herein, the variant Fc domain monomer (e.g., each variant Fc domain monomer of an Fc domain) comprises the amino acid sequence of SEQ ID NO: 52. In some embodiments, the variant Fc domain monomer comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 52.

[0094] In some embodiments of any of the aspects described herein, the variant Fc domain monomer (e.g., each variant Fc domain monomer of an Fc domain) comprises the amino acid sequence of SEQ ID NO: 56. In some embodiments, the variant Fc domain monomer comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 56.

[0095] In some embodiments of any of the aspects described herein, the variant Fc domain monomer (e.g., each variant Fc domain monomer of an Fc domain) comprises the amino acid sequence of SEQ ID NO: 57. In some embodiments, the variant Fc domain monomer comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 57.

[0096] In some embodiments of any of the aspects described herein, the variant Fc domain monomer (e.g., each variant Fc domain monomer of an Fc domain) comprises the amino acid sequence of SEQ ID NO: 58. In some embodiments, the variant Fc domain monomer comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 58.

[0097] definition To facilitate understanding of the present invention, a number of terms are defined below. Terms defined herein have meanings commonly understood by one of ordinary skill in the art relevant to the present invention. Terms such as "a," "an," and "the" are not intended to refer exclusively to the singular, but include the general class of which a specific example may be used for illustration. While terminology herein is used to describe particular embodiments of the present invention, their usage does not limit the invention except as set forth in the claims.

[0098] As used herein, the term "variant Fc domain monomer" refers to a variant Fc domain monomer that contains at least a hinge domain and a second and third antibody constant domain (C H 2 and C H3) or functional fragments thereof (e.g., fragments that are capable of (i) dimerizing with another variant Fc domain monomer to form a variant Fc domain, and (ii) binding to an Fc receptor). In some embodiments, a variant Fc domain monomer comprises at least the following quadruple mutation: C220S / M252Y / S254T / T256E. In some embodiments, a variant Fc domain monomer comprises at least the quadruple mutation: C220S / V309D / Q311H / N434S. In some embodiments, a variant Fc domain monomer has a mutation comprising C220S. A variant Fc domain monomer having any of the above amino acid substitutions may further comprise one or more (1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) additional mutations (e.g., amino acid deletions, additions, and / or substitutions) relative to a corresponding human wild-type Fc sequence, e.g., a wild-type human IgG sequence. The variant Fc domain monomer can be an IgG subtype (e.g., IgG1, IgG2a, or IgG2b) (e.g., IgG1). The variant Fc domain monomer does not include any portion of an immunoglobulin that can act as an antigen recognition region, e.g., a variable domain or a complementarity-determining region (CDR). In some embodiments, the variant Fc domain monomer includes between 10 and 20 (e.g., 11, 12, 13, 14, 15, 16, 17, 18, or 19) amino acid residues of a Fab region. In some embodiments, a variant Fc domain monomer (e.g., an IgG heavy chain, e.g., IgG1) comprises a region extending from either Asn201 or Glu216 (e.g., Asn201, Val202, Asn203, His204, Lys205, Pro206, Ser207, Asn208, Thr209, Lys210, Val211, Asp212, Lys213, Lys214, Val215, or Glu216) to the carboxyl terminus of the heavy chain, e.g., Gly446 or Lys447. The C-terminal Lys447 of the Fc region may or may not be present without affecting the structure or stability of the Fc region. The present disclosure specifically contemplates any of SEQ ID NOs: 1-29 and 31-52 that do not include the C-terminal Lys corresponding to Lys447.Variant Fc domain monomers may be expressed that include a C-terminal Lys447, which can then be proteolytically cleaved after expression of the polypeptide (e.g., variant Fc domain monomers are expressed using a nucleic acid construct encoding a variant Fc domain monomer that includes a C-terminal lysine residue). Variant Fc domain monomers may also be expressed without a C-terminal Lys447. The N-terminal Asn201 may be deamidated after expression of the polypeptide. The N-terminal Asn201 of a variant Fc domain monomer may or may not be present. The presence or absence of the N-terminal Asn201 and / or C-terminal Lys447 does not affect the structure or stability of the variant Fc domain monomer. The present disclosure specifically contemplates any of SEQ ID NOs: 1-29, 31-52, and 56-58 that do not include the N-terminal Asn201 residue. Unless otherwise specified herein, the numbering of amino acid residues in variant Fc domain monomers is according to the EU numbering system for antibodies, also referred to as the Kabat EU index, as described, for example, in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0099] As used herein, the term "variant Fc domain" refers to a dimer of two variant Fc domain monomers that is capable of binding, for example, to an Fc receptor. In a wild-type Fc domain, the two Fc domain monomers are linked together by two C H The three antibody constant domains dimerize through interactions between them, and in some embodiments, one or more disulfide bonds form between the hinge domains of the two dimerized Fc domain monomers.

[0100] The terms "Fab" or "Fragment antigen-binding," used interchangeably herein, refer to the region of an antibody that binds to an antigen. Fab is a technical term, and its meaning is known to those skilled in the art. The Fab region is composed of one constant domain and one variable domain from each of the heavy and light chains. Each heavy chain contains a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region may contain three domains: CH1, CH2, and / or CH3. Each light chain contains a light chain variable region (VL) and a light chain constant region (CL). The VH and VL regions can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). In antibodies, the heavy chain (e.g., the VH and CH regions) are linked to the Fc domain monomer by a hinge. The variant Fc domain monomers described herein may comprise between residues 10 and / or 20 of the Fab domain (e.g., residues 11, 12, 13, 14, 15, 16, 17, 18, or 19) and the hinge region. In certain embodiments, the N-terminus of the variant Fc domain monomer is any one of amino acid residues 198-205 (corresponding to residues of the Fab domain). In some embodiments, the N-terminus of the variant Fc domain monomer is amino acid residue 201 (e.g., Asn201). In certain embodiments, the N-terminus of the variant Fc domain monomer is amino acid residue 202 (e.g., Val202).

[0101] The term "covalently bonded" refers to two portions of a conjugate that are joined together by a covalent bond formed between two atoms in the two portions of the conjugate.

[0102] As used herein, a "surface-exposed amino acid," or a "solvent-exposed amino acid," e.g., a surface-exposed cysteine ​​or a surface-exposed lysine, refers to an amino acid that is accessible to the solvent surrounding a protein. A surface-exposed amino acid can be a naturally occurring or engineered variant (e.g., substitution or insertion) of a protein. In some embodiments, a surface-exposed amino acid is an amino acid that, when substituted, does not substantially change the three-dimensional structure of a protein.

[0103] The term "optionally substituted," as used herein, refers to having zero, one, or more substituents, e.g., 0 to 25, 0 to 20, 0 to 10, or 0 to 5 substituents. Substituents include, but are not limited to, alkyl, alkenyl, alkynyl, aryl, alkaryl, acyl, heteroaryl, heteroalkyl, heteroalkenyl, heteroalkynyl, heteroalkaryl, halogen, oxo, cyano, nitro, amino, alkamino, hydroxy, alkoxy, alkanoyl, carbonyl, carbamoyl, guanidinyl, ureido, amidinyl, any of the above groups or moieties, and heteroversions of any of the above groups or moieties. Substituents include, but are not limited to, F, Cl, methyl, phenyl, benzyl, OR, NR, SR, SOR, SO,R, OCOR, NRCOR, NRCONR, NRCOOR, OCONR, RCO, COOR, alkyl-OOCR, SO,R, CONR, SO,NR, NRSO,NR, CN, CF, OCF, SiR, and NO, where each R is independently H, alkyl, alkenyl, aryl, heteroalkyl, heteroalkenyl, or heteroaryl, and any two of the substituents on the same or adjacent atoms can be joined together to form a fused, optionally substituted, aromatic or non-aromatic, saturated or unsaturated ring containing 3 to 8 members, or any two of the substituents on the same atom can be joined to form an optionally substituted, aromatic or non-aromatic, saturated or unsaturated ring containing 3 to 8 members.

[0104] The term "amino acid," as used herein, refers to naturally occurring and non-naturally occurring amino acids.

[0105] The term "naturally occurring amino acid" as used herein means amino acids including Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val.

[0106] The term "non-naturally occurring amino acid," as used herein, refers to an alpha amino acid that is not naturally occurring or found in mammals. Examples of non-naturally occurring amino acids include D-amino acids; amino acids having an acetylaminomethyl group attached to the sulfur atom of cysteine; pegylated amino acids; amino acids of the formula NH(CH) nOmega amino acids with COOH (n is 2-6), neutral nonpolar amino acids such as sarcosine, t-butylalanine, t-butylglycine, N-methylisoleucine, and norleucine; oxymethionine; phenylglycine; citrulline; methionine sulfoxide; cysteic acid; ornithine; diaminoalanine; 3-hydroxy-D-proline; 2,4-diaminobutyric acid; 2-aminopentanoic acid; 2-aminooctanoic acid, 2-carboxypiperazine; piperazine-2-carboxylic acid, 2-amino-4-phenylbutanoic acid; 3-(2-naphthyl)alanine, and hydroxyproline. Other amino acids include α-aminobutyric acid, α-amino-α-methylbutyrate, aminocyclopropane-carboxylate, aminoisobutyric acid, aminonorbornyl-carboxylate, L-cyclohexylalanine, cyclopentylalanine, LN-methylleucine, LN-methylmethionine, LN-methylnorvaline, LN-methylphenylalanine, LN-methylproline, LN-methylserine, LN-methyltryptophan, D-ornithine, LN-methylethylglycine, L-norleucine, α-methyl-aminoisobutyrate, α-methylcyclohexylalanine, D-α-methylalanine, D-α-methylarginine, D-α-methylasparagine, D-α-methylaspartate, D-α-methylcysteine, D-α-methylglutamine, D-α-methylhistidine, D -α-methylisoleucine, D-α-methylleucine, D-α-methyllysine, D-α-methylmethionine, D-α-methylornithine, D-α-methylphenylalanine, D-α-methylproline, D-α-methylserine, DN-methylserine, D-α-methylthreonine, D-α-methyltryptophan, D-α-methyltyrosine, D-α-methylvaline, DN-methylalanine, DN-methylarginine, DN-methylasparagine, DN-methylaspartate, DN-methylcysteine, DN-methylglutamine, DN-methylglutamate, DN-methylhistidine, DN-methylisoleucine, DN-methylleucine, DN-methyllysine, N-methylcyclohexylalanine, DN-methylornithine, N-methylglycine, N-methylaminoisobutyrate,N-(1-methylpropyl)glycine, N-(2-methylpropyl)glycine, DN-methyltryptophan, DN-methyltyrosine, DN-methylvaline, γ-aminobutyric acid, Lt-butylglycine, L-ethylglycine, L-homophenylalanine, L-α-methylarginine, L-α-methylaspartate, L-α-methylcysteine, L-α-methylglutamine, L-α-methylhistidine, L-α-methylisoleucine, L-α-methylleucine, L-α-methylmethionine, L-α-methylnorvaline, L-α-methylphenylalanine L-α-methylserine, L-α-methyltryptophan, L-α-methylvaline, N-(N-(2,2-diphenylethyl)carbamylmethylglycine, 1-carboxy-1-(2,2-diphenyl-ethylamino)cyclopropane, 4-hydroxyproline, ornithine, 2-aminobenzoyl(anthraniloyl), D-cyclohexylalanine, 4-phenyl-phenylalanine, L-citrulline, α-cyclohexylglycine, L-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, L-thiazolidine-4-carboxylic acid, L -Homotyrosine, L-2-furylalanine, L-histidine (3-methyl), N-(3-guanidinopropyl)glycine, O-methyl-L-tyrosine, O-glycan-serine, meta-tyrosine, nor-tyrosine, LN,N',N''-trimethyllysine, homolysine, norlysine, N-glycan asparagine, 7-hydroxy-1,2,3,4-tetrahydro-4-fluorophenylalanine, 4-methylphenylalanine, bis-(2-picolyl)amine, pentafluorophenylalanine, indoline-2-carboxylic acid, 2-aminobenzoic acid, 3-amino-2-naphthoic acid, asymmetric dimethylarginine, L-tetrahydroisoquinoline-1-carboxylic acid, D-tetrahydroisoquinoline-1-carboxylic acid, 1-amino-cyclohexaneacetic acid, D / L-allylglycine, 4-aminobenzoic acid, 1-amino-cyclobutanecarboxylic acid, 2 or 3 or 4-aminocyclohexanecarboxylic acid, 1-amino-1-cyclopentanecarboxylic acid, 1-aminoindan-1-carboxylic acid, 4-amino-pyrrolidine-2-carboxylic acid, 2-aminotetralin-2-carboxylic acid, azetidine-3-carboxylic acid,4-Benzyl-pyrrolidine-2-carboxylic acid, tert-butylglycine, b-(benzothiazolyl-2-yl)-alanine, b-cyclopropylalanine, 5,5-dimethyl-1,3-thiazolidine-4-carboxylic acid, (2R,4S)4-hydroxypiperidine-2-carboxylic acid, (2S,4S) and (2S,4R)-4-(2-naphthylmethoxy)-pyrrolidine-2-carboxylic acid, (2S,4S) and (2S,4R)4-phenoxy-pyrrolidine-2-carboxylic acid, (2R,5S) and (2S,5R)-5-phenyl-pyrrolidine-2-carboxylic acid Carboxylic acid, (2S,4S)-4-amino-1-benzoyl-pyrrolidine-2-carboxylic acid, t-butylalanine, (2S,5R)-5-phenyl-pyrrolidine-2-carboxylic acid, 1-aminomethyl-cyclohexane-acetic acid, 3,5-bis-(2-amino)ethoxy-benzoic acid, 3,5-diamino-benzoic acid, 2-methylamino-benzoic acid, N-methylanthranilic acid, LN-methylalanine, LN-methylarginine, LN-methylasparagine, LN-methylaspartic acid, LN-methylcysteine, LN-methylglutamine, LN-methyl Glutamic acid, LN-methylhistidine, LN-methylisoleucine, LN-methyllysine, LN-methylnorleucine, LN-methylornithine, LN-methylthreonine, LN-methyltyrosine, LN-methylvaline, LN-methyl-t-butylglycine, L-norvaline, α-methyl-γ-aminobutyrate, 4,4'-biphenylalanine, α-methylcyclopentylalanine, α-methyl-α-naphthylalanine, α-methylpenicillamine, N-(4-aminobutyl)glycine, N-(2-aminoethyl)glycine, N-(3-amino Propyl)glycine, N-amino-α-methylbutyrate, α-naphthylalanine, N-benzylglycine, N-(2-carbamylethyl)glycine, N-(carbamylmethyl)glycine, N-(2-carboxyethyl)glycine, N-(carboxymethyl)glycine, N-cyclobutylglycine, N-cyclodecylglycine, N-cycloheptylglycine, N-cyclohexylglycine, N-cyclodecylglycine, N-cyclododecylglycine, N-cyclooctylglycine, N-cyclopropylglycine, N-cycloundecylglycine,N-(2,2-diphenylethyl)glycine, N-(3,3-diphenylpropyl)glycine, N-(3-guanidinopropyl)glycine, N-(1-hydroxyethyl)glycine, N-(hydroxyethyl)glycine, N-(imidazolylethyl)glycine, N-(3-indolylethyl)glycine, N-methyl-γ-aminobutyrate, DN-methylmethionine, N-methylcyclopentylalanine, DN-methylphenylalanine, DN-methylproline, DN-methylthreonine, N-(1-methylethyl)glycine, N-methyl-naphthyl L-α-methylalanine, N-methylpenicillamine, N-(p-hydroxyphenyl)glycine, N-(thiomethyl)glycine, penicillamine, L-α-methylalanine, L-α-methylasparagine, L-α-methyl-t-butylglycine, L-methylethylglycine, L-α-methylglutamate, L-α-methylhomophenylalanine, N-(2-methylthioethyl)glycine, L-α-methyllysine, L-α-methylnorleucine, L-α-methylornithine, L-α-methylproline, L-α-methylthreonine, L-α-methyltyrosine, L-N-methyl -Homophenylalanine, N-(N-(3,3-diphenylpropyl)carbamylmethylglycine, L-pyroglutamic acid, D-pyroglutamic acid, O-methyl-L-serine, O-methyl-L-homoserine, 5-hydroxylysine, α-carboxyglutamate, phenylglycine, L-pipecolic acid (homoproline), L-homoleucine, L-lysine (dimethyl), L-2-naphthylalanine, L-dimethyldopa or L-dimethoxy-phenylalanine, L-3-pyridylalanine, L-histidine (benzoyloxymethyl), N-cycloheptyl L-glycine, L-diphenylalanine, O-methyl-L-homotyrosine, L-β-homolysine, O-glycan-threonine, ortho-tyrosine, LN,N'-dimethyllysine, L-homoarginine, neotryptophan, 3-benzothienylalanine, isoquinoline-3-carboxylic acid, diaminopropionic acid, homocysteine, 3,4-dimethoxyphenylalanine, 4-chlorophenylalanine, L-1,2,3,4-tetrahydronorharman-3-carboxylic acid, adamantylalanine, symmetrical dimethylarginine, 3-carboxythiomorpholine,D-1,2,3,4-tetrahydronorharman-3-carboxylic acid, 3-aminobenzoic acid, 3-amino-1-carboxymethyl-pyridin-2-one, 1-amino-1-cyclohexanecarboxylic acid, 2-aminocyclopentanecarboxylic acid, 1-amino-1-cyclopropanecarboxylic acid, 2-aminoindan-2-carboxylic acid, 4-amino-tetrahydrothiopyran-4-carboxylic acid, azetidine-2-carboxylic acid, b-(benzothiazol-2-yl)-alanine, neopentylglycine, 2-carboxymethylpiperidine, b-cyclobutylalanine, allylglycine, diaminopropionic acid, homo-cyclohexylalanine, (2S,4R)-4 -hydroxypiperidine-2-carboxylic acid, octahydroindole-2-carboxylic acid, (2S,4R) and (2S,4R)-4-(2-naphthyl)pyrrolidine-2-carboxylic acid, nipecotic acid, (2S,4R) and (2S,4S)-4-(4-phenylbenzyl)pyrrolidine-2-carboxylic acid, (3S)-1-pyrrolidine-3-carboxylic acid, (2S,4S)-4-tritylmercapto-pyrrolidine-2-carboxylic acid, (2S,4S)-4-mercaptoproline, t-butylglycine, N,N-bis(3-aminopropyl)glycine, 1-amino-cyclohexane-1-carboxylic acid, N-mercaptoethylglycine, and selenocysteine. In some embodiments, the amino acid residue may be charged or polar. Charged amino acids include alanine, lysine, aspartic acid, or glutamic acid, or non-naturally occurring analogs thereof. Polar amino acids include glutamine, asparagine, histidine, serine, threonine, tyrosine, methionine, or tryptophan, or non-naturally occurring analogs thereof. In some embodiments, it is specifically contemplated that the terminal amino group in the amino acid may be an amide group or a carbamate group.

[0107] The terms "linker," "L," and the like, as used herein, refer to a covalent bond or connection between two or more components in a fusion protein or conjugate (e.g., between a therapeutic peptide agent and a variant Fc domain monomer to form a fusion protein, between two therapeutic agents, between a therapeutic agent and a fusion protein, between one or more therapeutic agents and a fusion protein, and between one or more therapeutic agents and a variant Fc domain monomer). In some embodiments, the linker is a bivalent linker, e.g., a linker that connects a therapeutic peptide agent and a variant Fc domain monomer, a linker that connects a therapeutic agent to a fusion protein, or a linker that connects a therapeutic agent to a variant Fc domain. In some embodiments, the conjugates described herein may contain a linker having a trivalent structure (e.g., a trivalent linker). A trivalent linker has three arms, each covalently attached to a component of the conjugate (e.g., a first arm conjugated to a first therapeutic agent, a second arm conjugated to a therapeutic agent, and a third arm conjugated to a fusion protein or variant Fc domain monomer). The linker may be a chemical linker known to those skilled in the art and described in detail herein. Chemical linkers can be used to link two small molecules (e.g., to form a dimer), to link a small molecule monomer or dimer to a polypeptide, or to link two polypeptides to form a fusion protein. Alternatively, the linker can be a peptide linker. Peptide linkers can also be used to link two small molecules, to link a small molecule monomer or dimer to a polypeptide, or to link a polypeptide to form a fusion protein.

[0108] Molecules that can be used as linkers contain at least two functional groups, which can be the same or different, such as two carboxylic acid groups, two amine groups, two sulfonic acid groups, a carboxylic acid group and a maleimide group, a carboxylic acid group and an alkyne group, a carboxylic acid group and an amine group, a carboxylic acid group and a sulfonic acid group, an amine group and a maleimide group, an amine group and an alkyne group, or an amine group and a sulfonic acid group. In a bivalent linker, the first functional group can form a covalent bond with a first component, and the second functional group can form a covalent bond with a second component. In some embodiments, where the linker is a trivalent linker, the two arms of the linker can contain two dicarboxylic acids, of which the first carboxylic acid can form a covalent bond with a first therapeutic agent in the conjugate, the second carboxylic acid can form a covalent bond with a second therapeutic agent in the conjugate, and the third arm of the linker can form a covalent bond with a variant Fc domain monomer or fusion protein in the conjugate. Examples of dicarboxylic acids are described further herein. In some embodiments, molecules containing one or more maleimide groups can be used as linkers, in which case the maleimide groups can form carbon-sulfur bonds with cysteines in components of the conjugate. In some embodiments, molecules containing one or more alkyne groups can be used as linkers, in which case the alkyne groups can form 1,2,3-triazole bonds with azides in components of the conjugate. In some embodiments, molecules containing one or more azide groups can be used as linkers, in which case the azide groups can form 1,2,3-triazole bonds with alkynes in components of the conjugate. In some embodiments, molecules containing one or more bis-sulfone groups can be used as linkers, in which case the bis-sulfone groups can form bonds with amine groups in components of the conjugate. In some embodiments, molecules containing one or more sulfonic acid groups can be used as linkers, in which case the sulfonic acid groups can form sulfonamide bonds with components of the conjugate. In some embodiments, molecules containing one or more isocyanate groups can be used as linkers, where the isocyanate groups can form urea bonds with components in the conjugate.In some embodiments, molecules containing one or more haloalkyl groups can be used as linkers, where the haloalkyl groups can form covalent bonds, e.g., C-N and C-O bonds, with components in the conjugate.

[0109] In some embodiments, a linker provides space, rigidity, and / or flexibility between two or more components. In some embodiments, a linker can be a bond, e.g., a covalent bond. The term "bond" refers to a chemical bond, e.g., an amide bond, a disulfide bond, a C-O bond, a C-N bond, an N-N bond, a C-S bond, or any type of bond formed by a chemical reaction, e.g., chemical conjugation. In some embodiments, a linker comprises 250 atoms or fewer. In some embodiments, a linker comprises 250 non-hydrogen atoms or fewer. In some embodiments, the backbone of a linker comprises 250 atoms or fewer. The "backbone" of a linker refers to the atoms in the linker that together form the shortest path from one portion of the conjugate to another portion of the conjugate. The atoms in the backbone of a linker are directly involved in connecting one portion of the conjugate to another portion of the conjugate. For example, a hydrogen atom bonded to a carbon in the backbone of a linker is not considered to be directly involved in connecting one portion of the conjugate to another portion of the conjugate.

[0110] In some embodiments, the linker can include a synthetic group, for example, derived from a synthetic polymer (e.g., a polyethylene glycol (PEG) polymer). In some embodiments, the linker can include one or more amino acid residues, such as D- or L-amino acid residues. In some embodiments, the linker can be residues of an amino acid sequence (e.g., 1 to 25 amino acids, 1 to 10 amino acids, 1 to 9 amino acids, 1 to 8 amino acids, 1 to 7 amino acids, 1 to 6 amino acids, 1 to 5 amino acids, 1 to 4 amino acids, 1 to 3 amino acids, 1 to 2 amino acids, or 1 amino acid sequence). In some embodiments, the linker is selected from the group consisting of one or more, e.g., 1 to 100, 1 to 50, 1 to 25, 1 to 10, 1 to 5, or 1 to 3, optionally substituted alkylene, optionally substituted heteroalkylene (e.g., PEG units), optionally substituted alkenylene, optionally substituted heteroalkenylene, optionally substituted alkynylene, optionally substituted heteroalkynylene, optionally substituted cycloalkylene, optionally substituted heterocycloalkylene, optionally substituted cycloalkenylene, optionally substituted heterocycloalkenylene, optionally substituted cycloalkynylene, optionally substituted heterocycloalkynylene, optionally substituted arylene, optionally substituted heteroarylene (e.g., pyridine), O, S, NR i (R imay include H, optionally substituted alkyl, optionally substituted heteroalkyl, optionally substituted alkenyl, optionally substituted heteroalkenyl, optionally substituted alkynyl, optionally substituted heteroalkynyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted cycloalkenyl, optionally substituted heterocycloalkenyl, optionally substituted cycloalkynyl, optionally substituted heterocycloalkynyl, optionally substituted aryl, or optionally substituted heteroaryl), P, carbonyl, thiocarbonyl, sulfonyl, phosphate, phosphoryl, or imino. For example, the linker may be one or more optionally substituted C1-C20 alkylene, optionally substituted C1-C20 heteroalkylene (e.g., PEG units), optionally substituted C2-C20 alkenylene (e.g., C2 alkenylene), optionally substituted C2-C20 heteroalkenylene, optionally substituted C2-C20 alkynylene, optionally substituted C2-C20 heteroalkynylene, optionally substituted C3-C 20 Cycloalkylene (e.g., cyclopropylene, cyclobutylene), optionally substituted C-C 20 Heterocycloalkylene, optionally substituted C4-C20 cycloalkenylene, optionally substituted C4-C20 heterocycloalkenylene, optionally substituted C8-C20 cycloalkynylene, optionally substituted C8-C20 heterocycloalkynylene, optionally substituted C5-C15 arylene (e.g., C6 arylene), optionally substituted C3-C 15 Heteroarylene (e.g., imidazole, pyridine), O, S, NR i (R i is H, optionally substituted C1-C20 alkyl, optionally substituted C1-C20 heteroalkyl, optionally substituted C2-C20 alkenyl, optionally substituted C2-C20 heteroalkenyl, optionally substituted C2-C20 alkynyl, optionally substituted C2-C20 heteroalkynyl, optionally substituted C3-C 20 Cycloalkyl, optionally substituted C-C 20Heterocycloalkyl, optionally substituted C4-C20 cycloalkenyl, optionally substituted C4-C20 heterocycloalkenyl, optionally substituted C8-C20 cycloalkynyl, optionally substituted C8-C20 heterocycloalkynyl, optionally substituted C5-C15 aryl, or optionally substituted C3-C 15 Heteroaryl), P, carbonyl, thiocarbonyl, sulfonyl, phosphate, phosphoryl, or imino.

[0111] As used herein, the term "chemical linker" includes any linker described herein that does not include a polypeptide. For example, a chemical linker can include a hydrocarbon chain (e.g., an optionally substituted alkylene, heteroalkylene, alkenylene, heteroalkenylene, alkynylene, or heteroalkynylene) optionally containing one or more heteroatoms. A chemical linker can include one or more cycloalkyl, heterocycloalkynyl, aryl, or heteroaryl rings within the linker backbone. A chemical linker can include a polyethylene glycol (PEG) polymer, e.g., PEG2-PEG. 50 , most preferably PEG2, PEG3, PEG4, PEG5, PEG6, PEG7, PEG8, PEG9, or PEG 10 The chemical linker may be a bond. As described in more detail herein (see, e.g., conjugation chemistry), the chemical linker may comprise at least two functional groups, which may be the same or different, such as two carboxylic acid groups, two amine groups, two sulfonic acid groups, a carboxylic acid group and a maleimide group, a carboxylic acid group and an alkyne group, a carboxylic acid group and an amine group, a carboxylic acid group and a sulfonic acid group, an amine group and a maleimide group, an amine group and an alkyne group, or an amine group and a sulfonic acid group. In a bivalent linker, for example, the first functional group may form a covalent bond with a first component, and the second functional group may form a covalent bond with a second component.

[0112] As used interchangeably herein, the terms "peptide linker" or "polypeptide linker" include any linker comprising two or more amino acid residues. For example, a peptide linker can comprise, e.g., 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 40 or more, or 50 or more amino acid residues joined by peptide bonds. The carboxy terminus of the peptide linker can be covalently conjugated (e.g., by a peptide bond) to a first moiety (e.g., a variant Fc domain monomer or a therapeutic peptide agent), and the amino terminus of the peptide linker can be covalently conjugated (e.g., by a peptide bond) to a second moiety (e.g., a variant Fc domain monomer or a therapeutic peptide agent), thereby conjugating the first and second moieties and providing space and / or flexibility between the first and second moieties. The peptide linker can be expressed from a polynucleotide construct or chemically synthesized and then chemically conjugated to the first and second moieties. Alternatively, the peptide linker can be expressed in tandem with a first polypeptide (e.g., a variant Fc domain monomer or a therapeutic peptide agent) and a second polypeptide (e.g., a variant Fc domain monomer or a therapeutic peptide agent), thereby linking the first and second polypeptides to form a fusion protein.

[0113] As used herein, the term "percent identity" refers to the percentage of amino acid residues in a candidate sequence, e.g., an Fc-IgG, or fragment thereof, that are identical to those in a reference sequence after aligning the sequences and, if necessary, introducing gaps to achieve maximum identity (i.e., gaps may be introduced into one or both of the candidate and reference sequences for optimal alignment, and non-homologous sequences may be ignored for comparison purposes). Alignment for purposes of determining percent identity can be achieved in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared. In some embodiments, the percent amino acid sequence identity for a given reference sequence, for a given candidate sequence with or against a given reference sequence (which may alternatively be expressed as a given candidate sequence having or containing some percent amino acid sequence identity for, with, or against a given reference sequence) is calculated as follows: 100 x (A / B ratio) (where A is the number of amino acid residues scored as identical in the alignment of the candidate and reference sequences, and B is the total number of amino acid residues in the reference sequence.) In some embodiments where the length of the candidate sequence is not equal to the length of the reference sequence, the percent amino acid sequence identity of the candidate sequence to the reference sequence will not be equal to the percent amino acid sequence identity of the reference sequence to the candidate sequence.

[0114] Two polynucleotide or polypeptide sequences are "identical" if the nucleotide or amino acid sequences in the two sequences are the same when aligned for maximum correspondence as described above. Comparison between two sequences is typically performed by comparing the sequences over a comparison window to identify and compare regions of sequence similarity. As used herein, a "comparison window" refers to a segment of at least about 15 contiguous positions, about 20 contiguous positions, about 25 contiguous positions, or more (e.g., about 30 to about 75 contiguous positions, or about 40 to about 50 contiguous positions), in which a sequence can be compared to a reference sequence over the same number of contiguous positions after the two sequences are optimally aligned.

[0115] As used herein, the term "fusion protein" refers to any conjugate comprising two or more covalently linked peptides, polypeptides, or proteins. The two or more peptides, polypeptides, or proteins may be covalently conjugated by any of the linkers described herein, including a linker, e.g., a chemical linker, a peptide linker, or a bond. For example, a fusion protein may comprise one or more therapeutic peptide agents and one or more variant Fc domain monomers. The one or more therapeutic peptide agents and one or more variant Fc domain monomers may be encoded by the same polynucleotide sequence (e.g., a single, operably linked, contiguous polynucleotide sequence) and expressed as a single polypeptide construct. Alternatively, the one or more therapeutic peptide agents and one or more variant Fc domain monomers may be encoded by separate polynucleotides (e.g., non-contiguous polynucleotide sequences, which may be on the same vector or separate vectors), expressed as separate polypeptide constructs, and then covalently conjugated by any of the linker and / or conjugation chemistries described herein. In some examples, the variant Fc domain monomers of the fusion protein may be conjugated to one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) small molecule therapeutic agents via a linker (e.g., any linker described herein).

[0116] As used herein, the term "pharmaceutical composition" refers to a medicinal or pharmaceutical formulation containing at least one active ingredient (e.g., a conjugate of Formula (1) or a fusion protein described herein) and one or more excipients and diluents to make the active ingredient suitable for the method of administration. Pharmaceutical compositions of the present disclosure contain pharmaceutically acceptable ingredients that are compatible with the conjugates (e.g., conjugates of Formula (1)) or fusion proteins described herein.

[0117] As used herein, the term "pharmaceutically acceptable carrier" refers to an excipient or diluent in a pharmaceutical composition. For example, a pharmaceutically acceptable carrier can be a vehicle capable of suspending or dissolving an active conjugate (e.g., a conjugate of Formula (1)) or fusion protein described herein. A pharmaceutically acceptable carrier must be compatible with the other ingredients of the formulation and not deleterious to the recipient. In the present disclosure, a pharmaceutically acceptable carrier must provide adequate pharmaceutical stability for the conjugate or fusion protein described herein. The nature of the carrier will vary depending on the mode of administration. For example, for oral administration, a solid carrier is preferred; for intravenous administration, an aqueous carrier (e.g., WFI and / or buffer) is generally used.

[0118] The term "pharmaceutically acceptable salt," as used herein, refers to a salt of a conjugate described herein (e.g., a conjugate of Formula (1)) that is suitable for use in the methods described herein without undue toxicity, irritation, and / or allergic reaction, within the scope of reasonable medical judgment. Pharmaceutically acceptable salts are well known in the art. For example, pharmaceutically acceptable salts are described in Pharmaceutical Salts: Properties, Selection, and Use (Eds. P.H. Stahl and C.G. Wermuth), Wiley-VCH, 2008. Salts can be prepared in situ during the final isolation and purification of the conjugates described herein or separately by reacting the free base group with a suitable organic acid.

[0119] The term "drug-to-antibody ratio" or "DAR" refers to the average number of small molecule drug moieties (e.g., the average number of small molecule drug monomers or dimers) conjugated to a variant Fc domain monomer or variant Fc domain described herein. In some embodiments described herein, the DAR is represented by "T" (e.g., as in formula (1)). As used herein, each therapeutic agent conjugated to a variant Fc domain corresponds to a DAR value of 1.0 (e.g., a "T" value of 1.0). The DAR may also be calculated as the average DAR for a population of molecules, such as a population of variant Fc domain conjugates. The DAR value may affect the efficacy, potency, pharmacokinetics, or toxicity of the drug.

[0120] As used herein, the term "antiviral agent" refers to any one of the conjugates described herein (e.g., any one of the conjugates of Formula (1)) that exhibit antiviral activity. The antiviral activity exhibited by the antiviral agent can be against any viral infection, such as viral meningitis, herpes simplex virus (HSV) 1, HSV2, Epstein-Barr virus, varicella-zoster virus, poliovirus, coxsackievirus, West Nile virus, La Crosse virus, western equine encephalitis, eastern equine encephalitis, Powassan virus, rabies virus, respiratory syncytial virus (RSV), dengue, betacoronavirus (e.g., COVID-19), Zika virus, or influenza virus. In some examples, the antiviral agent exhibits antiviral activity by interfering with viral binding, fusion, and / or entry into cells.

[0121] The term "antibacterial agent" refers to an agent used in the treatment of bacterial infections and / or preventing, stabilizing, or inhibiting bacterial growth, or killing bacteria. An antibacterial agent can be an agent that prevents bacterial invasion into a cell, tissue, or organ of a subject, inhibits bacterial growth in a cell, tissue, or organ of a subject, and / or kills bacteria present within a cell, tissue, or organ of a subject. In some instances, an antibacterial agent exhibits antibacterial activity by preventing bacterial binding, fusion, and / or invasion into cells. Examples of antibacterial agents are described in further detail herein.

[0122] "Viral infection" refers to the pathogenic growth of a virus (e.g., viral meningitis, herpes simplex virus (HSV) 1, HSV2, Epstein-Barr virus, varicella-zoster virus, poliovirus, coxsackievirus, West Nile virus, La Crosse virus, western equine encephalitis, eastern equine encephalitis, Powassan virus, rabies virus, respiratory syncytial virus (RSV), dengue, betacoronavirus (e.g., COVID-19), Zika virus, or influenza virus) in a host organism (e.g., a human subject). A viral infection can be any situation in which the presence of a viral population(s) is damaging to the host's body. Thus, a subject is "suffering" from a viral infection when an excessive amount of viral population(s) is present in or on the subject's body or when the viral population(s) is damaging the subject's cells or other tissues.

[0123] "Bacterial infection" refers to an infection in a host organism (e.g., a human subject) caused by bacteria (e.g., Acinetobacter spp. (Acinetobacter baumannii), Bacteroides distasonis, Bacteroides fragilis, Bacteroides ovatus, Bacteroides thetaiotaomicron, Bacteroides uniformis, Bacteroides vulgatus, Citrobacter freundii, Citrobacter koser, Clostridium clostridioforme, Clostridium perfringens, Enterobacter aerogenes, Enterobacter cloacae, Enterococcus faecalis, Enterococcus spp. (vancomycin-susceptible and -resistant isolates), Escherichia coli (including ESBL- and KPC-producing isolates), Eubacterium lentum, Fusobacterium spp., Haemophilus influenzae (including beta-lactamase-positive isolates), Haemophilus influenzae parainfluenzae, Klebsiella pneumoniae (including ESBL- and KPC-producing isolates), Klebsiella oxytoca (including ESBL- and KPC-producing isolates), Legionella pneumophilia, Moraxella catarrhalis, Morganella morganii, Mycoplasma spp., Peptostreptococcus spp.Bacterial infection refers to the pathogenic growth of bacteria (e.g., Porphyromonas asaccharolytica, Prevotella bivia, Proteus mirabilis, Proteus vulgaris, Providencia rettgeri, Providencia stuartii, Pseudomonas aeruginosa, Serratia marcescens, Streptococcus anginosus, Staphylococcus aureus (methicillin-susceptible and -resistant isolates), Staphylococcus epidermidis (methicillin-susceptible and -resistant isolates), Stenotrophomonas maltophilia, Streptococcus agalactiae, Streptococcus constellatus, Streptococcus pneumoniae (penicillin-susceptible and -resistant isolates), and Streptococcus pyogenes. A bacterial infection can be any situation in which the presence of a bacterial population(s) is damaging to the host's body. Thus, a subject is "suffering from" a bacterial infection when an excessive amount of bacterial population(s) is present in or on the subject's body, or when the presence of the bacterial population(s) is damaging to the subject's cells or other tissues.

[0124] "Fungal infection" refers to fungi in a host organism (e.g., a human subject) (e.g., Trichophyton species (e.g., T. ajelloi, T. concentricum, T. equinum, T. erinacei, T. flavescens, T. gloriae, T. interdigitale, T. megnini, T. mentagrophytes, T. phaseoliforme, T. rubrum, T. schoenleini, T. simii, T. soudanense, T. terrestre, T. tonsurans, T. vanbreuseghemii, T. verrucosum, T. violaceum, or T. yaoundei), Epidermophyton species (e.g., E. floccosum or E. stockdaleae), Candida species (e.g., C. albicans, C. parapsiliosis, C. krusei, C. tropicalis, C. glabrata, C. parapsilosis, C. lusitaniae, C. kefyr, C. guilliermondii, or C. dubliniensis), Microsporum species (e.g., M. canis, M. gypseum, M. audouini, M. gallinae, M. ferrugineum, M. distortum, M. nanum, M. cookie, or M. vanbreuseghemii), Epicoccum species (e.g., E. nigrum), Aspergillus species (e.g., A. sydowii, A. terreus, A. niger, A. terreus, A. fumigatus, A. flavus, A. clavatus, A. glaucus group, A. nidulans, A. oryzae, A. terreus, A. ustus, or A. versicolor), Paecilomyces species (e.g., P. lilacinus or P. variotii), Fusarium species (e.g., F. oxysporum, F. solani, or F. semitectum), Acremonium species (e.g., A. strictum, A. roseogiseum, A. cucurbitacearum, A. kiliense, A. curvatum, A.comptosporum, Ulocladium chartarum, A.alternatum, or Emercellopsis minima), Chaetomium species (e.g. C. atrobrunneum, C. funicola, C. globosum, or C. strumarium), Phoma species, Scopulariopsis species (e.g. S. brevicaul is, S.candida, S.koningii, S.acremonium, S.flava, S.cinerea, S.trigonospora, S.brumptii, S.chartarum, S.fusca, or S.asperula), Fungal infection refers to the pathogenic growth of Alternaria species (e.g., A. alternata, A. chartarum, A. dianthicola, A. geophilia, A. infectoria, A. stemphyloides, or A. teunissima) and Curvularia species (e.g., C. brachyspora, C. clavata, C. geniculata, C. lunata, C. pallescens, C. senegalensis, or C. verruculosa). A fungal infection can be any situation in which the presence of a fungal population(s) is damaging to the host's body. Thus, a subject is "suffering" from a fungal infection when excessive amounts of fungal population(s) are present in or on the subject's body, or when the presence of a fungal population(s) is damaging to the subject's cells or other tissues.

[0125] The terms "treating" or "to treat," as used herein, refer to the therapeutic treatment of a disorder (e.g., a respiratory disorder, liver disorder, central nervous system disorder, skin disorder, eye disorder, vascular disorder, or infection) in a subject. In some embodiments, the therapeutic treatment may slow the progression of the disorder, improve the subject's outcome, and / or eliminate the disorder. In some embodiments, the therapeutic treatment of a disorder in a subject may alleviate or ameliorate one or more symptoms or pathology associated with the disorder, lessen the severity of the disorder, stabilize (i.e., do not worsen) the state of the disorder, prevent the spread of the disorder, and / or delay or slow the progression of the disorder, compared to the state and / or pathology of the disorder in the absence of the therapeutic treatment.

[0126] As used herein, "combination therapy" or "administered in combination" means that two or more active agents are administered to a subject as part of a defined treatment regimen. The treatment regimen defines the dosage and periodicity of administration of each agent so that the effects of the separate agents on the subject overlap. In some embodiments, delivery of the conjugate and one or more agents is simultaneous or concurrent, and the conjugate and one or more agents may be formulated together. In some embodiments, the conjugate and one or more agents are not formulated together but are administered sequentially as part of a prescribed regimen. In some embodiments, the administration of the conjugate and one or more agents or treatments in combination is such that the reduction in symptoms or other parameters associated with a viral infection is greater than that which would be observed with one agent or treatment delivered alone or in the absence of the other. The effects of the conjugate and one or more agents may be partially additive, wholly additive, or greater than additive (e.g., synergistic). The sequential or substantially simultaneous administration of each therapeutic agent may be by any appropriate route, including, but not limited to, oral, intravenous, intramuscular, and direct absorption through mucosal tissue. The therapeutic agents can be administered by the same route or by different routes, for example, a conjugate or fusion protein described herein can be administered by intravenous injection, while a second therapeutic agent of the combination can be administered by another route, for example, orally.

[0127] The term "subject," as used herein, may be a human, a non-human primate, or other mammal, such as, but not limited to, a dog, cat, horse, cow, pig, turkey, goat, fish, monkey, chicken, rat, mouse, and sheep.

[0128] The term "therapeutically effective amount," as used herein, refers to an amount, e.g., a pharmaceutical dose, effective in inducing a desired effect in a subject or in treating a subject with a condition or disorder described herein (e.g., a respiratory disorder, liver disorder, central nervous system disorder, muscle disorder, skin disorder, eye disorder, vascular disorder, or an infectious disease (e.g., a viral infection, a fungal infection, or a bacterial infection)). It should also be understood herein that a "therapeutically effective amount" can be interpreted as an amount that provides a desired therapeutic and / or prophylactic effect, taken in one or more doses or by any dosage or route, and / or taken alone or in combination with other therapeutic agents (e.g., antiviral agents described herein). For example, in the context of administering a pharmaceutical composition (e.g., a conjugate or fusion protein of Formula (1) described herein) used to treat an infectious disease, an effective amount of the conjugate or fusion protein is an amount sufficient to prevent, delay, or reverse the progression of the infectious disease (e.g., a viral infection, a fungal infection, or a bacterial infection), e.g., compared to the response obtained without administration of the conjugate or fusion protein.

[0129] As used herein, the term "small molecule" refers to a low molecular weight compound (e.g., a chemical compound (e.g., an organic compound)) having less than 900 Da that can regulate biological processes with sizes on the order of 1 nm. In some examples, the therapeutic agent is a small molecule therapeutic agent. In some examples, the small molecule agent is between about 300 and about 700 Da (e.g., about 325 Da, about 350 Da, about 375 Da, about 400 Da, about 425 Da, about 450 Da, about 475 Da, about 500 Da, about 525 Da, about 550 Da, about 575 Da, about 600 Da, about 625 Da, about 650 Da, or about 675 Da).

[0130] The term "about" as used herein indicates a deviation of up to ±5%. For example, about 10% refers to 9.5% to 10.5%.

[0131] Any value provided in a range of values ​​includes both the upper and lower limits, and any value included within the limits.

[0132] Other features and advantages of the conjugates described herein will be apparent from the following detailed description and claims. [Brief explanation of the drawings]

[0133] [Figure 1] 1 is a graph showing the plasma levels of a conjugate comprising a small molecule conjugated to a variant Fc domain monomer with a C220S / M252Y / S254T / T256E quadruple mutation (SEQ ID NO: 10) (2mpk IV) compared to a conjugate comprising the same small molecule conjugated to an Fc domain with a C220S mutation (SEQ ID NO: 21) (2mpk IV) in a non-human primate PK study. The study was performed as described in Example 4. [Figure 2] 1 is a graph showing plasma concentration levels of a small molecule conjugated to a variant Fc domain monomer with a C220S mutation (SEQ ID NO: 21) compared to epithelial lining fluid (ELF) levels of the same conjugate in mice. The study was performed as described in Example 5. [Figure 3] 1 is a graph showing plasma levels of a conjugate comprising a small molecule conjugated to a variant Fc domain monomer with a C220S / M252Y / S254T / T256E quadruple mutation (SEQ ID NO: 10) compared to a conjugate comprising the same small molecule conjugated to an Fc domain with a C220S mutation (SEQ ID NO: 21) in a mouse PK study. This study was performed as described in Example 6. [Figure 4] 1 is a graph showing plasma concentration levels of Fc domain monomers (SEQ ID NOs: 53-55) in a mouse PK study. The graph shows that Fc domain plasma levels increase with increasing molecular weight (SEQ ID NO: 53 > SEQ ID NO: 55 > SEQ ID NO: 54). This study was performed as described in Example 7. [Figure 5]1 is a graph showing the mean Fc plasma levels of Fc domain monomers (SEQ ID NOs: 53, 56, and 58) in a mouse PK study. This study was performed as described in Example 7. DETAILED DESCRIPTION OF THE INVENTION

[0134] The present disclosure provides Fc domain monomers, conjugates comprising Fc domain monomers, and fusion proteins comprising Fc domain monomers, where the Fc domain monomers are mutant variants of a parent Fc polypeptide (e.g., an IgG1 or IgG2 polypeptide). The Fc domain monomers may contain one or more mutations that contribute to increased half-life and / or efficacy. The one or more mutations may also minimize aggregation during manufacturing, thereby increasing productivity and reducing costs. The Fc domain monomers may also be optimized for size (e.g., measured by kDa or amino acid residues) to maximize tissue distribution to tissues of interest and / or minimize renal clearance.

[0135] In particular, the invention features variant Fc domain monomers comprising an amino acid mutation at position 220 (e.g., C220S). The invention features variant Fc domain monomers comprising amino acid mutations at positions 220, 252, 254, and / or 256 (e.g., C220S / M252Y / S254T / T256E mutations). The invention also includes variant Fc domain monomers comprising amino acid mutations at positions 220, 309, 311, and / or 434 (e.g., C220S / V309D / Q311H / N434S mutations). The invention also includes conjugates comprising one or more of the variant Fc domain monomers conjugated to one or more therapeutic agents. The invention further features fusion proteins comprising at least one therapeutic peptide agent and at least one variant Fc domain monomer or a conjugate thereof. The variant Fc domain monomers (e.g., of each of the two conjugates or two fusion proteins) can dimerize to form a variant Fc domain.

[0136] In some examples, the variant Fc domain monomers bind to FcγRs (e.g., FcRn, FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and FcγRIIIb) on immune cells, e.g., neutrophils, and activate phagocytosis and effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC), resulting in the phagocytosis and destruction of infectious agents (e.g., viruses, fungi, or bacteria). In other examples, the variant Fc domain monomers further comprise mutations that reduce or eliminate binding to FcγRs (e.g., FcRn, FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and FcγRIIIb) on immune cells, e.g., neutrophils, and are particularly useful for delivery of therapeutic agents (e.g., small molecule therapeutics and therapeutic peptide agents).

[0137] The variant Fc domain monomers and conjugates and fusion proteins thereof exhibit desirable tissue distribution, making such compositions useful in methods for treating disorders (e.g., respiratory disorders, liver disorders, central nervous system disorders, skin disorders, eye disorders, vascular disorders), inhibiting the spread of infection, and treating infectious diseases (e.g., viral, fungal, or bacterial infections).

[0138] I. Variant Fc Domain Monomers and Variant Fc Domains The variant Fc domain monomers consist of a hinge domain, C H 2 antibody constant domains, and C H 3 antibody constant domains. In some embodiments, the variant Fc domain monomer comprises the quadruple mutation C220S / M252Y / S254T / T256E. In some embodiments, the variant Fc domain monomer comprises the quadruple mutation C220S / V309D / Q311H / N434S. In another embodiment, the variant Fc domain monomer comprises the C220S mutation. The amino acid substitutions are relative to the wild-type Fc monomer amino acid sequence, e.g., wild-type human IgG1 or IgG2.

[0139] The variant Fc domain monomer can be of the immunoglobulin antibody isotype IgG. The variant Fc domain monomer can also be of any immunoglobulin antibody isotype (e.g., IgG1, IgG2a, or IgG2b). The variant Fc domain monomer can be of any immunoglobulin antibody allotype (e.g., IGHG1*01 (i.e., G1m(za)), IGHG1*07 (i.e., G1m(zax)), IGHG1*04 (i.e., G1m(zav)), IGHG1*03 (G1m(f)), IGHG1*08 (i.e., G1m(fa)), IGHG2*01, IGHG2*06, or IGHG2*02) (e.g., as described in Vidarsson et al. IgG subclasses and allotypes: from structure to effector function. Frontiers in Immunology. 5(520):1-17(2014)). The variant Fc domain monomers can also be from any species, e.g., human, murine, or mouse. A dimer of variant Fc domain monomers is a variant Fc domain that can bind to an Fc receptor, which is a receptor located on the surface of a leukocyte.

[0140] In some embodiments, the variant Fc domain monomer comprises one or more amino acid substitutions, additions, and / or deletions relative to a variant Fc domain monomer having the sequence of any one of SEQ ID NOS: 1-29, 31-52, or 56-58. In some embodiments, Asn297 in a variant Fc domain monomer in a conjugate described herein may be replaced by Ala to prevent N-linked glycosylation (see, e.g., SEQ ID NO: 4 (where the Asn297 to Ala substitution is represented by (*))).

[0141] In some embodiments, the variant Fc domain monomer or variant Fc domain of the invention is an aglycosylated Fc domain monomer or Fc domain (e.g., an Fc domain monomer or Fc domain that maintains engagement with an Fc receptor (e.g., FcRn)). For example, the Fc domain is an aglycosylated IgG1 variant (e.g., an IgG1 with amino acid substitutions at N297 and / or T299 of the glycosylation motif) that maintains engagement with an Fc receptor. Exemplary aglycosylated Fc domains and methods for making aglycosylated Fc domains are known in the art, as described, for example, in Sazinsky SLet et al., Aglycosylated immunoglobulin G1 variants productively engage activating Fc receptors, PNAS, 2008, 105(51):20167-20172, incorporated herein in its entirety.

[0142] The C-terminal Lys447 of the Fc region may or may not be present without affecting the structure or stability of the Fc region. The present disclosure particularly contemplates any of SEQ ID NOS: 1-29 and 31-52 that do not contain the C-terminal Lys corresponding to Lys447. The N-terminal Asn of the variant Fc domain monomer may or may not be present without affecting the structure or stability of the variant Fc domain monomer. The present disclosure particularly contemplates any of SEQ ID NOS: 1-29, 31-52, and 56-58 that do not contain the N-terminal Asn residue.

[0143] In some embodiments, the variant Fc domain monomer comprises an additional moiety attached to the N- or C-terminus of the variant Fc domain monomer, such as a purification peptide (e.g., a hexa-histidine peptide (HHHHHH (SEQ ID NO: 59)), or a signal sequence (e.g., the IL2 signal sequence MYRMQLLSCIALSLALVTNS (SEQ ID NO: 60)). In some embodiments, the variant Fc domain monomer in the conjugate is an antibody variable region of any type, e.g., a V H , V L , complementarity determining regions (CDRs), or hypervariable regions (HVRs).

[0144] In some embodiments, the variant Fc domain monomer has a sequence that is at least 95% identical (e.g., 97%, 99%, or 99.5% identical) to any one of SEQ ID NOs: 1-29, 31-52, and 56-58 shown below. In some embodiments, the variant Fc domain monomer has the sequence of any one of SEQ ID NOs: 1-29, 31-52, and 56-58 shown below.

[0145] SEQ ID NO: 1: Mature human IgG1 Fc, Cys to Ser substitution (#), YTE triple mutation (bold and underlined), X1 is Asp or Glu, X2 is Leu or Met, N-terminal Fab residues are underlined, hinge residues are italicized

[0146] TIFF0007762645000002.tif50170

[0147] SEQ ID NO: 2: Mature human IgG1 Fc, Cys to Ser substitution (#), YTE triple mutation (bold and underlined), allotype G1m(fa) (bold italics), N-terminal Fab residues are underlined, hinge residues are italics

[0148] TIFF0007762645000003.tif50170

[0149] SEQ ID NO: 3: Mature human IgG1 Fc, Cys to Ser substitution (#), YTE triple mutation (bold and underlined), allotype G1m(f) (bold italics), N-terminal Fab residues are underlined, hinge residues are italics

[0150] TIFF0007762645000004.tif50170

[0151] SEQ ID NO: 4: Mature human IgG1 Fc, Cys to Ser substitution (#), YTE triple mutation (bold and underlined), Asn to Ala substitution (*), X1 is Asp or Glu, X2 is Leu or Met, N-terminal Fab residues are underlined, hinge residues are italicized

[0152] TIFF0007762645000005.tif50170

[0153] SEQ ID NO: 5: Mature human IgG1 Fc, Cys to Ser substitution (#), YTE triple mutation (bold and underlined), allotype G1m(fa) (bold italics), Asn to Ala substitution (*), N-terminal Fab residues are underlined, hinge residues are italics

[0154] TIFF0007762645000006.tif50170

[0155] SEQ ID NO: 6: Mature human IgG1 Fc, Cys to Ser substitution (#), YTE triple mutation (bold and underlined), allotype G1m(f) (bold italics), Asn to Ala substitution (*), N-terminal Fab residues are underlined, hinge residues are italics

[0156] TIFF0007762645000007.tif50170

[0157] SEQ ID NO: 7: Mature human IgG1 Fc, Cys to Ser substitution (#), YTE triple mutation (bold and underlined), X6 is Asp or Glu, X7 is Leu or Met, Z1 is Asn or absent, Z2 is Asn or Ala, Z3 is Lys or absent, N-terminal Fab residues are underlined, hinge residues are italicized

[0158] TIFF0007762645000008.tif50170

[0159] SEQ ID NO: 8: Mature human IgG1 Fc, Cys to Ser substitution (#), YTE triple mutation (bold and underlined), allotype G1m(fa) (bold italics), N-terminal Fab residues are underlined, hinge residues are italics

[0160] TIFF0007762645000009.tif50170

[0161] SEQ ID NO: 9: Mature human IgG1 Fc, Cys to Ser substitution (#), YTE triple mutation (bold and underlined), allotype G1m(f) (bold italics), N-terminal Fab residues are underlined, hinge residues are italics

[0162] TIFF0007762645000010.tif50170

[0163] SEQ ID NO: 10: Mature human IgG1 Fc, Cys to Ser substitution (#), YTE triple mutation (bold and underlined), allotype G1m(fa) (bold italics), N-terminal Fab residues are underlined, hinge residues are italics

[0164] TIFF0007762645000011.tif50170

[0165] SEQ ID NO: 11: Mature human IgG1 Fc, Cys to Ser substitution (#), YTE triple mutation (bold and underlined), allotype G1m(f) (bold italics), N-terminal Fab residues are underlined, hinge residues are italics

[0166] TIFF0007762645000012.tif50170

[0167] SEQ ID NO: 12: Mature human IgG1 Fc, Cys to Ser substitution (#), YTE triple mutation (bold and underlined), allotype G1m(fa) (bold italics), N-terminal Fab residues are underlined, hinge residues are italics

[0168] TIFF0007762645000013.tif50170

[0169] SEQ ID NO: 13: Mature human IgG1 Fc, Cys to Ser substitution (#), YTE triple mutation (bold and underlined), allotype G1m(f) (bold italics), N-terminal Fab residues are underlined, hinge residues are italics

[0170] TIFF0007762645000014.tif50170

[0171] SEQ ID NO: 14: Mature human IgG1 Fc, Cys to Ser substitution (#), YTE triple mutation (bold and underlined), allotype G1m(fa) (bold italics), Asn to Ala substitution (*), N-terminal Fab residues are underlined, hinge residues are italics

[0172] TIFF0007762645000015.tif50170

[0173] SEQ ID NO: 15: Mature human IgG1 Fc, Cys to Ser substitution (#), YTE triple mutation (bold and underlined), allotype G1m(f) (bold italics), Asn to Ala substitution (*), N-terminal Fab residues are underlined, hinge residues are italics

[0174] TIFF0007762645000016.tif50170

[0175] SEQ ID NO: 16: Mature human IgG1 Fc, Cys to Ser substitution (#), YTE triple mutation (bold and underlined), allotype G1m(fa) (bold italics), Asn to Ala substitution (*), N-terminal Fab residues are underlined, hinge residues are italics

[0176] TIFF0007762645000017.tif50170

[0177] SEQ ID NO: 17: Mature human IgG1 Fc, Cys to Ser substitution (#), YTE triple mutation (bold and underlined), allotype G1m(f) (bold italics), Asn to Ala substitution (*), N-terminal Fab residues are underlined, hinge residues are italics

[0178] TIFF0007762645000018.tif50170

[0179] SEQ ID NO: 18: Mature human IgG1 Fc, Cys to Ser substitution (#), YTE triple mutation (bold and underlined), allotype G1m(fa) (bold italics), Asn to Ala substitution (*), N-terminal Fab residues are underlined, hinge residues are italics

[0180] TIFF0007762645000019.tif50170

[0181] SEQ ID NO: 19: Mature human IgG1 Fc, Cys to Ser substitution (#), YTE triple mutation (bold and underlined), allotype G1m(f) (bold italics), Asn to Ala substitution (*), N-terminal Fab residues are underlined, hinge residues are italics

[0182] TIFF0007762645000020.tif50170

[0183] SEQ ID NO: 20: Mature human IgG1 Fc, Cys to Ser substitution (#), X4 is Asp or Glu, X5 is Leu or Met; Z1 is Asn or absent, Z3 is Lys or absent, N-terminal Fab residues are underlined, hinge residues are italicized

[0184] TIFF0007762645000021.tif50170

[0185] SEQ ID NO: 21: Mature human IgG1 Fc, Cys to Ser substitution (#), allotype G1m (fa) (bold italics), N-terminal Fab residues are underlined, hinge residues are italics

[0186] TIFF0007762645000022.tif50170

[0187] SEQ ID NO: 22: Mature human IgG1 Fc, Cys to Ser substitution (#), allotype G1m(f) (bold italics), N-terminal Fab residues are underlined, hinge residues are italics

[0188] TIFF0007762645000023.tif50170

[0189] SEQ ID NO: 23: Mature human IgG1 Fc, Cys to Ser substitution (#), X1 is Met or Tyr, X2 is Ser or Thr, X3 is Thr or Glu, X6 is Asp or Glu, X7 is Leu or Met, Z1 is Asn or absent, Z2 is Asn or Ala, Z3 is Lys or absent, N-terminal Fab residues are underlined, hinge residues are italicized

[0190] TIFF0007762645000024.tif50170

[0191] SEQ ID NO: 24: Mature human IgG1 Fc, Cys to Ser substitution (#), X1 is Met or Tyr, X2 is Ser or Thr, X3 is Thr or Glu, X6 is Asp or Glu, X7 is Leu or Met, Z2 is Asn or Ala, N-terminal Fab residues are underlined, hinge residues are italicized

[0192] TIFF0007762645000025.tif50170

[0193] SEQ ID NO: 25: Mature human IgG1 Fc, Cys to Ser substitution (#), X1 is Met or Tyr, X2 is Ser or Thr, X3 is Thr or Glu, X4 is Asp or Glu, X5 is Leu or Met, Z2 is Asn or Ala, N-terminal Fab residues are underlined, hinge residues are italicized

[0194] TIFF0007762645000026.tif50170

[0195] SEQ ID NO: 26: Mature human IgG1 Fc, Cys to Ser substitution (#), X1 is Met or Tyr, X2 is Ser or Thr, X3 is Thr or Glu, X4 is Asp or Glu, X5 is Leu or Met, Z2 is Asn or Ala, N-terminal Fab residues are underlined, hinge residues are italicized

[0196] TIFF0007762645000027.tif50170

[0197] SEQ ID NO: 27: Mature human IgG1 Fc, Cys to Ser substitution (#), X1 is Met or Tyr, X2 is Ser or Thr, X3 is Thr or Glu, X6 is Asp or Glu, X7 is Leu or Met, Z2 is Asn or Ala, N-terminal Fab residues are underlined, hinge residues are italicized

[0198] TIFF0007762645000028.tif50170

[0199] SEQ ID NO: 28: Mature human IgG1 Fc, Cys to Ser substitution (#), X1 is Met or Tyr, X2 is Ser or Thr, X3 is Thr or Glu, Z2 is Asn or Ala, N-terminal Fab residues are underlined, hinge residues are italicized

[0200] TIFF0007762645000029.tif50170

[0201] SEQ ID NO: 29: Mature human IgG1 Fc, Cys to Ser substitution (#), X1 is Met or Tyr, X2 is Ser or Thr, X3 is Thr or Glu, Z2 is Asn or Ala, N-terminal Fab residues are underlined, hinge residues are italicized

[0202] TIFF0007762645000030.tif50170

[0203] SEQ ID NO: 30: Mature human IgG1 Fc with mouse heavy chain MIgG Vh signal sequence (bold), Cys to Ser substitution (#), allotype G1m(fa) (bold italics), N-terminal Fab residues are underlined, hinge residues are italicized

[0204] TIFF0007762645000031.tif50170

[0205] SEQ ID NO: 31: Mature human Fc IgG1, Z1 is Asn or absent, Z2 is Lys or absent, J1 is Cys or Ser, X1 is Met or Tyr, X2 is Ser or Thr, X3 is Thr or Glu, Z2 is Asn or Ala, X4 is Leu or Asp, X5 is Gln or His, X6 is Asp or Glu, X7 is Leu or Met, X8 is Met or Leu, X9 is Asn or Ser, N-terminal Fab residues are underlined, hinge residues are italicized

[0206] TIFF0007762645000032.tif50170

[0207] SEQ ID NO: 32: Mature human Fc IgG1, Cys to Ser substitution (#), Z1 is Asn or absent, Z3 is Lys or absent, Z2 is Asn or Ala, X4 is Leu or Asp, X5 is Gln or His, X6 is Asp or Glu, X7 is Leu or Met, X8 is Met or Leu, X9 is Asn or Ser, N-terminal Fab residues are underlined, hinge residues are italicized

[0208] TIFF0007762645000033.tif50170

[0209] SEQ ID NO: 33: Mature human Fc IgG1, Cys to Ser substitution (#), DHS triple mutation (bold and underlined), Z1 is Asn or absent, Z3 is Lys or absent, Z2 is Asn or Ala, X6 is Asp or Glu, X7 is Leu or Met, X8 is Met or Leu, X9 is Asn or Ser, N-terminal Fab residues are underlined, hinge residues are italicized

[0210] TIFF0007762645000034.tif50170

[0211] SEQ ID NO: 34: Mature human Fc IgG1, Cys to Ser substitution (#), DHS triple mutation (bold and underlined), Z2 is Asn or Ala, X6 is Asp or Glu, X7 is Leu or Met, N-terminal Fab residues are underlined, hinge residues are italicized

[0212] TIFF0007762645000035.tif50170

[0213] SEQ ID NO: 35: Mature human Fc IgG1, Cys to Ser substitution (#), DHS triple mutation (bold and underlined), X6 is Asp or Glu, X7 is Leu or Met, N-terminal Fab residues are underlined, hinge residues are italicized

[0214] TIFF0007762645000036.tif50170

[0215] SEQ ID NO: 36: Mature human Fc IgG1, Cys to Ser substitution (#), DHS triple mutation (bold and underlined), allotype G1m(fa) (bold italics), N-terminal Fab residues are underlined, hinge residues are italics

[0216] TIFF0007762645000037.tif50170

[0217] SEQ ID NO: 37: Mature human Fc IgG1, Cys to Ser substitution (#), DHS triple mutation (bold and underlined), allotype G1m(f) (bold italics), N-terminal Fab residues are underlined, hinge residues are italics

[0218] TIFF0007762645000038.tif50170

[0219] SEQ ID NO: 38: Mature human Fc IgG1, Cys to Ser substitution (#), DHS triple mutation (bold and underlined), allotype G1m(fa) (bold italics), N-terminal Fab residues are underlined, hinge residues are italics

[0220] TIFF0007762645000039.tif50170

[0221] SEQ ID NO: 39: Mature human Fc IgG1, Cys to Ser substitution (#), DHS triple mutation (bold and underlined), allotype G1m(f) (bold italics), N-terminal Fab residues are underlined, hinge residues are italics

[0222] TIFF0007762645000040.tif50170

[0223] SEQ ID NO: 40: Mature human Fc IgG1, Cys to Ser substitution (#), DHS triple mutation (bold and underlined), allotype G1m(fa) (bold italics), N-terminal Fab residues are underlined, hinge residues are italics

[0224] TIFF0007762645000041.tif50170

[0225] SEQ ID NO: 41: Mature human Fc IgG1, Cys to Ser substitution (#), DHS triple mutation (bold and underlined), allotype G1m(f) (bold italics), N-terminal Fab residues are underlined, hinge residues are italics

[0226] TIFF0007762645000042.tif50170

[0227] SEQ ID NO: 42: Mature human Fc IgG1, Cys to Ser substitution (#), DHS triple mutation (bold and underlined), allotype G1m(fa) (bold italics), N-terminal Fab residues are underlined, hinge residues are italics

[0228] TIFF0007762645000043.tif50170

[0229] SEQ ID NO: 43: Mature human Fc IgG1, Cys to Ser substitution (#), DHS triple mutation (bold and underlined), allotype G1m(f) (bold italics), N-terminal Fab residues are underlined, hinge residues are italics

[0230] TIFF0007762645000044.tif50170

[0231] SEQ ID NO: 44: Mature human Fc IgG1, Cys to Ser substitution (#), Asn to Ala substitution (*), DHS triple mutation (bold and underlined), X6 is Asp or Glu, X7 is Leu or Met, N-terminal Fab residues are underlined, hinge residues are italicized

[0232] TIFF0007762645000045.tif50170

[0233] SEQ ID NO: 45: Mature human Fc IgG1, Cys to Ser substitution (#), Asn to Ala substitution (*), DHS triple mutation (bold and underlined), allotype G1m(fa) (bold italics), N-terminal Fab residues are underlined, hinge residues are italics

[0234] TIFF0007762645000046.tif50170

[0235] SEQ ID NO: 46: Mature human Fc IgG1, Cys to Ser substitution (#), Asn to Ala substitution (*), DHS triple mutation (bold and underlined), allotype G1m(f) (bold italics), N-terminal Fab residues are underlined, hinge residues are italics

[0236] TIFF0007762645000047.tif50170

[0237] SEQ ID NO: 47: Mature human Fc IgG1, Cys to Ser substitution (#), Asn to Ala substitution (*), DHS triple mutation (bold and underlined), allotype G1m(fa) (bold italics), N-terminal Fab residues are underlined, hinge residues are italics

[0238] TIFF0007762645000048.tif50170

[0239] SEQ ID NO: 48: Mature human Fc IgG1, Cys to Ser substitution (#), Asn to Ala substitution (*), DHS triple mutation (bold and underlined), allotype G1m(f) (bold italics), N-terminal Fab residues are underlined, hinge residues are italics

[0240] TIFF0007762645000049.tif50170

[0241] SEQ ID NO: 49: Mature human Fc IgG1, Cys to Ser substitution (#), Asn to Ala substitution (*), DHS triple mutation (bold and underlined), allotype G1m(fa) (bold italics), N-terminal Fab residues are underlined, hinge residues are italics

[0242] TIFF0007762645000050.tif50170

[0243] SEQ ID NO: 50: Mature human Fc IgG1, Cys to Ser substitution (#), Asn to Ala substitution (*), DHS triple mutation (bold and underlined), allotype G1m(f) (bold italics), N-terminal Fab residues are underlined, hinge residues are italics

[0244] TIFF0007762645000051.tif50170

[0245] SEQ ID NO: 51: Mature human Fc IgG1, Cys to Ser substitution (#), Asn to Ala substitution (*), DHS triple mutation (bold and underlined), allotype G1m(fa) (bold italics), N-terminal Fab residues are underlined, hinge residues are italics

[0246] TIFF0007762645000052.tif50170

[0247] SEQ ID NO: 52: Mature human Fc IgG1, Cys to Ser substitution (#), Asn to Ala substitution (*), DHS triple mutation (bold and underlined), allotype G1m(f) (bold italics), N-terminal Fab residues are underlined, hinge residues are italics

[0248] TIFF0007762645000053.tif50170

[0249] SEQ ID NO: 53: Mature human Fc IgG1, added N-terminal ISAMVRS amino acid residues (italics), C-terminal G4S linker (italics), C-terminal myc-tag (underlined), allotype G1m(f) (bold italics)

[0250] TIFF0007762645000054.tif50170

[0251] SEQ ID NO: 54: Mature human Fc IgG1, with added N-terminal ISAMVRS amino acid residues (italics), allotype G1m(fa) (bold italics)

[0252] TIFF0007762645000055.tif43170

[0253] SEQ ID NO: 55: Mature human Fc IgG1, added N-terminal amino acid residues (italics), hinge residues are in italics, allotype G1m(fa) (bold italics)

[0254] TIFF0007762645000056.tif43170

[0255] SEQ ID NO: 56: Mature human IgG1 Fc, Cys to Ser substitution (#), allotype G1m (fa) (bold italics), N-terminal Fab residues are underlined, hinge residues are italics

[0256] TIFF0007762645000057.tif50170

[0257] SEQ ID NO: 57: Mature human IgG1 Fc, Cys to Ser substitution (#), allotype G1m(f) (bold italics), N-terminal Fab residues are underlined, hinge residues are italics

[0258] TIFF0007762645000058.tif50170

[0259] SEQ ID NO: 58: Mature human IgG1 Fc, Cys to Ser substitution (#), M428L, N434S (bold / underlined), allotype G1m(fa) (bold italics), N-terminal Fab residues are underlined, hinge residues are italics

[0260] TIFF0007762645000059.tif50170

[0261] As defined herein, a variant Fc domain is a C HThe dimerized variant Fc domain comprises two variant Fc domain monomers dimerized by interactions between three antibody constant domains and one or more disulfide bonds formed between the hinge domains of the two dimerized variant Fc domain monomers. In some examples, the variant Fc domain forms a minimal structure that binds to an Fc receptor, e.g., an Fc-gamma receptor (i.e., an Fcγ receptor (FcγR)), an Fc-alpha receptor (i.e., an Fcα receptor (FcαR)), an Fc-epsilon receptor (i.e., an Fcε receptor (FcεR)), and / or an embryonic Fc receptor (FcRn). In some embodiments, the Fc domains of the present invention bind to an Fcγ receptor (e.g., FcRn, FcγRI (CD64), FcγRIIa (CD32), FcγRIIb (CD32), FcγRIIIa (CD16a), FcγRIIIb (CD16b)), and / or FcγRIV and / or fetal Fc receptor (FcRn).

[0262] In some embodiments, variant Fc domains or variant Fc domain monomers of the invention are engineered to have improved binding to the fetal Fc receptor (FcRn). Improved binding to FcRn may increase the half-life of a conjugate or fusion protein containing the Fc domain, for example, the variant Fc domain monomer or variant Fc domain may increase the half-life of the conjugate by 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500% or more compared to a conjugate having a corresponding Fc domain without the C220S / M252Y / S254T / T256E, C220S / V309D / Q311H / N434S, C220S, or additional mutations that improve FcRn binding. As used herein, an amino acid "corresponding to" a particular amino acid residue (e.g., in a particular SEQ ID NO:) should be understood to include any amino acid residue that one of skill in the art would understand to align with the particular residue (e.g., in a particular sequence). For example, any one of SEQ ID NOs: 1-3, 8-13, or 20-29 can be mutated to include an N297 (e.g., N297A) mutation by mutating the "corresponding residue" in the amino acid sequence.

[0263] In some examples, the variant Fc domains or variant Fc domain monomers of the invention are engineered to reduce or eliminate binding to Fc receptors, e.g., Fc-gamma receptors (i.e., Fcγ receptors (FcγR)), Fc-alpha receptors (i.e., Fcα receptors (FcαR)), Fc-epsilon receptors (i.e., Fcε receptors (FcεR)), and / or fetal Fc receptors (FcRn). In some embodiments, the Fc domains of the invention bind to Fcγ receptors (e.g., FcRn, FcγRI (CD64), FcγRIIa (CD32), FcγRIIb (CD32), FcγRIIIa (CD16a), FcγRIIIb (CD16b)), and / or FcγRIV and / or fetal Fc receptors (FcRn), and are particularly useful for delivery of therapeutic agents (e.g., small molecule therapeutics and therapeutic peptide agents).

[0264] In some embodiments, a variant Fc domain or variant Fc domain monomer of the invention has the sequence of any one of SEQ ID NOs: 1-29 and 31-52, and may further comprise an additional amino acid (Xaa)x at the N-terminus and / or an additional amino acid (Xaa)z at the C-terminus, where each Xaa is independently any amino acid, and x and z are integers equal to or greater than zero, typically less than 100, preferably less than 10, and more preferably 0, 1, 2, 3, 4, or 5.

[0265] Immune cell activation Fc-gamma receptors (FcγRs) bind to the Fc portion of immunoglobulin G (IgG) and play an important role in immune activation and regulation. For example, the Fc domain of IgG in immune complexes (ICs) engages with FcγRs with high avidity, thereby inducing a signaling cascade that controls immune cell activation. The human FcγR family contains several activating receptors (FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and FcγRIIIb) and one inhibitory receptor (FcγRIIb). FcγR signaling is mediated by intracellular domains that contain immunotyrosine-based activation motifs (ITAMs) for activating FcγRs and immunotyrosine-based inhibitory motifs (ITIMs) for the inhibitory receptor FcγRIIb. In some embodiments, FcγR binding by the Fc domain results in ITAM phosphorylation by Src family kinases; this activates Syk family kinases, triggering downstream signaling, including the PI3K and Ras pathways.

[0266] In some examples, in the conjugates and fusion proteins described herein, the portion of the conjugate or fusion protein comprising a therapeutic agent monomer or dimer binds to a surface-exposed target of an infectious pathogen (e.g., a viral particle, a fungus, or a bacterium), while the variant Fc domain portion of the conjugate or fusion protein binds to FcγR (e.g., FcRn, FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and FcγRIIIb) on immune cells, activating phagocytosis and effector function, e.g., antibody-dependent cell-mediated cytotoxicity (ADCC), resulting in phagocytosis and destruction of the infectious pathogen by the immune cell and further enhancing the anti-pathogenic (e.g., antiviral, antifungal, or antibacterial) activity of the conjugate. Examples of immune cells that can be activated by the conjugates described herein include, but are not limited to, macrophages, neutrophils, eosinophils, basophils, lymphocytes, follicular dendritic cells, natural killer cells, and mast cells.

[0267] Half-life Biological half-life (t 1 / 2) is the time it takes for a therapeutic agent to decrease its maximum concentration by half. Improving the half-life of a therapeutic agent can reduce the effective dose. Many variables affect half-life, from patient variables (e.g., age, blood, diet, excess fluid, lack of fluid, gender, drug history, kidney function, liver function, obesity, pre-existing conditions, etc.) to variables specific to the therapeutic agent (e.g., therapeutic formulation, pharmacokinetics, method of administration, drug clearance (e.g., kidney, liver, or lung), tissue distribution and accumulation, size, charge, pKa of the therapeutic agent, etc.). For peptide therapeutics, a short plasma half-life is generally due to rapid renal clearance and enzymatic degradation that occurs during systemic circulation. Peptide or protein modifications can lead to extended plasma half-life times. In some examples, variant Fc domains or fusion proteins are engineered to increase the half-life of variant Fc domain monomers, conjugates, or fusion proteins. In some embodiments, the variant Fc domains or variant Fc domain monomers of the invention are engineered to have improved binding to the fetal Fc receptor (FcRn). Improved binding to FcRn may increase the half-life of a conjugate or fusion protein containing the Fc domain, for example, the variant Fc domain monomer or variant Fc domain may increase the half-life of the conjugate by 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500% or more compared to a conjugate having a corresponding Fc domain without mutations, e.g., C220S / M252Y / S254T / T256E, C220S / V309D / Q311H / N434S, or additional mutations that improve FcRn binding. In some examples, the variant Fc domain monomer is engineered to contain at least 220 residues.

[0268] Renal clearance Many therapeutic peptides have short half-lives (minutes) in vivo due to their size. The rapid clearance and short half-life of peptides limit their successful development into drugs. One of the major causes of rapid clearance of peptides from the systemic circulation is renal clearance. Glomeruli have a pore size of approximately 8 nm, and hydrophilic peptides with a MW <2-25 kDa are prone to rapid filtration through the glomerulus of the kidney. In some embodiments, the variant Fc domain monomers and fusion proteins described herein are greater than 20 kDa. In some embodiments, two variant Fc domain monomers and fusion proteins of a conjugate or fusion protein can dimerize to form a variant Fc domain. In some embodiments, the variant Fc domain monomer, conjugate, or fusion protein is engineered to reduce renal clearance. Decreased renal clearance may increase the half-life of a variant Fc domain monomer of a conjugate or fusion protein described herein, for example, a variant Fc domain may comprise at least about 200 amino acids (e.g., at least 200, at least 225, at least about 230, at least about 240, at least about 242, at least about 243, at least about 250, at least about 255, at least about 260, at least about 265, at least about 270, at least about 275, at least about 280, at least about 285, at least about 290, at least about 295, or at least about 300 amino acids).

[0269] tissue distribution After entering the systemic circulation, therapeutic agents are distributed to tissues throughout the body. Distribution is usually heterogeneous due to differences in blood perfusion, tissue binding, local pH, and cell membrane permeability. The rate at which a drug enters a tissue depends on the rate of blood flow to the tissue, the tissue mass, and the partitioning characteristics between blood and tissue. Distribution equilibrium between blood and tissue (where entry and elimination rates are equal) is reached more quickly in highly vascularized areas unless diffusion through cell membranes is the rate-limiting step. Size, shape, charge, target binding, FcRn and target binding mechanism, route of administration, and formulation affect tissue distribution.

[0270] In some examples, the variant Fc polypeptides are optimized for distribution to lung tissue. In some examples, the variant Fc domain monomers, conjugates, and fusion proteins have a concentration ratio of distribution in epithelial lining fluid that is at least 30% of the concentration of the polypeptide, conjugate, or fusion protein in plasma within 2 hours after administration. In certain embodiments, the concentration ratio is at least 45% within 2 hours after administration. In some embodiments, the concentration ratio is at least 55% within 2 hours after administration. In particular, the concentration ratio is at least 60% within 2 hours after administration. As shown in Example 5 and Figure 2, by 2 hours after injection, the ELF level of Conjugate 2 was surprisingly about 60% of the plasma exposure level as measured by AUC over the remaining time course, indicating near-instantaneous distribution of Conjugate 2 from plasma to ELF in the lung. This demonstrates that Conjugate 2 rapidly distributes to the lung and maintains a high concentration in the lung compared to the level in plasma.

[0271] In some embodiments, the variant Fc domain monomer comprises no more than 400 amino acid residues, no more than 350 amino acid residues, no more than 300 amino acid residues, or no more than 250 amino acid residues.

[0272] In some examples, the variant Fc polypeptide is optimized for distribution to liver, nervous (eg, CNS), muscle, skin, eye, or vascular tissue.

[0273] When an Fc polypeptide is preferentially distributed to one or more particular tissues, the polypeptide can be used to treat disorders of the corresponding tissue (eg, deliver a therapeutic agent to the tissue).

[0274] Fc domain monomer boundary The length of the variant Fc domain monomer (e.g., as determined by the N- and C-terminal boundaries) can be optimized to prevent kidney clearance and increase distribution to desired tissues (e.g., lung tissue). Antibodies are divided into two domains: an Fc (effector) domain and a fragment antigen-binding (Fab) domain, the latter containing the antigen-binding region. The present disclosure provides variant Fc domain monomers comprising a portion of a Fab domain at the N-terminus of the Fc domain. The inventors observed that smaller Fc constructs (e.g., Fc constructs lacking a portion of the Fab domain) exhibited reduced half-lives, likely due to renal excretion. To address this issue, the Fc constructs were iteratively extended by adding portions of the Fab domain back to the N-terminus until further increases in size no longer resulted in improvement (e.g., in mouse pharmacokinetic experiments). The present disclosure provides variant Fc domain monomers that are optimized (e.g., by length, mass, N-terminal, and / or C-terminal boundaries, in addition to mutational variants) to achieve the desired increased half-life and / or tissue distribution.

[0275] In some embodiments, the N-terminus of the variant Fc domain monomer comprises between 10 and 20 residues of the Fab domain (e.g., residues 11, 12, 13, 14, 15, 16, 17, 18, or 19). In certain embodiments, the N-terminus of the variant Fc domain monomer is any one of amino acid residues 198-205. In some embodiments, the N-terminus of the variant Fc domain monomer is amino acid residue 201 (e.g., Asn201). In certain embodiments, the N-terminus of the variant Fc domain monomer is amino acid residue 202 (e.g., Val202). In other embodiments, the C-terminus of the variant Fc domain monomer is any one of amino acid residues 437-447. In another embodiment, the C-terminus of the variant Fc domain monomer is amino acid residue 446 (e.g., Gly446). In some embodiments, the C-terminus of the variant Fc domain monomer is amino acid residue 447 (eg, Lys447).

[0276] Elongation of the construct required the addition of a portion of the hinge region containing a free cysteine ​​residue (C220), which created problems with thiol-mediated aggregation. To circumvent this problem, C220 was mutated to serine (C220S).

[0277] Therapeutic Delivery The large size of antibody molecules can make it difficult to transport targeting systems across cell membranes. In some instances, large targeting systems can be slowly cleared from the blood circulation, which can ultimately lead to bone marrow toxicity. Furthermore, the in vivo use of antibody-based targeting systems can be expensive and can lead to immunogenicity after repeated injections of such formulations. Antibody fragments smaller than intact antibodies have been successfully generated, but are still excessively large in many instances. Fragments can reach the extracellular space more easily than intact antibodies. In some instances, variant Fc domain monomers can be used in conjugates to deliver therapeutic agents. In some instances, the variant Fc domain forms a minimal structure that binds to Fc receptors, such as Fc-gamma receptors (i.e., Fcγ receptors (FcγR)), Fc-alpha receptors (i.e., Fcα receptors (FcαR)), Fc-epsilon receptors (i.e., Fcε receptors (FcεR)), and / or fetal Fc receptors (FcRn). In some embodiments, the Fc domains of the present invention bind to Fcγ receptors (e.g., FcRn, FcγRI (CD64), FcγRIIa (CD32), FcγRIIb (CD32), FcγRIIIa (CD16a), FcγRIIIb (CD16b)), and / or FcγRIV and / or fetal Fc receptor (FcRn). Fetal Fc receptor binding mediates internalization of the variant Fc domain monomer or its fusion protein conjugate, thereby delivering the therapeutic agent into the cell. Upon internalization, endocytic salvage pathways prevent degradation of the variant Fc domain monomer or its conjugate or fusion protein. In some examples, the variant Fc domain monomer of the variant Fc domain is engineered to reduce fetal Fc receptor binding, thereby reducing internalization into the cell and increasing the plasma concentration of the variant Fc domain conjugate or its fusion protein.

[0278] II. Conjugates of the Present Disclosure Provided herein are synthetic conjugates useful in treating the conditions or disorders described herein (e.g., respiratory disorders, liver disorders, central nervous system disorders, muscle disorders, skin disorders, eye disorders, vascular disorders, or infectious diseases (e.g., viral, fungal, or bacterial infections)). The conjugates disclosed herein (e.g., conjugates represented by Formula (1)) comprise a variant Fc domain conjugated to one or more therapeutic agents (e.g., one or more small molecule therapeutic agents).

[0279] Without being bound by theory, in some embodiments, the conjugates described herein bind to surface-exposed targets on infectious pathogens (e.g., viral particles, fungi, or bacteria) via interactions between the therapeutic agent in the conjugate and proteins on the surface of the infectious pathogen.

[0280] The conjugates of the invention comprise a therapeutic agent conjugated to a variant Fc domain or variant Fc domain monomer. The variant Fc domain in the conjugates described herein binds to FcγRs (e.g., FcRn, FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and FcγRIIIb) on immune cells. Binding of the variant Fc domain in the conjugates described herein to FcγRs on immune cells activates phagocytosis and effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC), resulting in the phagocytosis and destruction of infectious pathogens by immune cells, further enhancing the activity of the conjugate.

[0281] In some embodiments, the conjugates provided herein are represented by formula (1): In some embodiments, when n is 2, E (variant Fc domain monomers) dimerize to form a variant Fc domain.

[0282] In some embodiments, the variant Fc domain monomer of the conjugate comprises fewer than about 300 amino acid residues (e.g., fewer than about 300, fewer than about 295, fewer than about 290, fewer than about 285, fewer than about 280, fewer than about 275, fewer than about 270, fewer than about 265, fewer than about 260, fewer than about 255, fewer than about 250, fewer than about 245, fewer than about 240, fewer than about 235, fewer than about 230, fewer than about 225, or fewer than about 220 amino acid residues). In some embodiments, the variant Fc domain monomer of the conjugate is less than about 40 kDa (e.g., less than about 35 kDa, less than about 30 kDa, less than about 25 kDa).

[0283] In some embodiments, the variant Fc domain monomer of the conjugate comprises at least 200 amino acid residues (e.g., at least 210, at least 220, at least 230, at least 240, at least 250, at least 260, at least 270, at least 280, at least 290, or at least 300 amino acid residues). In some embodiments, the variant Fc domain monomer is at least 20 kDa (e.g., at least 25 kDa, at least 30 kDa, or at least 35 kDa).

[0284] In some embodiments, the variant Fc domain monomer of the conjugate comprises 200-400 amino acid residues (e.g., 200-250, 250-300, 300-350, 350-400, 200-300, 250-350, or 300-400 amino acid residues). In some embodiments, the variant Fc domain monomer of the conjugate is between 200 and 300 amino acid residues in length (e.g., between 210-300, 230-300, 250-300, 270-300, 290-300, 210-290, 220-280, 230-270, 240-260, or 245-255 amino acid residues). In some embodiments, the variant Fc domain monomer of the conjugate is between 20 and 40 kDa (e.g., 20-25 kDa, 25-30 kDa, 35-40 kDa, 20-30 kDa, 25-35 kDa, or 30-40 kDa). In some embodiments, the variant Fc domain monomer of the conjugate is between about 20 kDa and about 40 kDa in mass (e.g., 20 kDa-25 kDa, 25 kDa-30 kDa, 30 kDa-35 kDa, 35 kDa-40 kDa).

[0285] In some embodiments, each linker comprises a polyethylene glycol (PEG) linker comprising between about 2 and 10 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) PEG units. In some embodiments, at least one arm of the trivalent linker comprises a polyethylene glycol (PEG) linker comprising between about 2 and 10 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) PEG units.

[0286] In some embodiments, the conjugate is at least 40 kDa (e.g., at least 45 kDa, at least 50 kDa, at least 55 kDa, at least 60 kDa, at least 65 kDa, at least 70 kDa, at least 75 kDa, or at least 80 kDa). In some embodiments, the conjugate has a mass between about 40 kDa and about 80 kDa (e.g., 40 kDa to 50 kDa, 45 kDa to 55 kDa, 50 kDa to 60 kDa, 55 kDa to 65 kDa, 60 kDa to 70 kDa, 65 kDa to 75 kDa, or 70 kDa to 80 kDa).

[0287] In certain embodiments, the conjugate comprises between 230 and 250 amino acid residues linked to an average of between 1 and 10 (e.g., 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10) small molecules by a linker (e.g., a dimeric or trimeric linker (e.g., a linker comprising between 2 and 10 PEG units) linked to one or more (e.g., 1, 2, 3, 4, or more) small molecules). (e.g., 231 amino acid residues, 232 amino acid residues, 233 amino acid residues, 234 amino acid residues, 235 amino acid residues, 236 amino acid residues, 237 amino acid residues, 238 amino acid residues, 239 amino acid residues, 240 amino acid residues, 241 amino acid residues, 242 amino acid residues, 243 amino acid residues, 244 amino acid residues, 245 amino acid residues, 246 amino acid residues, 247 amino acid residues, 248 amino acid residues, 249 amino acid residues, or 250 amino acid residues).

[0288] The conjugates described herein can be synthesized using chemical synthesis techniques available in the art. If a functional group is not available for conjugation, the molecule can be derivatized using conventional chemical synthesis techniques well known in the art. In some embodiments, the conjugates described herein contain one or more chiral centers. The conjugates include isolated stereoisomers and mixtures of stereoisomers (including racemic mixtures) of varying chiral purity. They also encompass the various diastereomers, enantiomers, and tautomers that may be formed.

[0289] In the conjugates described herein, the curve connected to E indicates that one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) therapeutic agents may be attached to the variant Fc domain monomer. In some embodiments, when n is 1, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) therapeutic agents may be attached to the variant Fc domain monomer or variant Fc domain. In some embodiments, when n is 2, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) therapeutic agents may be attached to the variant Fc domain. The curves in the conjugates described herein should not be interpreted as single bonds between one or more therapeutic agents and atoms in the variant Fc domain. In some embodiments, when T is 1, one therapeutic agent may be attached to an atom in the variant Fc domain monomer or variant Fc domain. In some embodiments, when T is 2, two therapeutic agents may be attached to an atom in the variant Fc domain monomer or variant Fc domain.

[0290] As further described herein, the linker (e.g., L) in the conjugates described herein can be a branched structure. As further described herein, the linker (e.g., L) in the conjugates described herein can be a multivalent structure, e.g., a bivalent or trivalent structure having two or three arms, respectively. In some embodiments, when the linker has three arms, two of the arms can be attached to a first and second therapeutic agent, and the third arm can be attached to a variant Fc domain monomer or a variant Fc domain.

[0291] In a conjugate having a variant Fc domain covalently linked to one or more therapeutic agents, when n is 2, as represented by formula (1), two variant Fc domain monomers (each variant Fc domain monomer is represented by E) dimerize to form a variant Fc domain.

[0292] Monomeric conjugates of therapeutic agents linked to variant Fc domains In some embodiments, the conjugates described herein comprise a variant Fc domain monomer or variant Fc domain covalently linked to one or more monomers of a therapeutic agent. The conjugate of a variant Fc domain monomer and one or more monomers of a therapeutic agent may be formed by linking the variant Fc domain to each of the monomers of the therapeutic agent via a linker, e.g., any of the linkers described herein.

[0293] In conjugates having a variant Fc domain covalently linked to one or more monomers of a therapeutic agent described herein, the curve connected to E indicates that one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) monomers of the therapeutic agent can be linked to the variant Fc domain monomer or variant Fc domain. In some embodiments, when n is 1, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) monomers of the therapeutic agent can be linked to the variant Fc domain monomer. In some embodiments, when n is 2, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) monomers of a therapeutic agent may be attached to the variant Fc domain. The curves in the conjugates described herein should not be interpreted as single bonds between one or more monomers of a therapeutic agent and an atom in a variant Fc domain monomer or variant Fc domain. In some embodiments, when T is 1, one monomer of a therapeutic agent may be attached to an atom in a variant Fc domain monomer or variant Fc domain. In some embodiments, when T is 2, two monomers of a therapeutic agent may be attached to an atom in a variant Fc domain monomer or variant Fc domain. In some embodiments, the conjugated variant Fc domain is part of a fusion protein described herein.

[0294] In some embodiments, the first AL site is specifically conjugated to a lysine residue of E (e.g., the nitrogen atom of a surface-exposed lysine residue of E) and the second AL site is specifically conjugated to a cysteine ​​residue of E (e.g., the sulfur atom of a surface-exposed cysteine ​​residue of E). In some embodiments, the first AL site is specifically conjugated to a cysteine ​​residue of E (e.g., the sulfur atom of a surface-exposed cysteine ​​residue of E) and the second AL site is specifically conjugated to a lysine residue of E (e.g., the nitrogen atom of a surface-exposed lysine residue of E).

[0295] As further described herein, the linker (e.g., L) in a conjugate having a variant Fc domain monomer or variant Fc domain covalently linked to one or more therapeutic agents described herein may be a bivalent structure having two arms, one arm of which may be attached to a therapeutic agent and the other arm of which may be attached to a variant Fc domain monomer or variant Fc domain.

[0296] In conjugates having a variant Fc domain covalently linked to one or more monomers of a therapeutic agent, as described herein, when n is 2, two variant Fc domain monomers (each variant Fc domain monomer is represented by E) dimerize to form the variant Fc domain.

[0297] Dimeric conjugates of therapeutic agents linked to variant Fc domains In some embodiments, a conjugate described herein (e.g., a conjugate of Formula (1)) comprises a variant Fc domain monomer or variant Fc domain covalently linked to one or more dimers of a therapeutic agent. Conjugates of a variant Fc domain monomer and one or more dimers of a therapeutic agent can be formed by linking the variant Fc domain to each of the dimers of the therapeutic agent via a linker, e.g., a linker described herein. The first and second therapeutic agents are linked to each other by a linker, e.g., a linker described herein. In some embodiments where the therapeutic agent is a dimer, each therapeutic agent can be the same small molecule agent (e.g., a homodimer) or a different small molecule agent (e.g., a heterodimer).

[0298] In conjugates having a variant Fc domain covalently linked to one or more dimers of a therapeutic agent described herein, the curve connected to E indicates that one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) dimers of the therapeutic agent may be attached to the variant Fc domain monomer or variant Fc domain. In some embodiments, when n is 1, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) dimers of the therapeutic agent may be attached to the variant Fc domain monomer. In some embodiments, when n is 2, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) dimers of a therapeutic agent may be attached to the variant Fc domain. The curves in the conjugates described herein should not be interpreted as single bonds between one or more dimers of a therapeutic agent and an atom in a variant Fc domain monomer or variant Fc domain. In some embodiments, when T is 1, one dimer of a therapeutic agent may be attached to a variant Fc domain monomer or atom in a variant Fc domain. In some embodiments, when T is 2, two monomers of a therapeutic agent may be attached to a variant Fc domain monomer or atom in a variant Fc domain. In some embodiments, the variant Fc domain is part of a fusion protein described herein.

[0299] In some embodiments, the first AL site is specifically conjugated to a lysine residue of E (e.g., the nitrogen atom of a surface-exposed lysine residue of E) and the second AL site is specifically conjugated to a cysteine ​​residue of E (e.g., the sulfur atom of a surface-exposed cysteine ​​residue of E). In some embodiments, the first AL site is specifically conjugated to a cysteine ​​residue of E (e.g., the sulfur atom of a surface-exposed cysteine ​​residue of E) and the second AL site is specifically conjugated to a lysine residue of E (e.g., the nitrogen atom of a surface-exposed lysine residue of E).

[0300] As further described herein, the linker (e.g., L) in a conjugate having a variant Fc domain monomer or variant Fc domain covalently linked to one or more dimers of a therapeutic agent described herein can be a trivalent structure (e.g., a trivalent linker) having three arms, each covalently linked to a component of the conjugate (e.g., a first arm conjugated to a first therapeutic agent, a second arm conjugated to a therapeutic agent, and a third arm conjugated to a fusion protein or variant Fc domain monomer).

[0301] In conjugates having a variant Fc domain covalently linked to one or more dimers of a therapeutic agent, as described herein, when n is 2, two variant Fc domain monomers (each variant Fc domain monomer is represented by E) dimerize to form the variant Fc domain.

[0302] III. Fusion Proteins The present invention features fusion proteins comprising at least one variant Fc domain monomer conjugated to at least one (e.g., one or two) therapeutic peptide agents. An exemplary fusion protein of the invention comprises the structure: (P2-L2)n2-B-(L1-P1)n1, where B is a variant Fc domain monomer (e.g., an Fc domain monomer comprising the amino acid sequence of any one of SEQ ID NOS: 1-29, 31-52, and 56-58) or a conjugate thereof; P1 and P2 are each independently a therapeutic peptide agent; L1 and L2 are each independently a linker (e.g., a chemical linker or a peptide linker); n1 and n2 are each independently 0 or 1, and at least one of n1 and n2 is 1 (e.g., the fusion protein must comprise at least one therapeutic peptide agent).

[0303] In some embodiments, the fusion protein comprises one variant Fc domain monomer conjugated to one therapeutic peptide agent. For example, n1 is 1, n2 is 0, and the fusion protein comprises the structure: B-L1-P1. The variant Fc domain monomer and therapeutic peptide agent can be conjugated in any orientation. When C to N conjugation occurs, the variant Fc domain monomer and therapeutic peptide agent can be expressed as a single polypeptide construct including a polypeptide linker, or can be expressed separately and then conjugated via a polypeptide or chemical linker. When C to C or N to N conjugation occurs, the variant Fc domain monomer and therapeutic peptide agent can be expressed separately and then conjugated via, for example, a chemical or peptide linker. For example, a linker (L1) can be conjugated to the C-terminus of the variant Fc domain monomer (B) and the N-terminus of the therapeutic peptide agent (P1). Alternatively, the linker (L1) can be conjugated to the N-terminus of the variant Fc domain monomer (B) and the C-terminus of the therapeutic peptide agent (P1). Alternatively, the linker (L1) is conjugated to the N-terminus of the variant Fc domain monomer (B) and the N-terminus of the therapeutic peptide agent (P1). Alternatively, the linker (L1) is conjugated to the C-terminus of the variant Fc domain monomer (B) and the C-terminus of the therapeutic peptide agent (P1).

[0304] In some embodiments, the fusion protein comprises one variant Fc domain monomer conjugated to two therapeutic peptide agents. For example, n1 is 1, n2 is 1, and the fusion protein comprises the structure: P2-L2-B-L1-P1. As noted above, conjugation can occur in any orientation, and the fusion protein can be expressed as a single polypeptide construct or assembled by chemical conjugation. For example, linker (L2) can be conjugated to the C-terminus of the therapeutic peptide agent (P2) and the N-terminus of the variant Fc domain monomer (B), and linker (L1) can be conjugated to the C-terminus of the variant Fc domain monomer (B) and the N-terminus of the therapeutic peptide agent (P1). Alternatively, linker (L2) may be conjugated to the N-terminus of the therapeutic peptide agent (P2) and the N-terminus of the variant Fc domain monomer (B), and linker (L1) may be conjugated to the N-terminus of the therapeutic peptide agent (P1) and the C-terminus of the variant Fc domain monomer (B). Alternatively, linker (L2) may be conjugated to the C-terminus of the therapeutic peptide agent (P2) and the N-terminus of the variant Fc domain monomer (B), and linker (L1) may be conjugated to the C-terminus of the therapeutic peptide agent (P1) and the C-terminus of the variant Fc domain monomer (B).

[0305] The present disclosure also provides a conjugate comprising a first fusion protein selected from any of the therapeutic peptide agent-variant Fc domain monomer fusion proteins described herein; and a second fusion protein selected from any of the therapeutic peptide agent-variant Fc domain monomer fusion proteins described herein, wherein the variant Fc domain monomer (B) of the first fusion protein and the variant Fc domain monomer (B) of the second fusion protein dimerize to form a variant Fc domain monomer. In some embodiments, the first fusion protein and the second fusion protein have the same structure, and the conjugate is a homodimer.

[0306] IV. Linker A linker refers to a connection or connection between two or more components in a conjugate described herein (e.g., between two therapeutic agents in a conjugate described herein, between a therapeutic agent and a variant Fc domain monomer or variant Fc domain in a conjugate described herein, and between a dimer of two therapeutic agents and a variant Fc domain monomer or variant Fc domain in a conjugate described herein).

[0307] The linker can be a simple covalent bond, e.g., a peptide bond, a synthetic polymer, e.g., a polyethylene glycol (PEG) polymer, or any type of bond formed by a chemical reaction, e.g., chemical conjugation. When the linker is a peptide bond, a carboxylic acid group at the C-terminus of one protein domain can react with an amino group at the N-terminus of another protein domain in a condensation reaction to form a peptide bond. Specifically, the peptide bond can be formed by synthetic means via conventional organic chemical reactions well known in the art, or by natural production from a host cell in which a polynucleotide sequence encoding both proteins, e.g., DNA sequences of two variant Fc domain monomers in tandem, can be directly transcribed and translated into a continuous polypeptide encoding both proteins by the necessary molecular machinery in the host cell, e.g., DNA polymerase and ribosomes.

[0308] When the linker is a synthetic polymer, for example a PEG polymer, the polymer can be functionalized with reactive chemical groups at each end for reacting with the terminal amino acids at the connecting ends of the two proteins.

[0309] When a linker (excluding the above-mentioned peptide bond) is produced by a chemical reaction, a chemical functional group, such as an amine, a carboxylic acid, an ester, an azide, or other functional group commonly used in the art, can be synthetically bound to the C-terminus of one protein and the N-terminus of another protein, respectively. The two functional groups can then be reacted via synthetic chemical means to form a chemical bond, thereby connecting the two proteins to each other. Such chemical conjugation procedures are common to those skilled in the art.

[0310] Peptide Linker In the present invention, the linker (e.g., L1 or L2) between the therapeutic peptide agent and the variant Fc domain monomer is preferably 3 to 200 amino acids (e.g., 3 to 200, 3 to 180, 3 to 160, 3 to 140, 3 to 120, 3 to 100, 3 to 90, 3 to 80, 3 to 70, 3 to 60, 3 to 50, 3 to 45, 3 to 40, 3 to 35, 3 to 30, 3 to 25, 3 to 20, 3 to 15, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 3 to 6, 3 to 7, 3 to 8, 3 to 9, 3 to 10, 3 to 12, 3 to 14, 3 to 16, 3 to 18, 3 to 19, 3 to 20, 3 to 25, 3 to 20, 3 to 15, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 3 to 10, 3 to 12, 3 to 14, 3 to 16, 3 to 18, 3 to 19, 3 to 20, 3 to 25, 3 to 20, 3 to 15, 3 to 10, 3 to 12, 3 to 14, 3 to 16, 3 to 20, 3 to 25, 3 to 20, 3 to 25, 3 to 30, 3 to 35, 3 to 40, 3 to 40, 3 to 40, between 4, 4 to 200, 5 to 200, 6 to 200, 7 to 200, 8 to 200, 9 to 200, 10 to 200, 15 to 200, 20 to 200, 25 to 200, 30 to 200, 35 to 200, 40 to 200, 45 to 200, 50 to 200, 60 to 200, 70 to 200, 80 to 200, 90 to 200, 100 to 200, 120 to 200, 140 to 200, 160 to 200, or 180 to 200 amino acids). In some embodiments, the linker (e.g., L1 or L2) between the therapeutic peptide agent and the variant Fc domain monomer is at least 12 amino acids, e.g., 12 to 200 amino acids (e.g., 12 to 200, 12 to 180, 12 to 160, 12 to 140, 12 to 120, 12 to 100, 12 to 90, 12 to 80, 12 to 70, 12 to 60, 12 to 50, 12 to 40, 12 to 30, 12 to 20, 12 to 19, 12 to 18 , 12-17, 12-16, 12-15, 12-14, or 12-13 amino acids) (e.g., 14-200, 16-200, 18-200, 20-200, 30-200, 40-200, 50-200, 60-200, 70-200, 80-200, 90-200, 100-200, 120-200, 140-200, 160-200, 180-200, or 190-200 amino acids). In some embodiments, the linker (e.g., L1 or L2) between the therapeutic peptide agent and the variant Fc domain monomer is a polypeptide containing 12 to 30 amino acids (e.g., 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids).

[0311] Suitable peptide linkers are known in the art and include, for example, peptide linkers containing flexible amino acid residues such as glycine and serine. In some embodiments, the linker can contain a GS, GGS (SEQ ID NO: 61), GGGGS (SEQ ID NO: 62), GGSG (SEQ ID NO: 63), or SGGG (SEQ ID NO: 64) motif, e.g., multiple or repeated motifs. In some embodiments, the linker can contain 2 to 12 amino acids containing a GS motif, e.g., GS, GSGS (SEQ ID NO: 65), GSGSGS (SEQ ID NO: 66), GSGSGSGS (SEQ ID NO: 67), GSGSGSGSGS (SEQ ID NO: 68), or GSGSGSGSGSGS (SEQ ID NO: 69). In some other embodiments, the linker can contain 3 to 12 amino acids containing a GGS motif, e.g., GGS, GGSGGS (SEQ ID NO: 70), GGSGGSGGS (SEQ ID NO: 71), and GGSGGSGGSGGS (SEQ ID NO: 72). In still other embodiments, the linker can contain 4 to 12 amino acids including a GGSG (SEQ ID NO: 73) motif, e.g., GGSGGGSG (SEQ ID NO: 74), or GGSGGGSGGGSG (SEQ ID NO: 75). In other embodiments, the linker can contain a GGGGS (SEQ ID NO: 61) motif, e.g., GGGGSGGGGGSGGGGGS (SEQ ID NO: 76). In some embodiments, the linker is SGGGSGGGSGGGSGGGSGGG (SEQ ID NO: 77).

[0312] In a preferred embodiment, the peptide linkers (e.g., L1 and L2) are peptide linkers containing any one of the amino acid sequences of (GS)x, (GGS)x, (GGGGS)x, (GGSG)x, and (SGGG)x (where x is an integer of 1 to 50 (e.g., 1 to 40, 1 to 30, 1 to 20, 1 to 10, or 1 to 5)).

[0313] In some embodiments, the peptide linker contains only glycine residues, e.g., at least four glycine residues (e.g., 4 to 200, 4 to 180, 4 to 160, 4 to 140, 4 to 40, 4 to 100, 4 to 90, 4 to 80, 4 to 70, 4 to 60, 4 to 50, 4 to 40, 4 to 30, 4 to 20, 4 to 19, 4 to 18, 4 to 17, 4 to 16, 4 to 15, 4 to 14, 4 to 13, 4 to 12, 4 to 11, 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6 or 4 to 5 glycine residues) (e.g., 4 to 200, 6 to 200, 8 to 200, 10 to 200, 12 to 200, 14 to 200, 16 to 200, 18 to 200, 20 to 200, 30 to 200, 40 to 200, 50 to 200, 60 to 200, 70 to 200, 80 to 200, 90 to 200, 100 to 200, 120 to 200, 140 to 200, 160 to 200, 180 to 200, or 190 to 200 glycine residues). In some embodiments, a linker has between 4 and 30 glycine residues (e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 glycine residues). In some embodiments, a linker containing only glycine residues may be unglycosylated (e.g., O-linked glycosylation, also referred to as O-glycosylation) or may have a reduced level of glycosylation (e.g., reduced levels of O-glycosylation) (e.g., reduced levels of O-glycosylation with glycans such as xylose, mannose, sialic acid, fucose (Fuc), and / or galactose (Gal) (e.g., xylose)), for example, compared to a linker containing one or more serine residues.

[0314] In some embodiments, a linker containing only glycine residues may not be O-glycosylated (e.g., O-xylosylated) or may have a reduced level of O-glycosylation (e.g., a reduced level of O-xylosylation), for example, compared to a linker containing one or more serine residues.

[0315] In some embodiments, a linker containing only glycine residues may not undergo proteolysis or may have a reduced rate of proteolysis compared to, for example, a linker containing one or more serine residues.

[0316] In some embodiments, the linker may contain a GGGG (SEQ ID NO:78) motif, e.g., GGGGGGGG (SEQ ID NO:79), GGGGGGGGGGGG (SEQ ID NO:80), GGGGGGGGGGGGGGGG (SEQ ID NO:81), or GGGGGGGGGGGGGGGGGGGGGG (SEQ ID NO:82). In some embodiments, the linker may contain a GGGGG (SEQ ID NO:83) motif, e.g., GGGGGGGGGG (SEQ ID NO:84), GGGGGGGGGGGGGGGG (SEQ ID NO:85), or GGGGGGGGGGGGGGGGGGGG (SEQ ID NO:82). In some embodiments, the linker is GGGGGGGGGGGGGGGGGGGG (SEQ ID NO:82).

[0317] In other embodiments, the linker may also contain amino acids other than glycine and serine, for example, GENLYFQSGG (SEQ ID NO: 86), SACYCELS (SEQ ID NO: 87), RSIAT (SEQ ID NO: 88), RPACKIPNDLKQKVMNH (SEQ ID NO: 89), GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG (SEQ ID NO: 90), AAANSSIDLISVPVDSR (SEQ ID NO: 91), or GGSGGGSEGGGSEGGGSEGGGSEGGGSEGGGSGGGS (SEQ ID NO: 92).

[0318] Chemical Linkers In some embodiments, a linker provides space, rigidity, and / or flexibility between a therapeutic agent and a variant Fc domain monomer or variant Fc domain in the conjugates and fusion proteins described herein, or between two therapeutic agents in the conjugates described herein. In some embodiments, a linker can be a bond, e.g., a covalent bond, e.g., an amide bond, a disulfide bond, a C-O bond, a C-N bond, an N-N bond, a C-S bond, or any type of bond generated from a chemical reaction, e.g., chemical conjugation. In some embodiments, the linker (L shown in Formula (1)) is 250 or fewer atoms (e.g., 1 to 2, 1 to 4, 1 to 6, 1 to 8, 1 to 10, 1 to 12, 1 to 14, 1 to 16, 1 to 18, 1 to 20, 1 to 25, 1 to 30, 1 to 35, 1 to 40, 1 to 45, 1 to 50, 1 to 55, 1 to 60, 1 to 65, 1 to 70, 1 to 75, 1 to 80, 1 to 85, 1 to 90, 1 to 95, 1 to 100, 1 to 110, 1 to 120, 1 to 130, 1 to 140, 1 to 150, 1 to 160, 1 to 170, 1 to 180, 1 to 190, 1-200, 1-210, 1-220, 1-230, 1-240, or 1-250 atom(s); 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 28, 26, 24, 22, 20, 18, 16, 14, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 atom(s).In some embodiments, the linker (L) has 250 or fewer non-hydrogen atoms (e.g., 1 to 2, 1 to 4, 1 to 6, 1 to 8, 1 to 10, 1 to 12, 1 to 14, 1 to 16, 1 to 18, 1 to 20, 1 to 25, 1 to 30, 1 to 35, 1 to 40, 1 to 45, 1 to 50, 1 to 55, 1 to 60, 1 to 65, 1 to 70, 1 to 75, 1 to 80, 1 to 85, 1 to 90, 1 to 95, 1 to 100, 1 to 110, 1 to 120, 1 to 130, 1 to 140, 1 to 150, 1 to 160, 1 to 170, 1 to 180, 1 to 190, 1 to 200, 1-210, 1-220, 1-230, 1-240, or 1-250 non-hydrogen atom(s); 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 28, 26, 24, 22, 20, 18, 16, 14, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 non-hydrogen atom(s). In some embodiments, the backbone of the linker (L) is 250 atoms or less (e.g., 1 to 2, 1 to 4, 1 to 6, 1 to 8, 1 to 10, 1 to 12, 1 to 14, 1 to 16, 1 to 18, 1 to 20, 1 to 25, 1 to 30, 1 to 35, 1 to 40, 1 to 45, 1 to 50, 1 to 55, 1 to 60, 1 to 65, 1 to 70, 1 to 75, 1 to 80, 1 to 85, 1 to 90, 1 to 95, 1 to 100, 1 to 110, 1 to 120, 1 to 130, 1 to 140, 1 to 150, 1 to 160, 1 to 170, 1 to 180, 1 to 190 , 1 to 200, 1 to 210, 1 to 220, 1 to 230, 1 to 240, or 1 to 250 atom(s); 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 28, 26, 24, 22, 20, 18, 16, 14, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 atom(s). The "backbone" of a linker refers to the atoms in the linker that together form the shortest path from one portion of the conjugate to another portion of the conjugate. The atoms in the backbone of the linker are directly involved in connecting one part of the conjugate to another part of the conjugate.For example, hydrogen atoms attached to carbons in the backbone of a linker are not considered to be directly involved in connecting one portion of the conjugate to another portion of the conjugate.

[0319] Molecules that can be used to create linkers (L) contain at least two functional groups, e.g., two carboxylic acid groups. In some embodiments of trivalent linkers, the two arms of the linker can contain two dicarboxylic acids, where the first carboxylic acid can form a covalent bond with a first therapeutic agent in the conjugate, the second carboxylic acid can form a covalent bond with a second therapeutic agent in the conjugate, and the third arm of the linker can form a covalent bond (e.g., a CO bond) with a variant Fc domain monomer of a variant Fc domain in a conjugate or fusion protein described herein. In some embodiments of bivalent linkers, the bivalent linker can contain two carboxylic acids, where the first carboxylic acid can form a covalent bond with one component (e.g., a therapeutic agent) in the conjugate, and the second carboxylic acid can form a covalent bond (e.g., a C-S bond or a C-N bond) with another component (e.g., a variant Fc domain monomer or a variant Fc domain) in the conjugate.

[0320] In some embodiments, a dicarboxylic acid molecule may be used as a linker (e.g., a dicarboxylic acid linker). For example, in a conjugate containing a variant Fc domain monomer or variant Fc domain covalently linked to one or more dimers of a therapeutic agent, a first carboxylic acid in the dicarboxylic acid molecule may form a covalent bond with a hydroxyl or amine group of the first therapeutic agent, and a second carboxylic acid may form a covalent bond with a hydroxyl or amine group of the second therapeutic agent. In some instances where a reactive group (e.g., a carboxylic acid, hydroxyl, or amine) is not available on the therapeutic agent, a reactive group (e.g., a carboxylic acid, hydroxyl, or amine) may be introduced into the therapeutic agent in a manner that does not destroy the activity of the therapeutic agent.

[0321] In some embodiments, dicarboxylic acid molecules such as those described herein may be further functionalized to contain one or more additional functional groups, e.g., to provide a point of attachment (e.g., via a linker such as a PEG linker) to a variant Fc domain monomer, variant Fc domain, or fusion protein described herein.

[0322] In some embodiments, when a therapeutic agent is attached to a variant Fc domain monomer or variant Fc domain, the linking group may comprise a moiety comprising a carboxylic acid moiety and an amino moiety separated by 1 to 25 atoms.

[0323] In some embodiments, linking groups may include moieties containing carboxylic acid and amino moieties, such as those described herein, and may be further functionalized to contain one or more additional functional groups. Such linking groups may be further functionalized to provide a point of attachment (e.g., via a linker, such as a PEG linker) to, for example, a variant Fc domain monomer, variant Fc domain, or fusion protein described herein.

[0324] In some embodiments, when a therapeutic agent is attached to a variant Fc domain monomer or variant Fc domain, the linking group may comprise a moiety that includes two or more amino moieties (e.g., a diamino moiety) separated by 1 to 25 atoms.

[0325] In some embodiments, a linking group may comprise a diamino moiety, such as those described herein, and may be further functionalized to contain one or more additional functional groups. Such diamino linking groups may be further functionalized to provide a point of attachment (e.g., via a linker, such as a PEG linker) to, for example, a variant Fc domain monomer, variant Fc domain, or fusion protein described herein.

[0326] In some embodiments, molecules containing an azide group can be used to form linkers in which the azide group can undergo cycloaddition with an alkyne to form a 1,2,3-triazole bond. In some embodiments, molecules containing an alkyne group can be used to form linkers in which the alkyne group can undergo cycloaddition with an azide to form a 1,2,3-triazole bond. In some embodiments, molecules containing a maleimide group can be used to form linkers in which the maleimide group can react with a cysteine ​​to form a C-S bond. In some embodiments, molecules containing one or more sulfonic acid groups can be used to form linkers in which the sulfonic acid group can form a sulfonamide bond with a linking nitrogen in a therapeutic agent. In some embodiments, molecules containing one or more isocyanate groups can be used to form linkers in which the isocyanate group can form a urea bond with a linking nitrogen in a therapeutic agent. In some embodiments, molecules containing one or more haloalkyl groups can be used to form linkers in which the haloalkyl groups can form covalent bonds, e.g., C-N and C-O bonds, with a therapeutic agent.

[0327] In some embodiments, the linker (L) can include a synthetic group derived, for example, from a synthetic polymer (e.g., a polyethylene glycol (PEG) polymer). In some embodiments, the linker can include one or more amino acid residues. In some embodiments, the linker can be an amino acid sequence (e.g., 1 to 25 amino acids, 1 to 10 amino acids, 1 to 9 amino acids, 1 to 8 amino acids, 1 to 7 amino acids, 1 to 6 amino acids, 1 to 5 amino acids, 1 to 4 amino acids, 1 to 3 amino acids, 1 to 2 amino acids, or 1 amino acid sequence). In some embodiments, the linker (L) is one or more optionally substituted C1-C20 alkylene, optionally substituted C1-C20 heteroalkylene (e.g., PEG units), optionally substituted C2-C20 alkenylene (e.g., C2 alkenylene), optionally substituted C2-C20 heteroalkenylene, optionally substituted C2-C20 alkynylene, optionally substituted C2-C20 heteroalkynylene, optionally substituted C3-C20 cycloalkylene (e.g., cyclopropyl). cyclobutylene), optionally substituted C3-C20 heterocycloalkylene, optionally substituted C4-C20 cycloalkenylene, optionally substituted C4-C20 heterocycloalkenylene, optionally substituted C8-C20 cycloalkynylene, optionally substituted C8-C20 heterocycloalkynylene, optionally substituted C5-C15 arylene (e.g., C6 arylene), optionally substituted C2-C15 heteroarylene (e.g., imidazole, pyridine), O, S, NR i (R imay include H, optionally substituted C1-C20 alkyl, optionally substituted C1-C20 heteroalkyl, optionally substituted C2-C20 alkenyl, optionally substituted C2-C20 heteroalkenyl, optionally substituted C2-C20 alkynyl, optionally substituted C2-C20 heteroalkynyl, optionally substituted C3-C20 cycloalkyl, optionally substituted C3-C20 heterocycloalkyl, optionally substituted C4-C20 cycloalkenyl, optionally substituted C4-C20 heterocycloalkenyl, optionally substituted C8-C20 cycloalkynyl, optionally substituted C8-C20 heterocycloalkynyl, optionally substituted C5-C15 aryl, or optionally substituted C2-C15 heteroaryl), P, carbonyl, thiocarbonyl, sulfonyl, phosphate, phosphoryl, or imino.

[0328] Conjugation Chemistry Covalent conjugation of two or more components in a conjugate using a linker can be achieved using well-known organic chemistry synthesis techniques and methods. Complementary functional groups on two or more components can react with each other to form a covalent bond. Examples of complementary reactive functional groups include, but are not limited to, maleimide and cysteine, amine and activated carboxylic acid, thiol and maleimide, activated sulfonic acid and amine, isocyanate and amine, azide and alkyne, and alkene and tetrazine. Site-specific conjugation to a polypeptide (e.g., a variant Fc domain monomer, a variant Fc domain, or a fusion protein) can be achieved using techniques known in the art. Exemplary techniques for site-specific conjugation of small molecules to an Fc domain monomer of an Fc domain (e.g., a variant Fc domain monomer or variant Fc domain described herein) are provided in Agarwall, P., et al., Bioconjugate Chem. 26:176-192 (2015).

[0329] Other examples of functional groups capable of reacting with amino groups include, for example, alkylating and acylating agents. Representative alkylating agents include: (i) α-haloacetyl groups, such as XCHCO— (X=Br, Cl, or I); (ii) N-maleimide groups, which can react with amino groups via either a Michael-type reaction or acylation by addition to the ring carbonyl group; (iii) aryl halides, such as nitrohaloaromatic groups; (iv) alkyl halides; (v) aldehydes or ketones capable of forming Schiff bases with amino groups; (vi) epoxides, such as epichlorohydrin and bisoxiranes, which can react with amino, sulfhydryl, or phenolic hydroxyl groups; (vii) chlorine-containing s-triazines, which are reactive with nucleophiles, such as amino, sulfhydryl, and hydroxyl groups; (viii) aziridines, which are reactive with nucleophiles, such as amino groups, by ring opening; (ix) squaric acid diethyl ester; and (x) α-haloalkyl ethers.

[0330] Examples of amino-reactive acylating groups include, for example, (i) isocyanates and isothiocyanates; (ii) sulfonyl chlorides; (iii) acid halides; and (iv) activated esters, such as nitrophenyl esters or N-hydroxysuccinimidyl esters, or derivatives thereof (e.g., azido-PEG-PEG). 40 -NHS esters); (v) acid anhydrides, e.g., mixed, symmetric, or N-carboxyanhydrides; (vi) acyl azides; and (vii) imido esters. Aldehydes and ketones may be reacted with amines to form Schiff bases, which may be stabilized by reductive amination.

[0331] It is understood that certain functional group can be converted to other functional group before reaction, for example, to give additional reactivity or selectivity.The example of the method useful for this purpose includes: using reagent such as dicarboxylic anhydride to convert amine to carboxyl; using reagent such as N-acetylhomocysteine ​​thiolactone, S-acetylmercapto succinic anhydride, 2-iminothiolane or thiol-containing succinimidyl derivative to convert amine to thiol; using reagent such as α-haloacetate to convert thiol to carboxyl; using reagent such as ethyleneimine or 2-bromoethylamine to convert thiol to amine; using reagent such as carbodiimide, followed by diamine to convert carboxyl to amine; and using reagent such as tosyl chloride to convert alcohol to thiol, followed by transesterification with thioacetic acid and hydrolysis to thiol with sodium acetate.

[0332] In some embodiments, a linker of the invention (e.g., L) is conjugated to a variant Fc domain monomer (e.g., E) (e.g., by any of the methods described herein). In preferred embodiments of the invention, the linker is (a) a thiourea bond to a lysine of E (i.e., -NH(C=S)NH-); (b) a carbamate bond to a lysine of E (i.e., -NH(C=O)-O); (c) an amine bond via reductive amination between a lysine and E (i.e., -NHCH); (d) an amide to a lysine of E (i.e., -NH-(C=O)CH); (e) a cysteine-maleimide conjugate between a maleimide of the linker and a cysteine ​​of E; (f) a carbohydrate bond between the linker and E (e.g., a variant Fc domain monomer or variant Fc domain). (g) a re-bridged cysteine ​​conjugate in which the linker is conjugated to two cysteines of E; (h) an oxime bond between the linker and a carbohydrate of E (e.g., a glycosyl group of a variant Fc domain monomer or a variant Fc domain); (i) an oxime bond between the linker and an amino acid residue of E; (j) an azide bond between the linker and E; (k) direct acylation of the linker to E; or (l) conjugation by a thioether bond between the linker and E.

[0333] In some embodiments, the linker is conjugated to E, where the bond comprises the structure -NH(C=NH)X-, where X is O, HN, or a bond. In some embodiments, the linker is conjugated to E, where the bond between the remainder of the linker and E comprises the structure -NH(C=O)NH-.

[0334] In some embodiments, a linker (e.g., an active ester, such as a nitrophenyl ester or an N-hydroxysuccinimidyl ester, or a derivative thereof, such as a functionalized PEG linker (e.g., azido-PEG-PEG) 40-NHS ester) is between 0.5 and 10.0, e.g., 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 5.10, 5.11, 5.12, 5.13, 5.14, 5.15, 5.16, 5.17, 5.18, 5.19, 5.20, 5.21, 5.22, 5.23, 5.24, 5.25, 5.26, 5.27, 5.28, 5.29, 5.30, 5.31, 5.32, 5.33, 5.34, 5.35, 5.36, 5.37, 5.38, 5.39, 5.39, 5.39, 5.39, 5.39, 5.39, 5.39, 5.3 , 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0 (e.g., DAR). 40 The azide (e.g., Fc-(PEG-PEG)-azide) can be reacted with a modified therapeutic agent bearing a terminal alkyne linker (e.g., L) via click conjugation. During click conjugation, the azide (e.g., Fc-(PEG-PEG)-azide) can be reacted with a modified therapeutic agent bearing a terminal alkyne linker (e.g., L) via click conjugation. 40 Copper-catalyzed reaction of E with an alkyne (e.g., a modified therapeutic agent bearing a terminal alkyne linker (e.g., L)) forms a five-membered heteroatom ring. In some embodiments, the linker conjugated to E is a terminal alkyne and is conjugated to a modified therapeutic agent bearing a terminal azide. Those skilled in the art will readily recognize the final product from click chemistry conjugation.

[0335] V. Method The methods described herein include, for example, methods of protecting against or treating a condition or disorder described herein (e.g., a respiratory disorder, liver disorder, central nervous system disorder, muscle disorder, skin disorder, eye disorder, vascular disorder, or infectious disease (e.g., a viral infection, fungal infection, or bacterial infection) in a subject, and methods of preventing, stabilizing, or inhibiting the growth of an infectious disease pathogen (e.g., a viral particle, fungus, or bacteria). A method of treating a condition or disorder described herein (e.g., a respiratory disorder, liver disorder, central nervous system disorder, muscle disorder, skin disorder, eye disorder, vascular disorder, or infectious disease (e.g., a viral infection, fungal infection, or bacterial infection) in a subject comprises administering to the subject a conjugate described herein (e.g., a conjugate of Formula (1)), a fusion protein described herein, or a pharmaceutical composition thereof.

[0336] Viral infections The compounds and pharmaceutical compositions described herein (e.g., a conjugate of Formula (1) or a fusion protein described herein) can be used to treat viral infections (e.g., viral meningitis, herpes simplex virus (HSV) 1, HSV2, Epstein-Barr virus, varicella-zoster virus, poliovirus, coxsackievirus, West Nile virus, La Crosse virus, western equine encephalitis, eastern equine encephalitis, Powassan virus, rabies virus, respiratory syncytial virus (RSV), dengue, betacoronavirus (e.g., COVID-19), Zika virus, or influenza virus infections, e.g., influenza A, B, C, or parainfluenza).

[0337] Viral infection refers to the pathogenic growth of virus in a host organism (e.g., human subject).Viral infection can be any situation in which the presence of viral population(s) causes damage to the host's body.Therefore, a subject is "suffering" from viral infection when excessive viral population(s) are present in or on the subject's body, or when viral population(s) cause damage to the subject's cells or other tissues.

[0338] Influenza, commonly known as the flu, is an infectious disease caused by the influenza virus. Symptoms can range from mild to severe. The most common symptoms include high fever, runny nose, sore throat, muscle aches, headache, cough, and fatigue. These symptoms typically begin two days after exposure to the virus and most last less than a week. However, cough can persist for more than two weeks. Children may experience nausea and vomiting, but these are less common in adults. Complications of influenza can include viral pneumonia, secondary bacterial pneumonia, sinus infections, and worsening of preexisting health problems such as asthma or heart failure. Severe complications can occur in subjects with weakened immune systems, such as the young, the elderly, those with underlying illnesses that weaken the immune system, and those undergoing therapeutic treatments that result in a weakened immune system.

[0339] Three types of influenza viruses, namely, types A, B, and C, affect human subjects. The virus typically spreads through the air from coughing or sneezing. This is thought to occur primarily over relatively short distances. It can also spread by touching a surface contaminated with the virus and then touching the mouth or eyes. People can infect others before and while showing symptoms. Infection can be confirmed by testing the throat, sputum, or nose for the virus. Many rapid tests are available; however, people may still have an infection if the result is negative. A type of polymerase chain reaction that detects viral RNA can be used to diagnose influenza infection.

[0340] Viral infection can refer to the pathogenic growth of a virus (e.g., RSV, such as RSV A or RSV B) in a host organism (e.g., a human subject). Human respiratory syncytial virus (RSV) is a medium-sized (120-200 nm) enveloped virus containing a lipoprotein coat and a linear, negative-sense RNA genome (which must be converted to positive-sense RNA before translation). The former contains virally encoded F, G, and SH lipoproteins. The F and G lipoproteins are unique in that they target the cell membrane and are highly conserved among RSV isolates. Human RSV (HRSV) is divided into two antigenic subgroups, A and B, based on viral reactivity with monoclonal antibodies against the attachment (G) and fusion (F) glycoproteins. Subtype B is characterized as an asymptomatic strain of the virus experienced by the majority of the population. More severe clinical disease is associated with subtype A strains, which tend to predominate in most outbreaks.

[0341] Four of the viral genes encode intracellular proteins involved in genome transcription, replication, and particle budding: N (nucleoprotein), P (phosphoprotein), M (matrix protein), and L (a "large" protein containing an RNA polymerase catalytic motif). RSV genomic RNA forms a helical ribonucleoprotein (RNP) complex with the N protein, called the nucleocapsid, which serves as a template for RNA synthesis by the viral polymerase complex. The three-dimensional structure of a decameric circular ribonucleoprotein complex of the RSV nucleoprotein (N) bound to RNA has been determined at 3.3 Å resolution. This complex mimics one turn of the viral helical nucleocapsid complex. The crystal structure, combined with electron microscopy data, provides a detailed model for the RSV nucleocapsid.

[0342] Viral infections can refer to aseptic meningitis (AM), defined as inflammation of the subarachnoid space characterized by mononuclear cell pleocytosis and sterile CSF (cerebrospinal fluid) cultures. The primary cause of AM is viral infection (Ravel R: Clinical Laboratory Medicine: Clinical Application of Laboratory Data: Elsevier Health Sciences; 1994). Viral meningitis is common and often goes unreported. Nonpoliovirus enteroviruses (coxsackieviruses and echoviruses) account for 80-90% of cases of viral meningitis with a defined etiology (Atkinson P, Sharland M, Maguire H: Predominant enteroviral serotypes causing meningitis. Archives of Disease in Childhood 1998, 78:373-374).

[0343] Viral infections can refer to herpes simplex virus 1 (HSV1) or HSV2. HSV1 is the common cause of cold sores on the lips (herpes labialis) and sores on the cornea of ​​the eye (herpes simplex keratitis). HSV2 is the common cause of genital herpes. The distinction between the two is not absolute. Genital infections are sometimes caused by HSV1. Infections can also occur in other parts of the body, such as the brain (severe disease) or digestive tract. Disseminated infections can occur in newborns or people with weakened immune systems, especially those with HIV infection. HSV is highly contagious and can be spread by direct contact with sores and, sometimes even when no sores are visible, by contact with the mouth or genitals of someone with HSV infection.

[0344] Viral infections may refer to coxsackieviruses. Coxsackieviruses are non-enveloped, linear, positive-sense, single-stranded RNA viruses belonging to the Picornaviridae family and several related enteroviruses in the Enterovirus genus (which also includes polioviruses and echoviruses). Coxsackieviruses are one of the main causes of aseptic meningitis, but can also cause hand, foot, and mouth disease, as well as muscle, lung, and heart disease.

[0345] The present invention also provides a method of preventing, stabilizing, or inhibiting the growth of viral particles or preventing viral replication and spread, comprising contacting a virus or a site susceptible to viral growth with a conjugate described herein (e.g., a conjugate of any one of Formula (1)), a fusion protein described herein, or a pharmaceutical composition thereof. In some embodiments, the virus is a resistant strain of virus.

[0346] Additionally, the methods described herein also include methods of protecting against or treating a viral infection in a subject by administering to the subject a composition described herein (e.g., a conjugate of Formula (1)) or a fusion protein described herein in combination with a second therapeutic agent, e.g., an antiviral agent or an antiviral vaccine.

[0347] bacterial infection The compounds and pharmaceutical compositions described herein (e.g., a conjugate of Formula (1) or a fusion protein described herein) can be used to treat bacterial infections.

[0348] Bacterial infections can occur in a host organism (e.g., a human subject) due to the presence of bacteria (e.g., Acinetobacter spp. (Acinetobacter baumannii), Bacteroides distasonis, Bacteroides fragilis, Bacteroides ovatus, Bacteroides thetaiotaomicron, Bacteroides uniformis, Bacteroides vulgatus, Citrobacter freundii, Citrobacter koser, Clostridium clostridioforme, Clostridium perfringens, Enterobacter aerogenes, Enterobacter cloacae, Enterococcus faecalis, Enterococcus spp. (vancomycin-susceptible and -resistant isolates), Escherichia coli (including ESBL- and KPC-producing isolates), Eubacterium lentum, Fusobacterium spp., Haemophilus influenzae (including beta-lactamase-positive isolates), Haemophilus influenzae parainfluenzae, Klebsiella pneumoniae (including ESBL- and KPC-producing isolates), Klebsiella oxytoca (including ESBL- and KPC-producing isolates), Legionella pneumophilia, Moraxella catarrhalis, Morganella morganii, Mycoplasma spp., Peptostreptococcus spp.Bacterial infection refers to the pathogenic growth of bacteria such as Porphyromonas asaccharolytica, Prevotella bivia, Proteus mirabilis, Proteus vulgaris, Providencia rettgeri, Providencia stuartii, Pseudomonas aeruginosa, Serratia marcescens, Streptococcus anginosus, Staphylococcus aureus (methicillin-susceptible and -resistant isolates), Staphylococcus epidermidis (methicillin-susceptible and -resistant isolates), Stenotrophomonas maltophilia, Streptococcus agalactiae, Streptococcus constellatus, Streptococcus pneumoniae (penicillin-susceptible and -resistant isolates), and Streptococcus pyogenes. A bacterial infection can be any situation in which the presence of a bacterial population(s) is damaging to the host's body. Thus, a subject is "suffering from" a bacterial infection when an excessive amount of bacterial population is present in or on the subject's body, or when the presence of the bacterial population(s) is damaging to the subject's cells or other tissues.

[0349] Staphylococcus aureus is a major human pathogen, with an estimated 30% of humans being asymptomatic nasal carriers (Chambers and DeLeo 2009. Nat. Rev. Microbiol. 7:629-641). S. aureus causes skin, soft tissue, respiratory, bone, joint, and intravascular diseases. Life-threatening cases caused by S. aureus include bacteremia, endocarditis, sepsis, and toxic shock syndrome (Lowy 1998. N. Engl. J. Med. 339:520-532). Antibiotic resistance in S. aureus is becoming an increasingly urgent medical problem. Methicillin resistance in S. aureus is approaching epidemic levels (Chambers and DeLeo, supra; Grundmann et al., 2006. Lancet 368:874-885). It was estimated that 94,360 invasive MRSA infections occurred in the United States in 2005, and these infections were associated with death in 18,650 cases (Klevens et al., 2007. JAMA 298:1763-1771). S. epidermidis is part of the normal human epithelial flora but can cause infection when the skin or mucous membranes are broken.

[0350] Exemplary therapeutic agents that are effective against proliferating bacteria and thus may be conjugated to the Fc variants of the invention include β-lactams, such as penicillins (e.g., penicillin G, penicillin V, methicillin, oxacillin, cloxacillin, dicloxacillin, nafcillin, ampicillin, amoxicillin, carbenicillin, ticarcillin, mezlocillin, piperacillin, azlocillin, and temocillin), cephalosporins (e.g., cephalothin, cephapirin, cephradine, cephaloridine, cefazolin, cefamandole, , cefuroxime, cephalexin, cefprozil, cefaclor, loracarbef, cefoxitin, cefmetazole, cefotaxime, ceftizoxime, ceftriaxone, cefoperazone, ceftazidime, cefixime, cefpodoxime, ceftibuten, cefdinir, cefpirome, cefepime, BAL5788, and BAL9141), carbapenems (e.g., imipenem, ertapenem, and meropenem), and monobactams (e.g., aztreonam); β-lactamase inhibitors (e.g., clavulanate, sulbactam, and tazolam) bactams); aminoglycosides (e.g., streptomycin, neomycin, kanamycin, paromomycin, puromycin, gentamicin, tobramycin, amikacin, netilmicin, spectinomycin, sisomicin, dibecalin, and isepamicin); tetracyclines (e.g., tetracycline, chlortetracycline, demeclocycline, minocycline, oxytetracycline, methacycline, and doxycycline); macrolides (e.g., erythromycin, azithromycin, and clarithromycin) thromycin); ketolides (e.g., telithromycin, ABT-773); lincosamides (e.g., lincomycin and clindamycin); glycopeptides (e.g., vancomycin, oritavancin, dalbavancin, and teicoplanin); streptogramins (e.g., quinupristin and dalfopristin); sulfonamides (e.g., sulfanilamide, para-aminobenzoic acid, sulfadiazine, sulfisoxazole, sulfamethoxazole, and sulfasalidine); oxazolidinones (e.g., linezolid);Quinolones (e.g., nalidixic acid, oxolinic acid, norfloxacin, pefloxacin, enoxacin, ofloxacin, ciprofloxacin, temafloxacin, lomefloxacin, fleroxacin, grepafloxacin, sparfloxacin, trovafloxacin, clindafloxacin, gatifloxacin, moxifloxacin, gemifloxacin, and sitafloxacin); metronidazole; daptomycin; garenoxacin; ramoplanin; faropenem; polymyxins; tigecycline, AZD2563; and trimethoprim;

[0351] The methods described herein include, for example, methods of protecting against or treating an infectious disease (e.g., a bacterial infection) in a subject and methods of preventing, stabilizing, or inhibiting the growth of an infectious disease pathogen (e.g., bacteria). A method of treating an infectious disease (e.g., a bacterial infection) in a subject comprises administering to the subject a conjugate described herein (e.g., a conjugate of Formula (1)), a fusion protein described herein, or a pharmaceutical composition thereof. In some embodiments, the bacterial infection is caused by a resistant strain of bacteria. A method of preventing, stabilizing, or inhibiting bacterial growth or preventing bacterial replication and spread comprises contacting the bacterium or a site susceptible to bacterial growth with a conjugate described herein (e.g., a conjugate of any one of Formula (1)) or a pharmaceutical composition thereof.

[0352] Additionally, the methods described herein also include methods of protecting against or treating a bacterial infection in a subject by administering to the subject a conjugate described herein (e.g., a conjugate of Formula (1)) or a fusion protein described herein in combination with a second therapeutic agent, e.g., an antibacterial agent.

[0353] fungal infection The compounds and pharmaceutical compositions described herein (e.g., a conjugate of Formula (1) or a fusion protein described herein) can be used to treat fungal infections.

[0354] Fungal infections are caused by fungi (e.g., Trichophyton species (e.g., T. ajelloi, T. concentricum, T. equinum, T. erinacei, T. flavescens, T. gloriae, T. interdigitale, T. megnini, T. mentagrophytes, T. phaseoliforme, T. rubrum, T. schoenleini, T. simii, T. soudanense, T. terrestre, T. tonsurans, T. vanbreuseghemii, T. verrucosum, T. violaceum, or T. yaoundei), Epidermophyton species (e.g., E. floccosum or E. stockdaleae), Candida species (e.g., C. albicans, C. parapsiliosis, C. krusei, C. tropicalis, C. glabrata, C. parapsilosis, C. lusitaniae, C. kefyr, C. guilliermondii, or C. dubliniensis), Microsporum species (e.g., M. canis, M. gypseum, M. audouini, M. gallinae, M. ferrugineum, M. distortum, M. nanum, M. cookie, or M. vanbreuseghemii), Epicoccum species (e.g., E. nigrum), Aspergillus species (e.g., A. sydowii, A. terreus, A. niger, A. terreus, A. fumigatus, A. flavus, A. clavatus, A. glaucus group, A. nidulans, A. oryzae, A. terreus, A. ustus, or A. versicolor), Paecilomyces species (e.g., P. lilacinus or P. variotii), Fusarium species (e.g., F. oxysporum, F. solani, or F. semitectum), Acremonium species (e.g., A. strictum, A. roseogiseum, A. cucurbitacearum, A. kiliense, A. curvatum, A.comptosporum, Ulocladium chartarum, A.alternatum, or Emercellopsis minima), Chaetomium species (e.g. C. atrobrunneum, C. funicola, C. globosum, or C. strumarium), Phoma species, Scopulariopsis species (e.g. S. brevicau lis, S.candida, S.koningii, S.acremonium, S.flava, S.cinerea, S.trigonospora, S.brumptii, S.chartarum, S.fusca, or S.asperula) Fungal infection refers to the pathogenic growth of Alternaria species (e.g., A. alternata, A. chartarum, A. dianthicola, A. geophilia, A. infectoria, A. stemphyloides, or A. teunissima), and Curvularia species (e.g., C. brachyspora, C. clavata, C. geniculata, C. lunata, C. pallescens, C. senegalensis, or C. verruculosa). A fungal infection can be any situation in which the presence of a fungal population(s) is damaging to the host's body. Thus, a subject is "suffering" from a fungal infection when an excessive amount of fungal population is present in or on the subject's body, or when the presence of a fungal population(s) is damaging to the subject's cells or other tissues.

[0355] Fungi cause a variety of diseases in humans. Some fungi cause infections limited to the outermost layers of skin and hair (superficial mycoses), while others cause dermatomycoses by invading the keratinized layer of skin, hair, and nails and inducing pathological changes in the host. Subcutaneous mycoses cause infections in the epidermis, subcutaneous tissue, muscle, and fascia and are often chronic. Systemic mycoses occur primarily in the lungs and can cause secondary infections in other organ systems in the body. Patients with immune system deficiencies are often prone to developing opportunistic mycoses.

[0356] Dermatophytes, including Trichophyton rubrum and Trichophyton mentagrophytes, are responsible for fungal infections of the skin, or dermatophytosis. Tinea pedis is a skin infection that most commonly occurs between the toes, causing scaling, peeling, and itching of the affected skin. Blisters and cracked skin may also occur, resulting in exposed raw tissue, erythema, pain, swelling, and inflammation. The second type of tinea pedis, called moccasin tinea pedis, is characterized by chronic plantar erythema with slight scaling and diffuse hyperkeratosis, which may be asymptomatic or pruritic (e.g., an unpleasant, irritating sensation). Other types include inflammatory / bullous and ulcerative tinea pedis. The infection may spread to other areas of the body and manifest as ring-shaped, scaly patches with raised edges, pustules, and blisters on the trunk, arms, and legs (tinea corporis), a scaly rash on the palms and webs of the hands (tinea manubrium), erythematous lesions in the groin and pubic area (tinea rugosa), erythema, scaling, and pustules on the beard and neck area (tinea barbae or tinea faciei), or round, balding, scaly patches on the scalp (tinea capitis). Tinea versicolor, also known as pityriasis versicolor, is a common fungal infection of the skin that interferes with normal skin pigmentation, resulting in small, discolored patches. Tinea unguium is another term for dermatophyte infection of the nails. Secondary bacterial infections can develop from fungal infections.

[0357] Ringworm is very common, especially in children, and can spread through skin-to-skin contact and contact with contaminated items, such as hairbrushes, or by using the same toilet seat as an infected individual.Ringworm spreads easily because infected individuals are contagious even before they show symptoms of the disease.Participants in contact sports, such as wrestling, are at risk of contracting fungal infections through skin-to-skin contact.

[0358] Ringworm is mildly contagious. Ringworm is also a common infection in livestock, especially farm animals, dogs and cats, and even small pets such as hamsters or guinea pigs. Humans can contract ringworm (commonly called "ringworm") from these animals because of their close contact with them. Ringworm can also be transmitted from other humans through both direct contact and prolonged contact with shed skin fragments (e.g., from shared clothing or house dust).

[0359] The most common sign of tinea in humans is the appearance of one or more red, raised, itchy spots with sharp edges, similar to the herald rash of pityriasis rosea. These spots are often lighter in the center and have a ring-like appearance with surrounding hyperpigmentation caused by increased melanin. If the infected area involves the scalp or beard, then bald patches may become noticeable. The affected area may become itchy for a period of time.

[0360] Sometimes tinea infections can cause skin lesions on parts of the body distant from the actual infection. Such lesions are called "dermatophyte rashes." The lesions themselves do not contain fungus and usually disappear with treatment of the actual infection. The most common example is a rash on the hands caused by a fungal infection of the feet. Dermatophyte rash is essentially a systemic allergic reaction to the fungus.

[0361] Thus, fungi and yeasts, such as Microsporum species, Trichophyton species, Epidermophyton species, and Candida species, can cause persistent and difficult to treat infections.

[0362] Microsporum species include M. canis and M. gypseum. Microsporum is one of several fungal genera that cause dermatophytosis. Dermatophytosis is a general term used to define infection of hair, skin, or nails by any dermatophyte species. Like other dermatophytes, Microsporum has the ability to degrade keratin, thus allowing it to reside on the skin and its appendages and remain noninvasive. In particular, Microsporum spp. primarily infect hair and skin. Microsporum canis is a zoophilic fungal species that is the primary cause of ringworm in dogs and cats and causes sporadic dermatophytosis in humans, particularly tinea capitis in children with cats and dogs.

[0363] Skin infections caused by Trichophyton species occur primarily on the back of the neck, scalp, or beard. Symptoms of Trichophyton species infection include inflammatory scalp lesions, inflammatory neck lesions, inflammatory beard lesions, scarring, and permanent hair loss. Examples of Trichophyton species include T. rubrum, T. tonsurans, and T. mentagrophytes.

[0364] Trichophyton tonsurans is a human parasitic intradermal species of fungus that causes epidemic dermatophytosis in Europe, South America, and the United States. It infects some animals and requires thiamine for growth. It is the most common cause of tinea capitis in the United States, which causes black dots where hair breaks off on the skin surface. Trichophyton rubrum is the fungus that most commonly causes tinea pedis ("athlete's foot"), tinea cruris, and ringworm. Trichophyton rubrum is the most common dermatophyte that causes fungal infections of the fingernails. Most fungal skin infections are irritating and difficult to treat, but there have been reports of fatal fungal infections. In particular, Trichophyton mentagrophytes skin infections can migrate to the lymph nodes, testes, vertebrae, and CNS. Treatment with griseofulvin, amphotericin B, clotrimazole, and transfer factor was unsuccessful and ultimately led to the subject's death (Hironaga et al., J. Clin. Microbiol., 2003; 5298-5301). Trichophyton mentagrophytes is the second most common cause of fungal nail infections from the dermatophyte group.

[0365] The genus Epidermophyton contains two species: Epidermophyton floccosum and Epidermophyton stockdaleae. E. stockdaleae is known to be nonpathogenic, with E. floccosum being the only species causing infection in humans. E. floccosum is a common cause of dermatophytosis in otherwise healthy individuals. It infects the skin (tinea corporis, tinea pedis, and tinea pedis) and nails (onychomycosis). The infection is limited to the nonviable keratinized layer of the epidermis because the fungus lacks the ability to penetrate viable tissues in immunocompetent hosts. Disseminated infections caused by either dermatophyte are extremely rare because they are limited to infection of keratinized tissues.

[0366] However, invasive E. floccosum infections have been reported in immunocompromised patients with Behçet's syndrome. Like all forms of dermatophytosis, Epidermophyton floccosum infections are contagious and are usually transmitted by contact, particularly in public shower and gym facilities.

[0367] Candida species include C. albicans, C. parapsiliosis, and C. krusei. Patients with chronic mucocutaneous candidiasis can develop nail candidal infections. Candida species can invade nails already damaged by infection or trauma, causing infection in the periungual area and beneath the nail bed. The nail folds become erythematous, swollen, tender, and occasionally discharged. The disease causes onycholysis with loss of the nail cuticle, nail dystrophy, and discoloration around the lateral nail fold. In all forms of onychomycosis, the nails are variably damaged and deformed.

[0368] The methods described herein also include methods of protecting against or treating a fungal infection in a subject by administering to the subject a composition described herein (e.g., a conjugate of Formula (1)) or a fusion protein described herein in combination with an antifungal agent.

[0369] VI. Pharmaceutical Compositions Compositions comprising a variant Fc domain (e.g., a conjugate or fusion protein described herein) may be formulated in a pharmaceutical composition for use in the methods described herein. In some embodiments, a conjugate or fusion protein described herein may be formulated alone in a pharmaceutical composition. In some embodiments, a conjugate or fusion protein described herein may be formulated in combination with an antiviral agent, antiviral vaccine, antifungal agent, antibacterial agent, or therapeutic agent for the treatment of a disorder in a pharmaceutical composition. In some embodiments, the pharmaceutical composition comprises a conjugate described herein (e.g., a conjugate represented by Formula (1)) or a fusion protein described herein and a pharmaceutically acceptable carrier and excipient.

[0370] Acceptable carriers and excipients in pharmaceutical compositions are nontoxic to recipients at the dosages and concentrations employed. Acceptable carriers and excipients may include buffers such as phosphate, citrate, HEPES, and TAE, antioxidants such as ascorbic acid and methionine, preservatives such as hexamethonium chloride, octadecyldimethylbenzylammonium chloride, resorcinol, and benzalkonium chloride, proteins such as human serum albumin, gelatin, dextran, and immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acid residues such as glycine, glutamine, histidine, and lysine, and carbohydrates such as glucose, mannose, sucrose, and sorbitol.

[0371] Examples of other excipients include, but are not limited to, anti-adherents, binders, coating agents, compression aids, disintegrants, dyes, softeners, emulsifiers, fillers (diluents), film-forming or coating agents, flavors, fragrances, glidants (flow improvers), lubricants, adsorbents, suspending or dispersing agents, or sweeteners. Exemplary excipients include, but are not limited to, butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, croscarmellose, cross-linked polyvinylpyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropylmethylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methylparaben, microcrystalline cellulose, polyethylene glycol, povidone, pregelatinized starch, propylparaben, retinol palmitate, shellac, silicon dioxide, sodium carboxymethylcellulose, sodium citrate, sodium carboxymethylcellulose, starch (corn), stearic acid, stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, and xylitol.

[0372] The conjugates or fusion proteins described herein may have ionizable groups that allow them to be prepared as pharmaceutically acceptable salts. These salts may be acid addition salts with inorganic or organic acids, or, in the case of acidic forms of the conjugates herein, salts may be prepared from inorganic or organic bases. Often, conjugates or fusion proteins are prepared or used as pharmaceutically acceptable salts prepared as addition products of pharmaceutically acceptable acids or bases. Suitable pharmaceutically acceptable acids and bases, such as hydrochloric acid, sulfuric acid, hydrobromic acid, acetic acid, lactic acid, citric acid, or tartaric acid for forming acid addition salts and potassium hydroxide, sodium hydroxide, ammonium hydroxide, caffeine, and various amines for forming base salts, are well known in the art. Methods for preparing suitable salts are well established in the art.

[0373] Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxyethanesulfonate, lactobiolate, and cyclopentanesulfonate. Representative salts of alkali or alkaline earth metals include, but are not limited to, sodium, lithium, potassium, calcium, and magnesium salts, as well as non-toxic ammonium, quaternary ammonium, and amine cations, including, but not limited to, ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, and ethylamine.

[0374] Depending on the route of administration and dosage, the conjugate herein or its pharmaceutical composition used in the methods described herein is formulated into a suitable pharmaceutical composition to allow easy delivery.The conjugate (e.g., the conjugate of Formula (1)) or its pharmaceutical composition can be formulated to be administered intramuscularly, intravenously (e.g., as a sterile solution and in a solvent system suitable for intravenous use), intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, peritoneally, subcutaneously, subconjunctivally, intravesicularly, mucosally, intrapericardially, intraumbilically, intraocularly, orally (e.g., as a tablet, capsule, caplet, gelcap, or syrup), topically (e.g., as a cream, gel, lotion, or ointment), partially by inhalation, by injection, or by infusion (e.g., continuous infusion, partial perfusion that directly flushes target cells, catheter, lavage, in a cream, or lipid composition). Depending on the route of administration, the conjugates herein or pharmaceutical compositions thereof may be in the form of, for example, tablets, capsules, pills, powders, granules, suspensions, emulsions, solutions, gels, including hydrogels, pastes, ointments, creams, plasters, drenches, osmotic delivery devices, suppositories, enemas, injections, implants, sprays, preparations suitable for iontophoretic delivery, or aerosols. The compositions may be formulated according to conventional pharmaceutical practice.

[0375] The compositions described herein can be formulated in a variety of ways known in the art. For use in treating human and animal subjects, the conjugates described herein can be formulated as pharmaceutical or veterinary compositions. Depending on the subject (e.g., human) to be treated, the mode of administration, and the type of treatment desired, e.g., prophylaxis or therapy, the conjugates described herein are formulated in a manner consistent with these parameters. Summaries of such techniques can be found in Remington: The Science and Practice of Pharmacy, 22nd Edition, Lippincott Williams & Wilkins (2012); and Encyclopedia of Pharmaceutical Technology, 4th Edition, J. Swarbrick and J.C. Boylan, Marcel Dekker, New York (2013), each of which is incorporated herein by reference.

[0376] The formulations may be prepared in a manner suitable for systemic administration or local or regional administration. Systemic formulations include those designed for injection (e.g., intramuscular, intravenous, or subcutaneous injection), or may be prepared for transdermal, transmucosal, or oral administration. Formulations typically include a diluent and, in some cases, an adjuvant, a buffer, and a preservative. The conjugates may also be administered in liposomal compositions or as microemulsions. Systemic administration may also include relatively non-invasive methods, such as suppositories, transdermal patches, transmucosal delivery, and intranasal administration. Oral administration is also suitable for the conjugates herein. Suitable forms include syrups, capsules, and tablets, as understood in the art.

[0377] Pharmaceutical compositions can be administered parenterally in the form of injectable preparations. Pharmaceutical compositions for injection can be formulated using a sterile solution or any pharmaceutically acceptable liquid as a vehicle. The preparation can be prepared as a solid form or emulsion suitable for solution or suspension in liquid before injection. Pharmaceutically acceptable vehicles include, but are not limited to, sterile water, physiological saline, and cell culture media (e.g., Dulbecco's Modified Eagle's Medium (DMEM), α-Modified Eagle's Medium (α-MEM), F-12 medium). Such injectable compositions can also contain a predetermined amount of non-toxic auxiliary substances, such as wetting or emulsifying agents, pH buffers, e.g., sodium acetate and sorbitan monolaurate. Formulation methods are known in the art; see, for example, Pharmaceutical Preformulation and Formulation, 2nd Edition, M. Gibson, Taylor & Francis Group, CRC Press (2009).

[0378] The pharmaceutical composition can be prepared in the form of an oral formulation. Formulations for oral use include tablets containing the active ingredient(s) in a mixture with non-toxic pharmaceutically acceptable excipients. These excipients may be, for example, inert diluents or fillers (e.g., sucrose, sorbitol, sugars, mannitol, microcrystalline cellulose, starches including potato starch, calcium carbonate, sodium chloride, lactose, calcium phosphate, calcium sulfate, or sodium phosphate); granulating and disintegrating agents (e.g., cellulose derivatives including microcrystalline cellulose, starches including potato starch, croscarmellose sodium, alginates, or alginic acid); binders (e.g., sucrose, glucose, sorbitol, acacia, alginic acid, sodium alginate, gelatin, starch, pregelatinized starch, microcrystalline cellulose, magnesium aluminum silicate, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, ethylcellulose, polyvinylpyrrolidone, or polyethylene glycol); and lubricants, glidants, and antiadherents (e.g., magnesium stearate, zinc stearate, stearic acid, silica, hydrogenated vegetable oil, or talc). Formulations for oral use can also be provided as chewable tablets, or as hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent (e.g., potato starch, lactose, microcrystalline cellulose, calcium carbonate, calcium phosphate, or kaolin), or as soft gelatin capsules in which the active ingredient is mixed with water or an oil medium, such as peanut oil, liquid paraffin, or olive oil. Powders, granules, and pellets can be prepared using the ingredients described above under tablets and capsules in a conventional manner, for example, using a mixer, fluidized bed apparatus, or spray dryer.

[0379] Other pharmaceutically acceptable excipients for oral formulations include, but are not limited to, colorants, flavoring agents, plasticizers, humectants, and buffering agents.Formulations for oral use can also be provided as chewable tablets, or as hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent (e.g., potato starch, lactose, microcrystalline cellulose, calcium carbonate, calcium phosphate, or kaolin), or as soft gelatin capsules in which the active ingredient is mixed with water or an oil medium, such as peanut oil, liquid paraffin, or olive oil.Powder, granules, and pellets can be prepared using the ingredients described above under tablets and capsules in a conventional manner, for example, using a mixer, fluidized bed device, or spray dryer.

[0380] Dissolution- or diffusion-controlled release of the conjugates described herein (e.g., conjugates of Formula (1)) or pharmaceutical compositions thereof can be achieved by suitable coating of tablet, capsule, pellet, or granule formulations of the conjugate, or by incorporating the conjugate into a suitable matrix. Controlled-release coatings can include one or more of the coating materials described above and / or, for example, shellac, beeswax, glycowax, castor wax, carnauba wax, stearyl alcohol, glyceryl monostearate, glyceryl distearate, glycerol palmitostearate, ethyl cellulose, acrylic resin, dl-polylactic acid, cellulose acetate butyrate, polyvinyl chloride, polyvinyl acetate, vinylpyrrolidone, polyethylene, polymethacrylate, methyl methacrylate, 2-hydroxymethacrylate, methacrylate hydrogel, 1,3 butylene glycol, ethylene glycol methacrylate, and / or polyethylene glycol. In controlled release matrix formulations, the matrix material may also include, for example, hydrated methylcellulose, carnauba wax and stearyl alcohol, Carbopol 934, silicone, glyceryl tristearate, methyl acrylate-methyl methacrylate, polyvinyl chloride, polyethylene, and / or halogenated fluorocarbons.

[0381] The pharmaceutical composition may be formulated into a unit dosage form, if desired. The amount of the active ingredient, e.g., a conjugate described herein (e.g., a conjugate of Formula (1)), is contained in the pharmaceutical composition so as to provide a suitable dose within a specified range (e.g., a dose within the range of 0.01 to 100 mg / kg body weight).

[0382] VII. Route of Administration and Dosage In any of the methods described herein, the compositions described herein can be administered by any suitable route to treat or protect against an infection (e.g., a viral, fungal, or bacterial infection), or to prevent, stabilize, or inhibit the growth or spread of an infection (e.g., a viral, fungal, or bacterial infection). The compositions described herein can be administered to humans, domestic pets, livestock, or other animals with a pharmaceutically acceptable diluent, carrier, or excipient. In some embodiments, administering includes administering any of the conjugates (e.g., conjugates of Formula (1)) or compositions described herein intramuscularly, intravenously (e.g., as a sterile solution and in a solvent system suitable for intravenous use), intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, peritoneally, subcutaneously, subconjunctivally, intravesicularly, mucosally, intrapericardially, intraumbilically, intraocularly, orally (e.g., as a tablet, capsule, caplet, gelcap, or syrup), topically (e.g., as a cream, gel, lotion, or ointment), locally, by inhalation, by injection, or by infusion (e.g., continuous infusion, local perfusion directly flushing target cells, catheter, lavage, in a cream, or lipid composition). In some embodiments, when a second therapeutic agent, e.g., an antiviral agent, is also administered in addition to a conjugate described herein, the antiviral agent or pharmaceutical composition thereof may also be administered by any of the routes of administration described herein.

[0383] The dosage of a composition described herein (e.g., a conjugate of Formula (1)) or pharmaceutical composition thereof will depend on factors including the route of administration, the disease being treated (e.g., the extent and / or state of the infection (e.g., viral, fungal, or bacterial infection)), and the physical characteristics, e.g., age, weight, and general health of the subject. Typically, the amount of active agent contained in a single dose will be that amount that effectively prevents, delays, or treats the disorder without inducing significant toxicity. The pharmaceutical composition may comprise a dosage of a conjugate described herein ranging from 0.01 to 500 mg / kg (e.g., 0.01, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 mg / kg), in a more specific embodiment, from about 0.1 to about 30 mg / kg, and in an even more specific embodiment, from about 1 to about 30 mg / kg. In some embodiments, when a conjugate described herein (e.g., a conjugate of Formula (1)) and an antiviral agent or antiviral vaccine are administered in combination (e.g., substantially simultaneously in the same or separate pharmaceutical compositions, or separately in the same treatment regimen), the required dosage of the conjugate described herein may be less than the required dosage of the conjugate when used alone in a treatment regimen.

[0384] A composition described herein (e.g., a conjugate of Formula (1)) or pharmaceutical composition thereof can be administered to a subject in need thereof, for example, one or more times (e.g., 1 to 10 or more times; 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times) daily, weekly, monthly, biennially, yearly, or as medically necessary. Dosages can be provided in either single or multiple dosing regimens. The timing between administrations can decrease as the medical condition improves and increase as the patient's health declines. The dosage and frequency of administration can be adapted by a physician according to conventional factors, such as the extent of the subject's infection and different parameters.

[0385] VIII. Combination Therapy It is also understood that the conjugates, fusion proteins, and compositions of the present disclosure can be formulated and used in combination therapy, i.e., the conjugates, fusion proteins, and pharmaceutical compositions can be formulated with one or more other desired therapeutic agents or medical treatments, or administered simultaneously with, before, or after one or more other desired therapeutic agents or medical treatments. The particular combination of therapies (therapeutics or treatments) to use in a combination regimen will take into account the compatibility of the desired therapeutic agents and / or treatments and the desired therapeutic effect to be achieved. It is also understood that the therapies used may achieve a desired effect for the same disorder, or they may achieve different effects (e.g., control of any adverse effects). In a preferred embodiment, the conjugate or fusion protein and one or more other desired therapeutic agents are formulated in separate pharmaceutical compositions (e.g., formulated for different routes of administration). In some embodiments, the conjugate or fusion protein and one or more other desired therapeutic agents are administered simultaneously (e.g., at substantially the same time, e.g., within 5 minutes, 30 minutes, 1-6 hours, 1-12 hours, or within a day) or sequentially (e.g., at different times, e.g., one or more days apart). When the one or more other desired therapeutic agents and the conjugate or fusion protein are administered sequentially, the one or more other desired therapeutic agents are administered 1-50 (e.g., 1-15, 10-25, 20-35, 30-45, or 35-50) times after administration of the conjugate or fusion protein (e.g., administration 1 day, 2 days, 5 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 6 months, or 12 months or more after the conjugate or fusion protein).

[0386] antiviral agents In some embodiments, one or more antiviral agents may be administered in combination with a conjugate described herein (e.g., a conjugate of any one of Formula (1)) or a fusion protein described herein.

[0387] In some embodiments, the antiviral agent is selected from the group consisting of vidarabine, acyclovir, ganciclovir, valganciclovir, a nucleoside-analog reverse transcriptase inhibitor (e.g., AZT (zidovudine), ddI (didanosine), ddC (zalcitabine), d4T (stavudine), or 3TC (lamivudine)), a non-nucleoside reverse transcriptase inhibitor (e.g., (nevirapine or delavirdine), a protease inhibitor (saquinavir, ritonavir, indinavir, or nelfinavir), ribavirin, or an interferon). The foregoing list is meant to be exemplary of antiviral agents known to those skilled in the art for the treatment of infectious diseases and is not meant to limit the scope of the invention.

[0388] antiviral vaccines In some embodiments, any one of the conjugates described herein (e.g., a conjugate of Formula (1)) is administered in combination with an antiviral vaccine (e.g., a composition that elicits an immune response in a subject directed against a virus).

[0389] In some embodiments, the viral vaccine comprises an immunogen that elicits an immune response in a subject against influenza virus A, B, C, or parainfluenza virus. In some embodiments, the immunogen is an inactivated virus (e.g., the vaccine is a trivalent influenza vaccine containing purified and inactivated material of influenza virus A, B, C, or parainfluenza virus, or any combination thereof). In some embodiments, the vaccine is given as an intramuscular injection. In some embodiments, the vaccine is a live virus vaccine containing attenuated (weakened) live virus. In some embodiments, the vaccine is administered as a nasal spray.

[0390] antibacterial agents In some embodiments, one or more antibacterial agents may be administered in combination with a conjugate described herein (e.g., a conjugate of any one of Formula (1)) or a fusion protein described herein.

[0391] Antibacterial agents include amikacin, gentamicin, kanamycin, neomycin, netilmicin, tobramycin, paromomycin, streptomycin, spectinomycin, geldanamycin, herbimycin, rifaximin, loracarbef, ertapenem, doripenem, imipenem / cilastatin, meropenem, cefadroxil, cefazolin, cephalothin, cephalexin, cefaclor, cefamandole, cefoxitin, cefprozil, cefuroxime, cefixime, cefdinir, cefditoren, cefoperazone, cefotaxime, and cef Podoxime, ceftazidime, ceftibuten, ceftizoxime, ceftriaxone, cefepime, ceftaroline fosamil, ceftobiprole, teicoplanin, vancomycin, telavancin, dalbavancin, oritavancin, clindamycin, lincomycin, daptomycin, azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, troleandomycin, telithromycin, spiramycin, aztreonam, furazolidone, nitrofurantoin, linezolid, pozizolid, radezolid, trezolid Lidl, amoxicillin, ampicillin, azlocillin, carbenicillin, cloxacillin, dicloxacillin, flucloxacillin, mezlocillin, methicillin, nafcillin, oxacillin, penicillin g, penicillin v, piperacillin, penicillin g, temocillin, ticarcillin, amoxicillin clavulanate, ampicillin / sulbactam, piperacillin / tazobactam, ticarcillin / clavulanate, bacitracin, colistin, polymyxin b, ciprofloxacin, enoxacin, gatifloxacin, gemifloxacin, levofloxacin, Lomefloxacin, moxifloxacin, nalidixic acid, norfloxacin, ofloxacin, trovafloxacin, grepafloxacin, sparfloxacin, temafloxacin, mafenide, sulfacetamide, sulfadiazine, silver sulfadiazine, sulfadimethoxine, sulfamethizole, sulfamethoxazole, sulfanilimide, sulfasalazine, sulfisoxazole, trimethoprim-sulfamethoxazole (tmp-smx), sulfonamide chrysoidine, demeclocycline, doxycycline, minocycline,The antibiotic may be selected from the group consisting of oxytetracycline, tetracycline, clofazimine, dapsone, capreomycin, cycloserine, ethambutol (bs), ethionamide, isoniazid, pyrazinamide, rifampicin, rifabutin, rifapentine, streptomycin, arsphenamine, chloramphenicol, fosfomycin, fusidic acid, metronidazole, mupirocin, platensimycin, quinupristin / dalfopristin, thiamphenicol, tigecycline, tinidazole, and trimethoprim. The foregoing list is meant to be exemplary of antibiotics known to those skilled in the art for the treatment of infections and is not meant to limit the scope of the present invention.

[0392] antifungal agents In some embodiments, one or more antifungal agents may be administered in combination with a conjugate described herein (e.g., a conjugate of any one of Formula (1)) or a fusion protein described herein.

[0393] In some embodiments of the above-described combination therapy for treating an infection in a subject in need thereof, the antifungal is selected from the group consisting of rezafungin, amphotericin B, candicidin, filipin, hamycin, natamycin, nystatin, rimocidin, bifonazole, butoconazole, clotrimazole, econazole, fenticonazole, isoconazole, ketoconazole, luliconazole, miconazole, omoconazole, oxiconazole, sertaconazole, sulconazole, ticonazole, triamcinol, thiazolinone ... and undecylenic acid. The antifungal agents are selected from the group consisting of thiazole, albaconazole, efinaconazole, epoxiconazole, fluconazole, isavuconazole, itraconazole, posaconazole, propiconazole, ravuconazole, terconazole, voriconazole, thiazole, abafungin, amorolfine, butenafine, naftifine, terbinafine, anidulafungin, caspofungin, micafungin, ciclopirox, flucytosine, griseofulvin, tolnaftate, and undecylenic acid. The foregoing list is meant to be exemplary of antifungal agents known to those skilled in the art for the treatment of infections and is not meant to limit the scope of the invention. Hereinafter, an embodiment of the present invention will be described. [1] A variant Fc domain monomer, said variant Fc domain monomer comprising substitutions at position 220 and positions 252, 254, and 256 or positions 309, 311, and 434, numbering according to the EU index as Kabat, wherein the substitution at position 220 is serine, the substitution at position 252 is tyrosine, the substitution at position 254 is threonine, the substitution at position 256 is glutamic acid, the substitution at position 309 is aspartic acid, the substitution at position 311 is histidine, and the substitution at position 434 is serine. [2] The variant Fc domain monomer of [1], wherein the variant Fc domain monomer comprises substitutions at positions 220, 252, 254, and 256, numbering according to the EU index as Kabat, wherein the substitution at position 220 is serine, the substitution at position 252 is tyrosine, the substitution at position 254 is threonine, and the substitution at position 256 is glutamic acid. [3] The variant Fc domain monomer according to [2], wherein the variant Fc domain monomer is a variant of human IgG1 or human IgG2. [4] The variant Fc domain monomer of [2] or [3], wherein the substitution at position 220 is cysteine ​​to serine (C220S). [5] The variant Fc domain monomer according to any one of [2] to [4], wherein the substitution at position 252 is from methionine to tyrosine (M252Y). [6] The variant Fc domain monomer according to any one of [2] to [5], wherein the substitution at position 254 is from serine to threonine (S254T). [7] The variant Fc domain monomer according to any one of [2] to [6], wherein the substitution at position 256 is from threonine to glutamate (T256E). [8] The variant Fc domain monomer of [1], wherein the variant Fc domain monomer comprises substitutions at positions 220, 309, 311, and 434, numbering according to the EU index as Kabat, wherein the substitution at position 220 is serine, the substitution at position 309 is aspartic acid, the substitution at position 311 is histidine, and the substitution at position 434 is serine. [9] The variant Fc domain monomer according to [8], wherein the variant Fc domain monomer is a variant of human IgG1 or human IgG2.

[10] The variant Fc domain monomer of [8] or [9], wherein the substitution at position 220 is cysteine ​​to serine (C220S).

[11] The variant Fc domain monomer according to any one of [8] to

[10] , wherein the substitution at position 309 is from valine to aspartic acid (V309D).

[12] The variant Fc domain monomer according to any one of [8] to

[11] , wherein the substitution at position 311 is from glutamine to histidine (Q311H).

[13] The variant Fc domain monomer according to any one of [8] to

[12] , wherein the substitution at position 434 is from asparagine to serine (N434S).

[14] The variant Fc domain monomer according to any one of [1] to

[13] , wherein the variant Fc domain monomer comprises fewer than 300 amino acid residues.

[15] The variant Fc domain monomer according to any one of [1] to

[14] , wherein the variant Fc domain monomer comprises at least 200 amino acid residues.

[16] The variant Fc domain monomer according to any one of [1] to

[15] , wherein the variant Fc domain monomer comprises an amino acid sequence at least 90% identical to the sequence of SEQ ID NOs: 1 to 52, or a region thereof.

[17] A variant Fc domain monomer comprising a serine at amino acid position 220, wherein the amino acid numbering is according to the EU index as Kabat, and wherein the variant Fc domain monomer is between 200 and 300 amino acid residues in length.

[18] The variant Fc domain monomer according to

[17] , wherein the variant Fc domain monomer has a length of between 240 and 255 amino acid residues.

[19] A variant Fc domain monomer comprising a serine at amino acid position 220, wherein the amino acid numbering is according to the EU index as Kabat, and wherein the variant Fc domain monomer has a mass between about 20 kDa and about 40 kDa.

[20] The variant Fc domain monomer according to

[19] , wherein the variant Fc domain monomer has a mass between approximately 25 kDa and 28 kDa.

[21] The variant Fc domain monomer according to any one of

[17] to

[19] , wherein the variant Fc domain monomer is a variant of human IgG1 or human IgG2.

[22] The variant Fc domain monomer according to

[21] , wherein the variant Fc domain monomer is a variant of human IgG1.

[23] A variant Fc domain monomer according to any one of

[17] to

[22] , wherein the N-terminus of the variant Fc domain monomer comprises between 10 and 20 residues of the Fab domain.

[24] The variant Fc domain monomer according to

[23] , wherein the N-terminus of the variant Fc domain monomer comprises the N-terminus of any one of amino acid residues 198 to 205.

[25] The variant Fc domain monomer according to

[24] , wherein the variant Fc domain monomer comprises an N-terminus of amino acid residue Asn201.

[26] The variant Fc domain monomer according to

[24] , wherein the variant Fc domain monomer comprises an N-terminus of amino acid residue Val202.

[27] The variant Fc domain monomer according to any one of

[17] to

[26] , wherein the variant Fc domain monomer comprises the C-terminus of any one of amino acid residues 437 to 447.

[28] The variant Fc domain monomer according to

[27] , wherein the variant Fc domain monomer comprises a C-terminus of amino acid residue Gly446.

[29] The variant Fc domain monomer according to

[27] , wherein the variant Fc domain monomer comprises a C-terminus of amino acid residue Lys447.

[30] The variant Fc domain monomer of any one of

[17] to

[29] , further comprising substitutions at positions 252, 254, and 256, wherein the substitution at position 252 is methionine to tyrosine (M252Y), the substitution at position 254 is serine to threonine (S254T), and the substitution at position 256 is threonine to glutamate (T256E).

[31] The variant Fc domain monomer of any one of

[17] to

[29] , further comprising substitutions at positions 309, 311, and 434, wherein the substitution at position 309 is valine to aspartic acid (V309D), the substitution at position 311 is glutamine to histidine (Q311H), and the substitution at position 434 is asparagine to serine (N434S).

[32] A variant Fc domain monomer according to any one of

[17] to

[31] , comprising an amino acid sequence or region thereof that is at least 90% identical to the sequence of SEQ ID NOs: 20 to 52 or 56 to 58.

[33] A variant Fc domain comprising a dimer of variant Fc domain monomers independently selected from the variant Fc domain monomers described in any one of [1] to

[32] , wherein the variant Fc domain has a mass between about 50 kDa and about 70 kDa.

[34] A conjugate comprising a variant Fc domain monomer and at least one therapeutic agent, wherein said variant Fc domain monomer is covalently conjugated to said therapeutic agent by a linker.

[35] The conjugate has the formula (1): [C1] TIFF0007762645000060.tif46170 wherein each A is independently a therapeutic agent; each E comprises a variant Fc domain monomer according to any one of [1] to

[21] ; L is a linker; n is 1 or 2; T is an integer from 1 to 20; The curved line connected to E indicates that each LA is covalently bonded to E. The conjugate according to

[34] , which is represented by or a pharmaceutically acceptable salt thereof.

[36] The conjugate of

[35] , wherein the therapeutic agent is an antiviral agent, an antifungal agent, or an antibacterial agent.

[37] The conjugate according to

[36] , wherein the therapeutic agent is an antiviral agent.

[38] The conjugate according to

[37] , wherein the therapeutic agent is an antifungal agent.

[39] The conjugate according to

[37] , wherein the therapeutic agent is an antibacterial agent.

[40] A fusion protein comprising a variant Fc domain monomer and at least one polypeptide therapeutic agent, wherein the variant Fc domain monomer is covalently conjugated to the polypeptide therapeutic agent by a linker.

[41] The fusion protein has the structure: (P 2 -L 2 ) n2 -B-(L 1 -P 1 ) n1 (wherein B is a variant Fc domain monomer according to any one of [1] to

[33] or a conjugate according to any one of

[34] to

[39] ; P 1 and P 2 are each independently a polypeptide therapeutic agent; L 1 and L 2 are each independently a linker; n 1 and n 2 are each independently 0 or 1, and n 1 and n 2 At least one of is 1) The fusion protein according to

[40] , comprising:

[42] n 1 is 1 and n 2 is 0, and the fusion protein has the structure: BL 1 -P 1 The fusion protein according to

[41] , comprising:

[43] The linker (L 1 ) is attached to the C-terminus of the Fc domain monomer (B) and the polypeptide therapeutic agent (P 1 The fusion protein according to

[42] , wherein the fusion protein is conjugated to the N-terminus of

[44] The linker (L 1 ) is attached to the N-terminus of the Fc domain monomer (B) and the polypeptide therapeutic agent (P 1 The fusion protein according to

[43] , wherein the C-terminus of

[45] L 1 The fusion protein according to any one of

[42] to

[44] , wherein the linker is a peptide containing between 2 and 200 amino acids.

[46] L 1 is a peptide linker containing between 5 and 25 amino acids.

[47] L 1 (GS) x , (GGS) x , (GGGGS) x , (GGSG) x , (SGGG) x (x is an integer of 1 to 10).

[48] ​​B, L 1 , and P 1 The fusion protein according to any one of

[42] to

[47] , which is expressed as a single polypeptide chain.

[49] The linker (L 1 ) is attached to the N-terminus of the Fc domain monomer (B) and the polypeptide therapeutic agent (P 1 The fusion protein according to

[42] , wherein the fusion protein is conjugated to the N-terminus of

[50] The linker (L 1 ) is attached to the C-terminus of the Fc domain monomer (B) and the polypeptide therapeutic agent (P 1 The fusion protein according to

[42] , wherein the C-terminus of

[51] L 1 B and P 1 The fusion protein according to any one of

[42] to

[44] ,

[49] , and

[50] , comprising a chemical linker covalently conjugated to each of the above.

[52] B and P 1 are expressed as separate polypeptide chains, and then 1 The fusion protein according to any one of

[42] to

[44] ,

[49] , and

[50] , wherein the fusion protein is covalently conjugated to

[53] n 1 is 1 and n2 and the fusion protein has the structure: P 2 -L 2 -BL 1 -P 1 The fusion protein according to

[42] , comprising:

[54] The linker (L 2 ) is a polypeptide therapeutic agent (P 2 and the N-terminus of the Fc domain monomer (B), The linker (L 1 ) is attached to the C-terminus of the Fc domain monomer (B) and the polypeptide therapeutic agent (P 1 ) conjugated to the N-terminus of The fusion protein described in

[53] .

[55] L 1 and L 2 and each of the peptide linkers is independently selected and contains between 2 and 200 amino acids.

[56] L 1 and L 2

[55] A fusion protein according to

[55] , wherein each of the linkers is an independently selected peptide linker containing between 5 and 25 amino acids.

[57] L 1 and L 2 are respectively (GS) x , (GGS) x , (GGGGS) x , (GGSG) x , (SGGG) x (x is an integer of 1 to 10).

[58] P 2 、L 2 , B, L 1 , and P 1 The fusion protein according to any one of

[54] to

[57] , wherein the fusion protein and the fusion protein are expressed together as a single polypeptide chain.

[59] The linker (L 2 ) is a polypeptide therapeutic agent (P 2 ) and the N-terminus of the Fc domain monomer (B), The linker (L 1 ) is a polypeptide therapeutic agent (P 1 and the C-terminus of the Fc domain monomer (B), The fusion protein described in

[54] .

[60] The linker (L 2 ) is a polypeptide therapeutic agent (P 2 and the N-terminus of the Fc domain monomer (B), The linker (L 1 ) is a polypeptide therapeutic agent (P 1 ) and the C-terminus of the Fc domain monomer (B), The fusion protein described in

[54] .

[61] L 2 B and P 2 and a chemical linker covalently conjugated to each of L 1 B and P 1

[54] ,

[55] ,

[59] , or

[60] , comprising a chemical linker covalently conjugated to each of:

[62] P 2 , B, and P 1 are expressed as separate polypeptide chains, and P 2 and B are then respectively, L 2 covalently conjugated to P 1 and B are then respectively, L 1 5. The fusion protein of any one of

[54] ,

[55] ,

[59] , or

[60] , wherein the fusion protein is covalently conjugated to

[63] The variant Fc domain monomer according to any one of [1] to

[33] , the conjugate according to any one of

[34] to

[39] , or the fusion protein according to any one of

[40] to

[62] , wherein the Fc domain monomer dimerizes to form an Fc domain.

[64] A pharmaceutical composition comprising a variant Fc domain monomer according to any one of [1] to

[33] , a conjugate according to any one of

[34] to

[39] , a fusion protein according to any one of

[40] to

[62] , or an Fc domain according to

[63] , and a pharmaceutically acceptable carrier.

[65] A method for treating or preventing a respiratory disorder in a subject, the method comprising administering to the subject the pharmaceutical composition described in

[64] .

[66] The method according to

[65] , wherein the respiratory disorder is an infectious disease.

[67] The method according to

[66] , wherein the infectious disease is a viral infection.

[68] The method according to

[67] , wherein the viral infection is selected from the group including RSV, influenza, dengue, betacoronavirus, and Zika virus.

[69] The method according to

[66] , wherein the infection is a bacterial infection.

[70] The method of

[65] , wherein the respiratory disorder is selected from the group consisting of chronic obstructive pulmonary disease (COPD), chronic bronchitis, cystic fibrosis, bronchiectasis, and pneumonia.

[71] The method according to any one of

[65] to

[70] , wherein the ratio of the concentration of the polypeptide, the conjugate, or the fusion protein in the epithelial lining fluid is at least 30% of the concentration of the polypeptide, the conjugate, or the fusion protein in the plasma within 2 hours after administration.

[72] The method according to

[71] , wherein the ratio of the concentrations is at least 45% within 2 hours after administration.

[73] The method according to

[71] or

[72] , wherein the ratio of concentrations is at least 55% within 2 hours after administration.

[74] The method according to any one of

[71] to

[73] , wherein the concentration ratio is at least 60% within 2 hours after administration.

[75] A method for treating or preventing liver damage in a subject, the method comprising administering to the subject the pharmaceutical composition described in

[64] .

[76] The method according to

[75] , wherein the liver disorder is an infectious disease.

[77] The method according to

[76] , wherein the infectious disease is a viral infection.

[78] The method according to

[76] , wherein the viral infection is selected from the group consisting of hepatitis A, hepatitis B, and hepatitis C.

[79] The method according to

[75] , wherein the liver disorder is selected from the group consisting of primary cholangitis, primary sclerosing cholangitis, hepatocellular carcinoma, cholangiocarcinoma, hepatocellular adenoma, non-alcoholic fatty liver disease (NAFLD), acute liver failure, and cirrhosis.

[80] A method for treating or preventing a central nervous system (CNS) disorder in a subject, the method comprising administering to the subject the pharmaceutical composition described in

[64] .

[81] The method according to

[80] , wherein the CNS disorder is an infectious disease.

[82] The method according to

[81] , wherein the infectious disease is a viral infection.

[83] The method of

[82] , wherein the viral infection is selected from the group including viral meningitis, herpes simplex virus (HSV) 1, HSV2, Epstein-Barr virus, varicella-zoster virus, poliovirus, coxsackievirus, West Nile virus, La Crosse virus, western equine encephalitis, eastern equine encephalitis, Powassan virus, or rabies virus.

[84] The method of

[80] , wherein the CNS disorder is selected from the group consisting of cancer, Alzheimer's disease, Parkinson's disease, epilepsy, multiple sclerosis, schizophrenia, and meningitis.

[85] A method for treating or preventing a muscle disorder in a subject, the method comprising administering to the subject the pharmaceutical composition described in

[64] .

[86] The method according to

[85] , wherein the muscle disorder is cancer or myositis.

[87] The method according to

[86] , wherein the myositis is caused by injury, infection, or immune disorder.

[88] A method for treating or preventing a skin disorder in a subject, the method comprising administering to the subject the pharmaceutical composition described in

[64] .

[89] The method of

[88] , wherein the skin disorder is selected from the group including eczema, psoriasis, acne, rosacea, cold sores, cellulitis, basal cell carcinoma, squamous cell carcinoma, and melanoma.

[90] A method for treating or preventing an eye disorder in a subject, the method comprising administering to the subject the pharmaceutical composition described in

[64] .

[91] The method of

[90] , wherein the eye disorder is selected from age-related macular degeneration, cataracts, and glaucoma.

[92] A method for treating or preventing a vascular disorder in a subject, the method comprising administering to the subject the pharmaceutical composition described in

[64] .

[93] A method for treating or preventing an infectious disease in a subject, the method comprising administering to the subject the pharmaceutical composition described in

[64] .

[94] The method according to

[93] , wherein the infection is a viral infection, a bacterial infection, or a fungal infection. [Example]

[0394] The following examples are presented to provide one of ordinary skill in the art with a description of how the compositions and methods described herein may be used, made, and evaluated, and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention.

[0395] Example 1. General procedure for the synthesis of azido-Fc Preparation of PEG4-azide NHS ester solution (0.050 M) in DMF / PBS: 16.75 mg of PEG4-azide NHS ester was dissolved in 0.100 mL of DMF at 0° C. and diluted to 0.837 mL by adding PBS 1x buffer at 0° C. This solution was used to prepare other PEG4-azide Fc with various DAR values ​​by adjusting the equivalent volume of this PEG4-azide NHS ester PBS solution.

[0396] Pretreatment of h-IgG1 Fc (107.2 mg, MW approximately 57891 Da, 1.852 μmol in 8.800 mL of PBS, pH 7.4): The Fc solution was transferred to four centrifugal concentrators (30,000 MWCO, 15 mL), diluted to 15 mL with PBSx1 buffer, and concentrated to a volume of approximately 1.5 mL. The residue was diluted 1:10 in PBS pH 7.4 and concentrated again. This washing procedure was repeated a total of four times, followed by dilution to 8.80 mL.

[0397] Preparation of PEG4-Azide Fc: A 0.050 M PEG4-Azide NHS ester PBS buffer solution (0.593 mL, 29.6 μmol, 16 equivalents) was added to the above solution of h-IgG1 Fc (SEQ ID NO: 21; the C-terminal Lys is proteolytically cleaved after expression), and the mixture was subjected to rotary shaking at ambient temperature for 2 hours. The solution was concentrated to a volume of approximately 1.5 mL using four centrifugal concentrators (30,000 MWCO, 15 mL). The crude mixture was diluted 1:10 with PBS pH 7.4 and concentrated again. This washing procedure was repeated a total of three times. The concentrated Fc-PEG4-Azide was diluted to 8.80 mL with pH 7.4 PBS buffer and prepared for click conjugation. The purified material was quantified using a NANODROP™ UV-visible spectrophotometer (using the extinction coefficient calculated based on the amino acid sequence of h-IgG1). After purification, the yield was quantitative.

[0398] Example 2. Synthesis of Conjugate 1 (Fc Domain Containing C220S / M252Y / S254T / T256E Mutations) Preparation of click reagent solution: 0.0050 M CuSO in PBS buffer solution: 10.0 mg CuSO was dissolved in 12.53 mL PBS, then 5.00 mL of this CuSO solution was taken and 43.1 mg BTTAA (CAS#1334179-85-9) and 247.5 mg sodium ascorbate were added to obtain a click reagent solution (0.0050 M CuSO, 0.020 M BTTAA, and 0.25 M sodium ascorbate).

[0399] To a solution of azide-functionalized Fc (Example 1; 65.5 mg, 10.0 mL, 1.13 μmol, azide DAR approximately 5.9, SEQ ID NO: 10) in a 15 mL centrifuge tube was added an alkyne-derivatized small molecule virus inhibitor (22.7 mg, 15.2 μmol, 3.0 equivalents per azide of Fc). After gentle stirring to dissolve all solids, the mixture was treated with the click reagent solution (1.80 mL). The resulting mixture was gently rotated at ambient temperature for 12 hours. It was purified by affinity chromatography on a Protein A column followed by size-exclusion chromatography. Maldi TOF analysis of the purified final product yielded an average mass of 66,420 Da (DAR = 5.8). Yield: 57 mg with 98% purity.

[0400] Example 3. Synthesis of Conjugate 2 (Fc Domain Containing the C220S Mutation) This conjugate was prepared similarly to Conjugate 1 (Example 2) with PEG4-Azide-Fc (SEQ ID NO: 21, prepared as in Example 1) and a small molecule viral inhibitor. Maldi TOF analysis of the purified final product gave an average mass of 62,927 Da (DAR=4.2).

[0401] Example 4. Comparative 30-day non-human primate PK study of conjugate 1 and conjugate 2 following IV administration A non-human primate (NHP) PK study was conducted by BTS Research (San Diego, CA) using male and female 5- to 9-year-old cynomolgus monkeys (weight range: 3.5-8.5 kg). NHPs were injected IV with 2 mg / kg of test article (0.4 mL / kg dose volume). Animals were housed under standard IACUC-approved housing conditions. At appropriate times, animals were exsanguinated (via the femoral or cephalic vein) and blood was collected in K2EDTA tubes to prevent clotting. Collected blood was centrifuged (2,000 x g, 10 minutes), and plasma was collected for time-course analysis of test article concentrations. Plasma concentrations of conjugate 1 (C220S / M252Y / S254T / T256E) and conjugate 2 (C220S) at each time point were measured by sandwich ELISA. Briefly, test articles were captured on Fc-coated plates and then detected using an HRP-conjugated anti-human IgG-Fc antibody. Protein concentrations were calculated in GraphPad Prism using 4PL nonlinear regression of the conjugate 1 or conjugate 2 standard curves. Curves comparing conjugate 1 and conjugate 2 are shown in Figure 1. Conjugate 1 demonstrated a significantly improved terminal half-life of approximately 45 days compared to approximately 10 days for conjugate 2. The AUC for conjugate 1 was twice that of conjugate 2 (Table 2).

[0402] [Table 1]

[0403] Example 5. 14-day mouse PK study of plasma and epithelial lining fluid (ELF) concentrations of conjugate 2 Female BALB / c mice from Charles River Laboratories were acclimated 5 days prior to the start of the study. Animals were housed 3-6 per cage and allowed free access to food and water. All procedures were performed in accordance with NeoSome IACUC policies and guidelines. Mice were injected subcutaneously (SC) with 20 mg / kg of test article (10 mL / kg dose volume). At selected time points, three mice were euthanized by CO2 inhalation. Blood was collected via cardiac puncture into K2EDTA tubes for plasma retention. After blood collection, bronchoalveolar lavage (BAL) was performed by exposing the trachea, inserting a 23G tubing adapter, and performing two 0.5 mL flushes with sterile 1X PBS pH 7.4. The volume of collected fluid was recorded and retained. After the BAL procedure was completed, lungs were removed, weighed, and stored at -80°C. Aliquots of plasma and BAL fluid (BALF) were decanted, and samples were stored at -80°C for use in urea quantification assays. The collected BALF was centrifuged at 12,000 RPM for 5 minutes at room temperature to pellet alveolar macrophages, and both the pellet and supernatant were stored at -80°C until transport to the sponsor. Plasma concentrations of conjugate 2 at each time point were measured by indirect ELISA, as described in detail above. Briefly, conjugate 2 molecules were captured on small molecule virus target-coated plates and then detected using HRP-conjugated anti-human IgG Fcγ-specific F(ab')2. The same ELISA was performed on the collected BALF as described above. Conjugate 2 plasma concentrations were calculated in GraphPad Prism using 4PL nonlinear regression of the conjugate 2 standard curve. ELF volume and conjugate 2 concentration in the ELF were determined using urea as a dilution marker as previously described (Rennard et al., 1986 J Appl Physiol 60:532-538). A curve comparing conjugate 2 to ELF levels is shown in Figure 2.By 2 hours post-injection, epithelial lining fluid (ELF) levels of conjugate 2 were approximately 60% of the plasma exposure level (AUC) over the remainder of the time course, indicating near-immediate distribution of conjugate 2 from plasma to ELF in the lung (Figure 2, Table 2).

[0404] [Table 2]

[0405] Example 6. 7-day mouse PK study comparing SC administration of conjugate 1 and conjugate 2 Mouse PK studies were conducted using 6-week-old male CD-1 mice. Mice were injected SC with 10 mg / kg of test article (10 mL / kg dose volume). Animals were housed under standard IACUC-approved housing conditions. At appropriate times, animals were non-terminally bled (by retroorbital, cheek, or tail vein), and blood was collected in K2EDTA tubes to prevent clotting. Collected blood was centrifuged (2,000 x g for 10 minutes), and plasma was collected for time-course analysis of test article concentration. Plasma concentrations of conjugate 2 at each time point were measured by indirect ELISA, as described in detail above. Briefly, conjugate 2 molecules were captured on small molecule virus target-coated plates and then detected using HRP-conjugated anti-human IgG Fcγ-specific F(ab')2. Protein concentrations were calculated in GraphPad Prism using 4PL nonlinear regression of the conjugate 2 standard curve. Curves comparing the 7-day PK profiles of conjugate 2 and conjugate 1 are shown in Figure 3. The plasma exposure level of conjugate 2 (C220S) is approximately 50% higher than that of conjugate 1 (C220S / M252Y / S254T / T256E). Compared to WT human IgG1, the half-life of the human IgG1 YTE Fc variant is known to be reduced in mice due to improved mouse FcRn binding at neutral pH, which negates the improved binding to mouse FcRn at acidic pH (Dall'Acqua et al. 2002 J Immunol 169:5171-5180).

[0406] Example 7. 7-day mouse PK study comparing IV administration of Fc with different molecular weights Mouse PK studies were conducted using 6-week-old male CD-1 mice. Mice were injected intravenously (IV) via the tail vein with 5 mg / kg of test article (5 mL / kg dose volume). Animals were housed under standard IACUC-approved housing conditions. At appropriate times, animals were non-terminally bled (by retroorbital, cheek, or tail vein), and blood was collected in K2EDTA tubes to prevent clotting. Collected blood was centrifuged (2,000 x g, 10 minutes), and plasma was harvested for analysis of test article concentration over time. Fc plasma concentrations at each time point were measured by Fc-capture ELISA as follows: Nunc Maxisorp 96-well plates (cat no. 12-565-136, Fisher Scientific) were coated overnight at 4°C with 0.1 μg / 100 μL / well goat anti-human IgG (Fcγ fragment-specific; cat no. 109-005-098, Jackson Immunoresearch) in carbonate buffer (cat no. C3041, MilliporeSigma). Plates were washed five times with 300 μL / well of PBST and blocked with 200 μL / well of 5% nonfat dry milk (cat no. 9999S, Cell Signaling) in PBST for 1 hour with shaking at room temperature. Three-fold serial dilutions of plasma samples were plated at 100 μL / well and incubated for 2 hours with shaking at room temperature (sample diluent: 2.5% nonfat dry milk in PBS 0.025% Tween 20 + naive mouse plasma final concentration of 1:900). An Fc standard curve ranging from 0.03 to 55 ng / mL was run in duplicate on each plate. After a 2-hour incubation, the plate was washed five times with 300 μL / well of PBST. The test article (Fc) bound to the capture antibody on the plate was then probed with 100 μL / well of HRP-conjugated anti-human IgG Fc F(ab')2 (cat. no. 709-036-098, Jackson Immunoresearch) diluted 1:2,000 in sample diluent for 1 hour with agitation at room temperature. The plate was then washed eight times with 300 μL / well of PBST and developed with 100 μL / well of TMB substrate reagent (cat. no. 555214, BD) for 7–8 minutes.The reaction was stopped with 100 μL / well of 1N H2SO4, and absorbance was read at 450 nm using an EnSpire multimode plate reader (PerkinElmer). Test article concentrations in plasma samples were interpolated using GraphPad Prism Version 8 after nonlinear regression analysis (sigmoidal, 4PL analysis) of the standard curve. The resulting mean plasma concentrations were then used to calculate the overall AUC for each plasma concentration-time profile.

[0407] Mouse PK studies were performed to optimize PK (by reducing clearance) based on the length and molecular weight of the Fc domain monomer (Tables 3 and 4 and Figures 4 and 5). Slower clearance was observed for the longer Fc domain (Fc domain homodimer of SEQ ID NO: 53, MW: 58,272 Da) containing extended N- and C-terminal affinity tags containing non-germline amino acids. Removal of the potentially immunogenic N- and C-terminal extensions from the Fc domain monomer (Fc domain homodimer of SEQ ID NO: 54, MW: 53,743 Da) resulted in a smaller Fc domain that was rapidly cleared from mouse plasma, presumably via kidney filtration. To improve PK parameters to resemble those seen with a larger Fc domain with non-endogenous N- and C-terminal extensions, six amino acid residues from the endogenous IgG1 sequence were included at the N-terminus of an Fc domain monomer (Fc domain homodimer of SEQ ID NO: 55, MW: 55,031 Da), which exhibited improved Fc domain PK parameters (i.e., reduced clearance), although the Fc N-terminal extension was unable to restore PK values ​​to those seen with an Fc domain containing unnecessary N- and C-terminal tags (Fc domain homodimer of SEQ ID NO: 53) (data shown in Table 3 and Figure 4).

[0408] [Table 3]

[0409] SEQ ID NO: 53: Mature human Fc IgG1, added N-terminal ISAMVRS amino acid residues (italics), C-terminal G4S linker (italics), C-terminal myc-tag (underlined), allotype G1m(f) (bold italics)

[0410] TIFF0007762645000064.tif50170

[0411] SEQ ID NO: 54: Mature human Fc IgG1, with added N-terminal ISAMVRS amino acid residues (italics), allotype G1m(fa) (bold italics)

[0412] TIFF0007762645000065.tif43170

[0413] SEQ ID NO: 55: Mature human Fc IgG1, added N-terminal amino acid residues (italics), hinge residues are in italics, allotype G1m(fa) (bold italics)

[0414] TIFF0007762645000066.tif43170

[0415] To develop a new Fc domain that closely resembles the endogenous IgG1 domain with PK parameters similar to those of the Fc of SEQ ID NO: 53, further mouse PK studies were conducted using an Fc domain monomer (Fc domain homodimer of SEQ ID NO: 56, MW: 58,154 Da) containing endogenous amino acids on the N-terminus extending into the Fab region of the antibody. The study showed that the Fc domain monomer containing amino acid residues extending into the Fab region of the antibody demonstrated surprising improvements in PK parameters (data shown in Table 4 and Figure 5). Overall, improved plasma exposure levels were observed with increasing molecular weight. Specifically, the AUC of the Fc domain with a molecular weight of 55,031 Da (homodimer of SEQ ID NO: 55) was higher than that of the Fc domain with a molecular weight of 53,743 Da (homodimer of SEQ ID NO: 54). Furthermore, the addition of N-terminal Fab residues to generate an Fc domain with a molecular weight of 58,154 Da (SEQ ID NO: 56) further improved the AUC of the Fc domain. The inclusion of additional Fab residues at the N-terminus of the Fc domain monomer is believed to introduce unwanted features, including unpaired cysteines, hydrophobic regions, and secondary structure of the endogenous Fab region, which may adversely affect solution properties and promote aggregation, without significantly reducing clearance from plasma.

[0416] [Table 4]

[0417] SEQ ID NO: 56: Mature human IgG1 Fc, Cys to Ser substitution (#), allotype G1m (fa) (bold italics), N-terminal Fab residues are underlined, hinge residues are italics

[0418] TIFF0007762645000068.tif50170

[0419] SEQ ID NO: 58: Mature human IgG1 Fc, Cys to Ser substitution (#), M428L, N434S (bold / underlined), allotype G1m(fa) (bold italics), N-terminal Fab residues are underlined, hinge residues are italics

[0420] TIFF0007762645000069.tif50170

Claims

1. A variant human IgG1 Fc domain monomer comprising an amino acid sequence or a region thereof that is at least 90% identical to a sequence of SEQ ID NOs: 20-52 or 56-58 and that comprises a serine at amino acid position 220, said variant Fc domain monomer is between 200 and 300 amino acid residues in length, or said variant Fc domain monomer has a mass between about 20 kDa and about 40 kDa; the N-terminus of the variant Fc domain monomer comprises between 10 and 20 residues of a Fab domain; and the N-terminus of the variant Fc domain monomer is any one of amino acid positions 198-205; where the amino acid numbering is according to the EU index as per Kabat The variant Fc domain monomer.

2. 2. A conjugate comprising the variant Fc domain monomer of claim 1 and at least one therapeutic agent, wherein the variant Fc domain monomer is covalently conjugated to the therapeutic agent by a linker.

3. The conjugate has the formula (1): 【Chemistry 1】 (In the formula, each A is independently a therapeutic agent; each E comprises said variant Fc domain monomer; L is a linker; n is 1 or 2; T is an integer from 1 to 20; and The curved line connected to E indicates that each L-A is covalently bonded to E. The conjugate of claim 2, represented by: or a pharmaceutically acceptable salt thereof.

4. A fusion protein comprising the variant Fc domain monomer of claim 1 and at least one polypeptide therapeutic agent, wherein the variant Fc domain monomer is covalently conjugated to the polypeptide therapeutic agent by a linker.

5. 10. The variant Fc domain monomer of claim 1, the conjugate of claim 2 or 3, or the fusion protein of claim 4, wherein the Fc domain monomer dimerizes to form an Fc domain.

6. A pharmaceutical composition comprising a variant Fc domain monomer according to claim 1, a conjugate according to claim 2 or 3, a fusion protein according to claim 4 or an Fc domain according to claim 5, and a pharmaceutically acceptable carrier.

7. 7. The pharmaceutical composition of claim 6 for use in treating or preventing a respiratory disorder, liver disorder, central nervous system disorder, muscle disorder, skin disorder, eye disorder or vascular disorder in a subject.

8. 7. The pharmaceutical composition of claim 6 for use in treating or preventing an infectious disease in a subject.

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