Immunoreceptor inhibitory proteins and related methods

Abstract

Provided herein are immunoreceptor inhibitory proteins and compositions (e.g., pharmaceutical compositions) comprising the same; as well as methods of making the immunoreceptor inhibitory proteins and compositions. The immunoreceptor inhibitory proteins provided herein are useful in pharmaceutical compositions and methods of use (including, e.g., inhibiting binding of one or more TNFRSF member to one or more TNFLSF member (e.g., binding of TNFα to TNFR1 and / or TNFR2; and / or binding of LTα to TNFR1 and / or TNFR2).

Description

Attorney Docket No.62801.71WO01 IMMUNORECEPTOR INHIBITORY PROTEINS AND RELATED METHODS RELATED APPLICATIONS

[0001] This application claims priority to U.S. Serial No.: 63 / 648,381, filed May 16, 2024; U.S. Serial No.: 63 / 671,552, filed July 15, 2024; U.S. Serial No.: 63 / 684,945, filed August 20, 2024; and U.S. Serial No.: 63 / 715,927, filed November 4, 2024; the entire contents of each of which is incorporated herein by reference. 1. FIELD

[0002] This disclosure relates to immunoreceptor inhibitory proteins that bind to one or more TNF superfamily (TNFSF) member (e.g., TNTNFα, LTα) and nucleic acid molecules encoding the same. The disclosure further relates to methods of making and utilizing the same. 2. BACKGROUND

[0003] The TNFSF comprises 19 ligands and 29 receptors that regulate multiple cellular functions, including e.g., immune responses, cell proliferation, cell survival, cell differentiation, and programmed cell death. Exemplary TNFSF ligands and exemplary cognate receptors, include, e.g., TNFα and TNFR1 / TNFR2; FasL and Fas; LIGHT and LIGHTR and LTβR; and TL1A and DR3. A subset of TNFSF ligands are known to interact with more than one TNFSF receptor (e.g., TNFα is known to interact with both TNFR1 and TNFR2). The intracellular domains and signaling properties of the various TNFSF receptors are known to vary. For example, a subset of TNFSF receptors comprise a death domain; others comprise one or more TRAF interacting motif (TIM); while other subsets of TNFSF receptors do not contain functional intracellular signaling domains or motifs. 3. SUMMARY

[0004] Provided herein are, inter alia, immunoreceptor inhibitory proteins and nucleic acid molecules encoding the same; fusions and conjugates comprising the immunoreceptor inhibitory proteins; methods of manufacturing; pharmaceutical compositions; and methods of use including e.g., methods of inhibiting binding of TNFα to TNFR1 and / or TNFR2, inhibiting signaling of TNFR1 and / or TNFR2 (including, e.g., signaling mediated through the binding of TNFα to TNFR1 and / or TNFR2), and modulating (e.g., suppressing) an immune response, as well as diagnostics.Attorney Docket No.62801.71WO01

[0005] Accordingly, in one aspect provided herein are proteins (e.g., isolated and / or recombinant proteins) comprising an amino acid sequence at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any protein set forth in Table 2 or set forth in any one of SEQ ID NOS: 7-16.

[0006] In some embodiments, the amino acid sequence of the protein comprises an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any protein set forth in Table 2 or set forth in any one of SEQ ID NOS: 7-16. In some embodiments, the amino acid sequence of the protein comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any protein set forth in Table 2 or set forth in any one of SEQ ID NOS: 7-16.

[0007] In some embodiments, the amino acid sequence of the protein comprises an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any protein set forth in Table 2 or set forth in any one of SEQ ID NOS: 7-11. In some embodiments, the amino acid sequence of the protein comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any protein set forth in Table 2 or set forth in any one of SEQ ID NOS: 7-11.

[0008] In specific embodiments, the amino acid sequence of the protein comprises an amino acid sequence at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 7 or 12. In specific embodiments, the amino acid sequence of the protein comprises an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 7 or 12. In specific embodiments, the amino acid sequence of the protein comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 7 or 12. In specific embodiments, the amino acid sequence of the protein comprises the amino acid sequence set forth in SEQ ID NO: 7 or 12.

[0009] In specific embodiments, the amino acid sequence of the protein comprises an amino acid sequence at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 7. In specific embodiments, the amino acid sequence of the protein comprises an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to theAttorney Docket No.62801.71WO01 amino acid sequence set forth in SEQ ID NO: 7. In specific embodiments, the amino acid sequence of the protein comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 7. In specific embodiments, the amino acid sequence of the protein comprises the amino acid sequence set forth in SEQ ID NO: 7.

[0010] In some embodiments, the protein exhibits anti-inflammatory properties (e.g., upon administration to a subject). In some embodiments, the protein specifically binds human TNFα (hTNFα); and / or specifically binds human LTα (hLTα). In some embodiments, the protein inhibits binding of hTNFα to human TNFR1 (hTNFR1) and / or human TNFR2 (hTNFR2). In some embodiments, the protein inhibits binding of hLTα to human TNFR1 (hTNFR1) and / or human TNFR2 (hTNFR2).

[0011] In some embodiments, the protein comprises a homologous or heterologous signal peptide (e.g., operably connected to the N-terminus of the protein).

[0012] In some embodiments, the protein operably connected to a heterologous moiety (e.g., described herein). In some embodiments, the heterologous moiety is a protein, peptide, small molecule, nucleic acid molecule (e.g., DNA, RNA, DNA / RNA hybrid molecule), lipid, or synthetic polymer. In some embodiments, the heterologous moiety is a protein.

[0013] In one aspect, provided herein are conjugates comprising a protein described herein operably connected to a heterologous moiety (e.g., described herein).

[0014] In one aspect, provided herein are radioligands comprising a protein described herein operably connected to a radionuclide.

[0015] In one aspect, provided herein are fusion proteins comprising a protein described herein operably connected to a heterologous protein.

[0016] In some embodiments, the heterologous protein comprises an antibody. In some embodiments, the antibody specifically binds a cytokine. In some embodiments, the cytokine is an interleukin. In some embodiments, the interleukin is interleukin 23 (IL-23) (e.g., human IL-23).

[0017] In some embodiments, the heterologous protein comprises a half-life extension protein.

[0018] In some embodiments, the heterologous protein comprises an immunoglobulin (Ig) (e.g., a human Ig (hIg)) Fc region. In some embodiments, the Ig (e.g., hIg) Fc region comprises at least a portion of a hinge region, a CH2 region, and a CH3 region. In some embodiments, the Ig (e.g., hIg) Fc region comprises a hinge region, a CH2 region, and a CH3 region. In someAttorney Docket No.62801.71WO01 embodiments, the Ig is a hIg. In some embodiments, the hIg is a human IgG (hIgG). In some embodiments, the hIgG is hIgG1 or hIgG4.

[0019] In some embodiments, the Ig (e.g., hIg) Fc region comprises one or more amino acid substitutions relative to a reference Ig (e.g., hIg) Fc region that reduces or abolishes one or more of the following effector functions relative to the reference Ig (e.g., hIg) Fc region: antibody dependent cell mediated cytotoxicity (ADCC), complement dependent cytotoxicity (CDC), and / or affinity to one or more human Fc receptor (e.g., an Fcγ receptor (e.g., FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and / or FcγRIIIb (e.g., FcγRI, FcγIIa, and / or FcγIIIa))). In some embodiments, the Ig (e.g., hIg) Fc region does not substantially mediate ADCC, does not substantially mediate CDC, and / or does not bind to one or more human Fc receptor (e.g., an Fcγ receptor (e.g., FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and / or FcγRIIIb (e.g., FcγRI, FcγIIa, and / or FcγIIIa))).

[0020] In some embodiments, the Ig is an hIgG4 and the amino acid sequence of the Fc region comprises an amino acid substitution at amino acid position S228, an amino acid substitution at amino acid position F234, and / or an amino acid substitution at amino acid position L235, numbering according to the EU index of Kabat. In some embodiments, the Ig is hIgG4 and the amino acid sequence of the Fc region comprises a proline at amino acid position S228, an alanine at amino acid position F234, and / or an alanine at amino acid position L235, numbering according to EU index of Kabat. In some embodiments, the Ig is hIgG1 and the amino acid sequence of the Fc region comprises an amino acid substitution at amino acid position L234, and / or an amino acid substitution at amino acid position L235, numbering according to the EU index of Kabat. In some embodiments, the Ig is hIgG1 and the amino acid sequence of the Fc region comprises an alanine at amino acid position L234 and / or an alanine at amino acid position L235, numbering according to the EU index of Kabat. In some embodiments, the Ig is hIgG1 and the amino acid sequence of the Fc region comprises an alanine at amino acid position L234, an alanine at amino acid position L235, and / or a glycine at position P329 numbering according to the EU index of Kabat.

[0021] In some embodiments, the Ig (e.g., hIg) Fc region comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any protein set forth in any one of Tables 4-7 or set forth in any one of SEQ ID NOS: 22-95 or 123-124. In some embodiments, the Ig (e.g., hIg) Fc region comprises an amino acid sequence comprising a set of amino acid variations set forth in any one of Tables 10-12.Attorney Docket No.62801.71WO01

[0022] In some embodiments, the protein described herein is directly operably connected to the heterologous protein through a peptide bond.

[0023] In some embodiments, the protein described herein is indirectly operably connected to the heterologous protein through a peptide linker. In some embodiments, the amino acid sequence of the peptide linker comprises or consists of glycine or glycine and serine amino acid residues. In some embodiments, the amino acid of the peptide linker comprises or consists of (a) the amino acid sequence set forth in any one of SEQ ID NOS: 96-105; or (b) the amino acid sequence set forth in any one of SEQ ID NOS: 96-105 comprising or consisting of 1, 2, or 3 amino acid substitutions.

[0024] In some embodiments, the fusion protein comprises from N- to C-terminus: the protein described herein and the heterologous protein. In some embodiments, the fusion protein comprises from N- to C-terminus: the protein described herein, a peptide linker, and the heterologous protein. In some embodiments, the fusion protein comprises from N- to C-terminus: a signal peptide, the protein described herein, a peptide linker, and the heterologous protein. In some embodiments, the fusion protein comprises from N- to C-terminus: the heterologous protein and the protein described herein. In some embodiments, the fusion protein comprises from N- to C-terminus: the heterologous protein, a peptide linker, and the protein described herein. In some embodiments, the fusion protein comprises from N- to C-terminus: a signal peptide, the heterologous protein, a peptide linker, and the protein described herein.

[0025] In some embodiments, the amino acid sequence of the fusion protein is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 106-120 or set forth in Table 9. In some embodiments, the amino acid sequence of the fusion protein is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 106-120.

[0026] In specific embodiments, the amino acid sequence of the fusion protein is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 106-108. In specific embodiments, the amino acid sequence of the fusion protein is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth inAttorney Docket No.62801.71WO01 any one of SEQ ID NOS: 106-108. In specific embodiments, the amino acid sequence of the fusion protein is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 106-108. In specific embodiments, the amino acid sequence of the fusion protein comprises the amino acid sequence set forth in any one of SEQ ID NOS: 106- 108.

[0027] In one aspect, provided herein are fusion proteins comprising a first protein and a second protein, wherein the first protein comprises a first Ig (e.g., hIg) Fc region operably connected to a first protein described herein; and wherein the second protein comprises a second Ig (e.g., hIg) Fc region operably connected to a second protein described herein.

[0028] In some embodiments, the first Fc region and the second Fc region associate to form a dimer. In some embodiments, the first protein comprises an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of the second protein.

[0029] In some embodiments, the first Ig (e.g., hIg, mIg) Fc region comprises at least a portion of a hinge region, a CH2 region, and a CH3 region; and the second Ig (e.g., hIg) Fc region comprises at least a portion of a hinge region, a CH2 region, and a CH3 region. In some embodiments, the first Ig (e.g., hIg) Fc region comprises a hinge region, a CH2 region, and a CH3 region; and the second Ig (e.g., hIg) Fc region comprises a hinge region, a CH2 region, and a CH3 region. In some embodiments, the Ig of the first Ig Fc region is a hIg and the Ig of the second Ig Fc region is a hIg. In some embodiments, the hIg of the first hIg Fc region is a hIgG and the hIg of the second hIg Fc region is a hIgG. In some embodiments, the hIgG of the first hIg Fc region is hIgG4 and the hIgG of the first hIg Fc region is hIgG4. In some embodiments, the hIgG of the first hIg Fc region is hIgG1 and the hIgG of the first hIg Fc region is hIgG1.

[0030] In some embodiments, the first Ig (e.g., hIg) Fc region and the second Ig (e.g., hIg) Fc region each comprises one or more amino acid substitutions relative to a reference Ig (e.g., hIg) Fc region that reduces or abolishes one or more of the following effector functions relative to the reference Ig (e.g., hIg) Fc region: ADCC, CDC, and / or binding affinity to one or more Fc receptor (e.g., an Fcγ receptor (e.g., FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and / or FcγRIIIb (e.g., FcγRI, FcγIIa, and / or FcγIIIa))). In some embodiments, the fusion protein does not substantially mediate ADCC, does not substantially mediate CDC, and / or does not bind to one or more Fc receptor (e.g., an Fcγ receptor (e.g., FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and / or FcγRIIIb (e.g., FcγRI, FcγIIa,Attorney Docket No.62801.71WO01 and / or FcγIIIa))).

[0031] In some embodiments, the Ig of the first Ig Fc region and the second Ig Fc region is hIgG4 and the amino acid sequence of the first Fc region and the second Fc region each comprise an amino acid substitution at amino acid position S228, an amino acid substitution at amino acid position F234, and / or an amino acid substitution at amino acid position E235, numbering according to the EU index of Kabat. In some embodiments, the Ig of the first Ig Fc region and the second Ig Fc region is hIgG4 and the amino acid sequence of the first Fc region and the second Fc region each comprise a proline at amino acid position S228, an alanine at amino acid position F234, and / or an alanine at amino acid position E235, numbering according to EU index of Kabat. In some embodiments, the Ig of the first Ig Fc region and the second Ig Fc region is hIgG1 and the amino acid sequence of the first Fc region and the second Fc region each comprise an amino acid substitution at amino acid position L234, and / or an amino acid substitution at amino acid position L235, numbering according to the EU index of Kabat. In some embodiments, the Ig of the first Ig Fc region and the second Ig Fc region is hIgG1 and the amino acid sequence of the first Fc region and the second Fc region each comprise an alanine at amino acid position L234 and / or an alanine at amino acid position L235, numbering according to the EU index of Kabat. In some embodiments, the Ig of the first Ig Fc region and the second Ig Fc region is hIgG1 and the amino acid sequence of the first Fc region and the second Fc region each comprise a proline (or alanine) at amino acid position L234 and / or a proline (or alanine) at amino acid position L235, numbering according to the EU index of Kabat. In some embodiments, the Ig of the first Ig Fc region and the second Ig Fc region is hIgG1 and the amino acid sequence of the first Fc region and the second Fc region each comprise a proline (or alanine) at amino acid position L234 a proline (or alanine) at amino acid position L235, and / or a glycine at amino acid position P329, numbering according to the EU index of Kabat.

[0032] In some embodiments, the Ig (e.g., hIg) Fc region comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any protein set forth in any one of Tables 4-7 or set forth in any one of SEQ ID NOS: 22-95 or 123-124. In some embodiments, the Ig (e.g., hIg) Fc region comprises an amino acid sequence comprising a set of amino acid variations set forth in any one of Tables 10-12.

[0033] In some embodiments, the first Ig (e.g., hIg) Fc region comprises an amino acidAttorney Docket No.62801.71WO01 sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of the second Ig (e.g., hIg) Fc region.

[0034] In some embodiments, the first protein comprises from N- to C-terminus: the first Ig (e.g., hIg) Fc region and the first protein described herein; and the second protein comprises from N- to C-terminus: the second Ig (e.g., hIg) Fc region and the second protein described herein. In some embodiments, the first protein comprises from N- to C-terminus: the first Ig (e.g., hIg) Fc region, a first peptide linker, and the first protein described herein; and the second protein comprises from N- to C-terminus: the second Ig (e.g., hIg) Fc region, a second peptide linker, and the second protein described herein. In some embodiments, the first protein comprises from N- to C-terminus: the first protein described herein and the first Ig (e.g., hIg) Fc region; and the second protein comprises from N- to C-terminus: the second protein described herein and the second Ig (e.g., hIg) Fc region. In some embodiments, the first protein comprises from N- to C-terminus: the first protein described herein, a first peptide linker, and the first Ig (e.g., hIg) Fc region; and the second protein comprises from N- to C-terminus: the second protein described herein, a second peptide linker, and the second Ig (e.g., hIg) Fc region.

[0035] In some embodiments, the amino acid sequence of the first peptide linker and the second peptide linker each comprises or consists of glycine or glycine and serine amino acid residues. In some embodiments, the amino acid of the first peptide linker and the second peptide linker each comprises or consists of (a) the amino acid sequence set forth in any one of SEQ ID NOS: 96-105; or (b) the amino acid sequence set forth in any one of SEQ ID NOS: 96-105 comprising or consisting of 1, 2, or 3 amino acid substitutions.

[0036] In some embodiments, the amino acid sequence of the first protein is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 106-120 or set forth in Table 9; and the amino acid sequence of the first protein is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 106-120 or set forth in Table 9.

[0037] In some embodiments, the amino acid sequence of the first protein is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ IDAttorney Docket No.62801.71WO01 NOS: 106-120; and the amino acid sequence of the first protein is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 106-120

[0038] In one aspect, provided herein are immunogenic peptides or proteins comprising at least an immunogenic fragment of a protein described herein.

[0039] In some embodiments, the immunogenic peptide or protein does not specifically bind hTNFα or binds one or more of hTNFα with lower affinity relative to a reference protein described herein. In some embodiments, the immunogenic peptide or protein comprises a full-length protein described herein. In some embodiments, the immunogenic peptide or protein comprises an immunogenic fragment of a protein described herein.

[0040] In some embodiments, the immunogenic peptide or protein comprises at least about 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, or 130 amino acids. In some embodiments, the immunogenic peptide or protein comprises from about 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, or 130 amino acids.

[0041] In some embodiments, the amino acid sequence of the immunogenic peptide or protein comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acid variations (e.g., substitutions, additions, deletions) relative to a reference protein described herein.

[0042] In some embodiments, the immunogenic peptide or protein comprises an amino acid sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a contiguous stretch of at least about 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, or 130 amino acids set forth in any one of SEQ ID NOS: 7-16.

[0043] In some embodiments, the immunogenic peptide or protein comprises an amino acid sequence that, other than the one or more amino acid variation (e.g., substitution, addition, deletion), is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 7-16.

[0044] In some embodiments, the immunogenic peptide or protein is formulated with an adjuvant.

[0045] In one aspect, provided herein are isolated antibodies that specifically binds to a protein described herein.

[0046] In one aspect, provided herein are nucleic acid molecules encoding a protein described herein, a conjugate described herein, a radioligand described herein, a fusion protein describedAttorney Docket No.62801.71WO01 herein, an immunogenic peptide or protein described herein, or an antibody described herein.

[0047] In some embodiments, the nucleic acid molecule is an RNA (e.g., mRNA, circular RNA) molecule or a DNA molecule. In some embodiments, the nucleic acid molecule comprises a heterologous 5'-untranslated region (UTR), 3'-UTR, or both a 5'-UTR and 3'-UTR.In some embodiments, the nucleic acid molecule comprises a poly(A) sequence. In some embodiments, the nucleic acid molecule comprises a 5' cap structure. In some embodiments, the nucleic acid molecule comprises at least one variant nucleotide. In some embodiments, the sequence of the nucleic acid molecule is codon optimized.

[0048] In one aspect, provided herein are mRNAs molecules encoding a protein described herein, a conjugate described herein, a radioligand described herein, a fusion protein described herein, an immunogenic peptide or protein described herein, or an antibody described herein.

[0049] In some embodiments, the mRNA molecule comprises a heterologous 5'-untranslated region (UTR), 3'-UTR, or both a 5'-UTR and 3'-UTR.In some embodiments, the nucleic acid molecule comprises a poly(A) sequence. In some embodiments, the mRNA molecule comprises a 5' cap structure. In some embodiments, the mRNA molecule comprises at least one variant nucleotide. In some embodiments, the sequence of the mRNA molecule is codon optimized.

[0050] In one aspect, provided herein are vectors (e.g., expression vectors) comprising a nucleic acid molecule described herein or an mRNA molecule described herein.

[0051] In some embodiments, the vector is a viral vector or a non-viral vector (e.g., a plasmid).

[0052] In one aspect, provided herein are viral particles conjugated to a protein described herein, a conjugate described herein, a radioligand described herein, or a fusion protein described herein.

[0053] In one aspect, provided herein are cells (e.g., host cells) comprising a protein described herein, a conjugate described herein, a radioligand described herein, a fusion protein described herein, an immunogenic peptide or protein described herein, an antibody described herein, a nucleic acid molecule described herein, an mRNA molecule described herein, a vector described herein, a carrier described herein, a lipid nanoparticle described herein, a vaccine composition described herein, or a pharmaceutical composition described herein.

[0054] In one aspect, provided herein are cells (e.g., a therapeutic cells) (e.g., CAR cells) expressing and / or genetically encoding a protein described herein, a conjugate described herein, a radioligand described herein, or a fusion protein described herein (e.g., on the surface of the cell).Attorney Docket No.62801.71WO01

[0055] In one aspect, provided herein are carriers comprising a protein described herein, a conjugate described herein, a radioligand described herein, a fusion protein described herein, an immunogenic peptide or protein described herein, an antibody described herein, a nucleic acid molecule described herein, an mRNA molecule described herein, a vector described herein, a cell described herein, a vaccine composition described herein, or a pharmaceutical composition described herein.

[0056] In one aspect, provided herein are carriers conjugated to a protein described herein, a conjugate described herein, a radioligand described herein, or a fusion protein described herein.

[0057] In some embodiments, the carrier is a lipid nanoparticle, liposome, lipoplex, or nanoliposome.

[0058] In one aspect, provided herein are lipid nanoparticles comprising a protein described herein, a conjugate described herein, a radioligand described herein, a fusion protein described herein, an immunogenic peptide or protein described herein, an antibody described herein, a nucleic acid molecule described herein, an mRNA molecule described herein, a vector described herein, a cell described herein, a vaccine composition described herein, or a pharmaceutical composition described herein.

[0059] In one aspect, provided herein are lipid nanoparticles conjugated to a protein described herein, a conjugate described herein, a radioligand described herein, or a fusion protein described herein.

[0060] In one aspect, provided herein are vaccines composition comprising an immunogenic peptide or protein described herein (or a nucleic acid molecule encoding the same (or a vector encoding the nucleic acid molecule) or a carrier comprising any of the foregoing).

[0061] In one aspect, provided herein are pharmaceutical compositions comprising a protein described herein, a conjugate described herein, a radioligand described herein, a fusion protein described herein, an immunogenic peptide or protein described herein, an antibody described herein, a nucleic acid molecule described herein, an mRNA molecule described herein, a vector described herein, a carrier described herein, a lipid nanoparticle described herein, a vaccine composition described herein, or a cell described herein; and a pharmaceutically acceptable excipient.

[0062] In one aspect, provided herein are kits comprising a protein described herein, a conjugate described herein, a radioligand described herein, a fusion protein described herein, anAttorney Docket No.62801.71WO01 immunogenic peptide or protein described herein, an antibody described herein, a nucleic acid molecule described herein, an mRNA molecule described herein, a vector described herein, a carrier described herein, a lipid nanoparticle described herein, a vaccine composition described herein, a cell described herein, or a pharmaceutical composition described herein; and optionally instructions for using any one or more of the foregoing.

[0063] In one aspect, provided herein are methods of delivering a protein, fusion protein, conjugate, radioligand, nucleic acid molecule, mRNA molecule, expression vector, cell, carrier, lipid nanoparticle, immunogenic peptide or protein, an antibody, a vaccine composition, or pharmaceutical composition to a subject, the method comprising administering to the subject a protein described herein, a conjugate described herein, a radioligand described herein, a fusion protein described herein, an immunogenic peptide or protein described herein, an antibody described herein, a nucleic acid molecule described herein, an mRNA molecule described herein, a vector described herein, a carrier described herein, a lipid nanoparticle described herein, a vaccine composition described herein, a cell described herein, or a pharmaceutical composition described herein, to thereby deliver the protein, fusion protein, conjugate, radioligand, immunogenic peptide or protein, antibody, mRNA molecule, vector, cell, carrier, lipid nanoparticle, vaccine composition, or pharmaceutical composition to a subject.

[0064] In one aspect, provided herein are methods of inhibiting or reducing (e.g., preventing) binding of TNFα to TNFR1 and / or TNFR2 in a subject in need thereof, the method comprising administering to the subject a protein described herein, a conjugate described herein, a radioligand described herein, a fusion protein described herein, an immunogenic peptide or protein described herein, an antibody described herein, a nucleic acid molecule described herein, an mRNA molecule described herein, a vector described herein, a carrier described herein, a lipid nanoparticle described herein, a vaccine composition described herein, a cell described herein, or a pharmaceutical composition described herein, to thereby inhibit or reduce (e.g., prevent) binding of TNFα to TNFR1 and / or TNFR2 in the subject.

[0065] In one aspect, provided herein are methods of inhibiting reducing (e.g., preventing) signaling mediated by the binding of TNFα to TNFR1 and / or TNFR2 in a subject in need thereof, the method comprising administering to the subject a protein described herein, a conjugate described herein, a radioligand described herein, a fusion protein described herein, an immunogenic peptide or protein described herein, an antibody described herein, a nucleic acidAttorney Docket No.62801.71WO01 molecule described herein, an mRNA molecule described herein, a vector described herein, a carrier described herein, a lipid nanoparticle described herein, a vaccine composition described herein, a cell described herein, or a pharmaceutical composition described herein, to thereby inhibit or reduce (e.g., prevent) signaling mediated by the binding of TNFα to TNFR1 and / or TNFR2 in the subject.

[0066] In one aspect, provided herein are methods of suppressing or preventing a pro- inflammatory immune response in a subject in need thereof, the method comprising administering to the subject a protein described herein, a conjugate described herein, a radioligand described herein, a fusion protein described herein, an immunogenic peptide or protein described herein, an antibody described herein, a nucleic acid molecule described herein, an mRNA molecule described herein, a vector described herein, a carrier described herein, a lipid nanoparticle described herein, a vaccine composition described herein, a cell described herein, or a pharmaceutical composition described herein, to thereby suppress or prevent a pro-inflammatory immune response in the subject.

[0067] In one aspect, provided herein are methods of inducing or enhancing an immune response in a subject in need thereof, the method comprising administering to the subject (i) an immunogenic peptide or protein described herein (or a conjugate or a fusion protein thereof); (ii) a nucleic acid molecule encoding (i); (iii) a vector comprising (ii); (iv) a carrier comprising (i), (ii), or (iii); a vaccine composition comprising (i), (ii), (iii), or (iv); or a pharmaceutical composition comprising (i), (ii), (iii), (iv), or (v), to thereby induce or enhance an immune response in the subject.

[0068] In one aspect, provided herein are methods of vaccinating a subject in need thereof (e.g., against a viral infection), the method comprising administering to the subject (i) an immunogenic peptide or protein described herein (or a conjugate or a fusion protein thereof); (ii) a nucleic acid molecule encoding (i); (iii) a vector comprising (ii); (iv) a carrier comprising (i), (ii), or (iii); a vaccine composition comprising (i), (ii), (iii), or (iv); or a pharmaceutical composition comprising (i), (ii), (iii), (iv), or (v), to thereby vaccinate the subject in need thereof (e.g., against a virus).

[0069] In one aspect, provided herein are methods of determining the presence of a virus in a subject, the method comprising (a) obtaining the sample from a subject or providing a sample that has been obtained from a subject, and (b) determining the presence or absence of a proteinAttorney Docket No.62801.71WO01 described herein (or a fragment or variant thereof) or a nucleic acid molecule encoding a protein described herein (or the fragment or variant thereof) in the sample. In some embodiments, the method is an in vitro method. In some embodiments, the sample is a blood, cell, tissue, or saliva, or nasal swab. In some embodiments, an antibody described herein is utilized to determine the presence or absence of a protein described herein (or the fragment or variant thereof).

[0070] In one aspect, provided herein are methods of diagnosing a viral infection in a subject, the method comprising (a) obtaining a sample from a subject or providing a sample that has been obtained from a subject, (b) determining the presence or absence of a protein described herein (or a fragment or variant thereof) or a nucleic acid molecule encoding a protein described herein (or a fragment or variant thereof), and (c) diagnosing the subject as having the viral infection if a protein described herein (or a fragment or variant thereof) or a nucleic acid molecule encoding a protein described herein (or the fragment or variant thereof) is determined to be present in the sample in step (b). In some embodiments, the method is an in vitro method. In some embodiments, the sample is a blood, cell, tissue, or saliva, or nasal swab. In some embodiments, an antibody described herein is utilized to determine the presence or absence of a protein described herein (or the fragment or variant thereof).

[0071] In one aspect, provided herein are methods of treating a viral infection in a subject, the method comprising (a) receiving testing results that determined the presence of a protein described herein (or a fragment or variant thereof) or a nucleic acid molecule encoding a protein described herein (or the fragment or variant thereof) in a sample from the subject, (b) diagnosing the subject as having the viral infection, and (c) administering a therapeutic agent to treat the viral infection. In some embodiments, the sample is a blood, cell, tissue, or saliva, or nasal swab. In some embodiments, an antibody described herein is utilized to determine the presence or absence of a protein described herein (or the fragment or variant thereof).

[0072] In some embodiments, the subject is a human. 4. BRIEF DESCRIPTION OF THE FIGURES

[0073] FIG 1. is a graph showing the % binding ranges of 149 proteins (identified as being less than 110 amino acids in length) (including IIPs1-5) to hTNF (as determined by Luminex assay as described in Example 1). The graph is a representative of one experiment executed in duplicates. Controls included a reference anti-TNF antibody, a reference TNFRR2 extracellular domainAttorney Docket No.62801.71WO01 (ECD) Ig Fc fusion protein, a reference TL1A binding protein, IgG4 Ig Fc control, a secondary antibody control, and a mock control.

[0074] FIG 2. is a graph showing the % binding ranges of 149 proteins (identified as being less than 110 amino acids in length) (including IIPs1-5) to hLTα (as determined by Luminex assay as described in Example 1). The graph is a representative of one experiment executed in duplicates. Controls included a reference anti-TNF antibody, a reference TNFRR2 extracellular domain (ECD) Ig Fc fusion protein, a reference TL1A binding protein, IgG4 Ig Fc control, a secondary antibody control, and a mock control.

[0075] FIG. 3 is a line graph showing the percent binding of each indicated agent or control (IFP-1, IFP-2, IFP-3, IFP-4, IFP-5, reference anti-TNF antibody, a reference TNFRR2 extracellular domain (ECD) Ig Fc fusion protein (FP), a reference anti-LTα antibody, a reference TL1A binding protein, IgG4 Fc control, and mock control) to TNFα (as determined by ELISA according to Example 2).

[0076] FIG. 4 is a line graph showing the percent inhibition of TNFα mediated TNFR1 / 2 induced NFκB signaling by each indicated agent or control (IFP-1, IFP-2, IFP-3, IFP-4, IFP-5, reference anti-TNF antibody, a reference TNFRR2 extracellular domain (ECD) Ig Fc fusion protein, a reference TL1A binding protein, IgG4 Fc control, and mock control) (as determined according to Example 3).

[0077] FIG. 5 is a line graph showing the percent binding of each indicated agent or control (IFP-1, IFP-2, IFP-3, IFP-4, IFP-5, reference anti-TNF antibody, a reference TNFRR2 extracellular domain (ECD) Ig Fc fusion protein, a reference anti-LTα antibody, a reference TL1A binding protein, IgG4 Fc control, and mock control) to LTα (as determined by ELISA according to Example 2).

[0078] FIG. 6 is a line graph showing the percent inhibition of LTα mediated TNFR1 / 2 induced NFκB signaling by each indicated agent or control (IFP-1, IFP-2, IFP-3, IFP-4, IFP-5, reference anti-TNF antibody, a reference TNFRR2 extracellular domain (ECD) Ig Fc fusion protein, a reference anti-LTα antibody, a reference TL1A binding protein, IgG4 Fc control, and mock control) (as determined according to Example 3).

[0079] FIG. 7 is a line graph showing the percent inhibition of LTα mediated TNFR1 / 2 induced NFκB signaling by each indicated agent or control (IFP-1, IFP-2, IFP-3, IFP-4, IFP-5, reference anti-TNF antibody, a reference TNFRR2 extracellular domain (ECD) Ig Fc fusionAttorney Docket No.62801.71WO01 protein, a reference anti-LTα antibody, a reference TL1A binding protein, IgG4 Fc control, and mock control) (as determined according to Example 3 utilizing 1ng / mL LTα).

[0080] FIG. 8 is a line graph showing is a line graph showing the percent inhibition of LTα mediated TNFR1 / 2 induced NFκB signaling by each indicated agent or control (IFP-1, IFP-2, IFP- 3, IFP-4, IFP-5, reference anti-TNF antibody, a reference TNFRR2 extracellular domain (ECD) Ig Fc fusion protein, a reference anti-LTα antibody, a reference TL1A binding protein, IgG4 Fc control, and mock control) to TNFα (as determined according to Example 3 utilizing 10ng / mL LTα).

[0081] FIG.9A is a line graph showing the clinical disease score (as described in Example 4) at the indicated study day and indicated treatment group (IFP-1, hIgG4 Fc control, no disease control) in the CAIA mouse model of rheumatoid arthritis. FIG. 9B is a bar graph showing the clinical disease score area under the curve (as described in Example 4) at the indicated study day and indicated treatment group (IFP-1, hIgG4 Fc control) in the CAIA mouse model of rheumatoid arthritis.

[0082] FIG. 10A is a bar graph showing the level of IL-1β expression (pg IL-1β / mg of total protein) in the inflamed joints of CAIA mice at study day 14 in the indicated treatment group (IFP- 1 or hIgG4 Fc control). FIG.10B is a bar graph showing the level of IL-6 expression (pg IL-6 / mg of total protein) in the inflamed joints of CAIA mice at study day 14 in the indicated treatment group (IFP-1 or hIgG4 Fc control). FIG. 10C is a bar graph showing the level of KC / GRO expression (pg KC / GRO / mg of total protein) in the inflamed joints of CAIA mice at study day 14 in the indicated treatment group (IFP-1 or hIgG4 Fc control). FIG.10D is a bar graph showing the level of TNF expression (pg TNF / mg of total protein) in the inflamed joints of CAIA mice at study day 14 in the indicated treatment group (IFP-1 or hIgG4 Fc control).

[0083] FIG. 11A is a bar graph showing the level of IL-1β expression (pg IL-1β / mg of total protein) in the serum joints of CAIA mice at study day 14 in the indicated treatment group (IFP-1 or hIgG4 Fc control). FIG. 11B is a bar graph showing the level of IL-6 expression (pg IL-6 / mg of total protein) in the serum joints of CAIA mice at study day 14 in the indicated treatment group (IFP-1 or hIgG4 Fc control). FIG. 11C is a bar graph showing the level of KC / GRO expression (pg KC / GRO / mg of total protein) in the serum joints of CAIA mice at study day 14 in the indicated treatment group (IFP-1 or hIgG4 Fc control). FIG. 11D is a bar graph showing the level of TNF expression (pg TNF / mg of total protein) in the serum joints of CAIA mice at study day 14 in theAttorney Docket No.62801.71WO01 indicated treatment group (IFP-1 or hIgG4 Fc control).

[0084] FIG. 12 is a line graph showing the concentration (ng / mL) of IFP-1 in the serum of mice at the indicated number of days post intravenous administration of 3mg / kg IFP-1.

[0085] FIG. 13 is a bar graph showing the serum level of ICAM-1 (U / mL) in mice pre- administration and 24 hours post administration of an isotype control or IFP-1. 5. DETAILED DESCRIPTION

[0086] The inventors have, inter alia, identified and developed immunoreceptor inhibitory proteins that specifically bind to TNFSF ligand TNFα. Accordingly, the novel immunoreceptor inhibitory proteins disclosed herein may be useful for various methods, including, e.g., selectively inhibiting binding of TNFα to TNFR1 and / or TNFR2, inhibiting signaling of TNFR1 and / or TNFR2 (including, e.g., signaling mediated through the binding of TNFR1 and / or TNFR2 to TNFα), and modulating (e.g., suppressing) an immune response, as well as in diagnostic assays. As such, the current disclosure provides, inter alia, novel immunoreceptor inhibitory proteins, nucleic acid molecules encoding, the methods for utilizing the same. TABLE OF CONTENTS 5.1 Definitions 5.2 Immunoreceptor Inhibitory Proteins 5.3 Exemplary Properties of Immunoreceptor Inhibitory Proteins 5.4 Immunoreceptor Inhibitory Protein Fusions & Conjugates 5.4.1 Radioligands 5.4.2 Chimeric Antigen Receptors 5.4.3 Signal Peptides 5.4.4 Half-Life Extension Moieties 5.4.5 Ig Fusion Proteins 5.4.5.1 Antibody Fusion Proteins 5.4.5.2 Ig Fusion Proteins 5.4.5.3 Half-Life Extension 5.4.5.4 Ig Fc Effector Function 5.4.5.4(i) Reduced Effector FunctionAttorney Docket No.62801.71WO01 ins s ble Lipids s ntsAttorney Docket No.62801.71WO01 5.13 Pharmaceutical Compositions 5.14 Methods of Use 5.14.1 Methods of Delivery 5.14.2 Methods of Inhibiting or Reducing Binding of TNFα to TNFR1 5.14.3 Methods of Inhibiting or Reducing Signaling Mediated by TNFα Binding to TNFR1 5.14.4 Methods of Inhibiting or Reducing Binding of TNFα to TNFR2 5.14.5 Methods of Inhibiting or Reducing Signaling Mediated by TNFα Binding to TNFR2 5.14.6 Methods of Inhibiting or Reducing Binding of TNFα to TNFR1 and TNFR2 5.14.7 Methods of Inhibiting or Reducing Signaling Mediated by TNFα Binding to TNFR1 and TNFR2 5.14.8 Methods of Inhibiting or Reducing Binding of LTα to TNFR1 5.14.9 Methods of Inhibiting or Reducing Signaling Mediated by LTα Binding to TNFR1 5.14.10 Methods of Inhibiting or Reducing Binding of LTα to TNFR2 5.14.11 Methods of Inhibiting or Reducing Signaling Mediated by LTα Binding to TNFR2 5.14.12 Methods of Inhibiting or Reducing Binding of LTα to TNFR1 and TNFR2 5.14.13 Methods of Inhibiting or Reducing Signaling Mediated by LTα Binding to TNFR1 and TNFR2 5.14.14 Methods of Suppressing or Preventing a Pro-Inflammatory Immune Response 5.14.15 Methods of Inducing or Enhancing an Immune Response 5.14.16 Methods of Vaccinating a Subject 5.14.17 Diagnostic Methods 5.15 Kits 5.1 Definitions

[0087] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.Attorney Docket No.62801.71WO01

[0088] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the claimed subject matter belongs. It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of any subject matter claimed.

[0089] Use of the singular herein includes the plural unless specifically stated otherwise. For example, as used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Furthermore, use of the term “including” as well as other forms, such as “include,” “includes,” and “included,” is not limiting.

[0090] It is understood that wherever aspects are described herein with the language “comprising,” otherwise analogous aspects described in terms of “consisting of” and “consisting essentially of” are also provided.

[0091] The term “and / or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and / or” as used in a phrase such as “A and / or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Likewise, the term “and / or” as used in a phrase such as “A, B, and / or C” is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0092] As described herein, any concentration range, percentage range, ratio range or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated.

[0093] The term “about” refers to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. When particular values or compositions are provided herein, unless otherwise stated, the meaning of “about” should be assumed to be within an acceptable error range for that particular value or composition.

[0094] Where proteins and / or polypeptides are described herein, it is understood that nucleic acid molecules (e.g., RNA (e.g., mRNA) or DNA molecules) encoding the protein are also provided herein.Attorney Docket No.62801.71WO01

[0095] Where proteins, peptides, nucleic acid molecules, vectors, carriers, etc. are described herein, it is understood that isolated forms of the proteins, peptides, nucleic acid molecules, vectors, carriers, etc. are also provided herein.

[0096] Where proteins, peptides, nucleic acid molecules, etc. are described herein, it is understood that recombinant forms of the proteins, peptides, nucleic acid molecules, etc. are also provided herein.

[0097] Where polypeptides or sets of polypeptides are described herein, it is understood that proteins comprising the polypeptides or sets of polypeptides folded into their three-dimensional structure (i.e., tertiary or quaternary structure) are also provided herein and vice versa.

[0098] As used herein, the term “adjuvant” refers to a substance that causes stimulation of the immune system of a subject when administered to the subject.

[0099] As used herein, the term “administering” refers to the physical introduction of an agent, e.g., a therapeutic agent (or a precursor of the therapeutic agent that is metabolized or altered within the body of the subject to produce the therapeutic agent in vivo) or vaccine to a subject, using any of the various methods and delivery systems known to those skilled in the art. Administering can also be performed, for example, once, a plurality of times, and / or over one or more extended periods.

[0100] As used herein, the term “affinity” refers to the strength of the binding of one protein (e.g., a Ligand) to another protein (e.g., a Receptor). The affinity of a protein is measured by the dissociation constant Kd, defined as [Ligand] x [Receptor] / [Ligand-Receptor] where [Ligand- Receptor] is the molar concentration of the Ligand-Receptor complex, [Ligand] is the molar concentration of the unbound Ligand and [Receptor] is the molar concentration of the unbound Receptor. The affinity constant Ka is defined by 1 / Kd. Standard methods of measuring affinity are known to the person of ordinary skill in the art and described herein, see, e.g., § 5.3.

[0101] As used herein, the term “agent” is used generically to describe any macro or micro molecule. Exemplary agents include, but are not limited to, proteins, peptides, nucleic acid molecules (e.g., DNA molecules, RNA molecules), vectors, carriers, carbohydrates, lipids, synthetic polymers, etc.

[0102] As used herein, the term “antibody” or “antibodies” is used in the broadest sense and encompasses various immunoglobulin (Ig) (e.g., human Ig (hIg), murine Ig (mIg)) structures, including, but not limited to monoclonal antibodies, polyclonal antibodies, multispecific (e.g.,Attorney Docket No.62801.71WO01 bispecific, trispecific) antibodies, and antibody fragments so long as they exhibit the desired antigen-binding activity (i.e., antigen binding fragments or variants). The term antibody thus includes, for example, full-length antibodies; antigen-binding fragments of full-length antibodies; molecules comprising antibody CDRs, VH regions, and / or VL regions; and antibody-like scaffolds (e.g., fibronectins). Examples of antibodies include, without limitation, monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, camelized antibodies, intrabodies, affybodies, diabodies, tribodies, heteroconjugate antibodies, antibody-drug conjugates, single domain antibodies (e.g.,VHH, (VHH)2), single chain antibodies, single-chain Fvs (scFv; (scFv)2), Fab fragments (e.g., Fab, single chain Fab (scFab), F(ab’)2fragments, disulfide-linked Fvs (sdFv), Fc fusions (e.g., Fab-Fc, scFv-Fc, VHH-Fc, (scFv)2-Fc, (VHH)2-Fc), and antigen-binding fragments of any of the above, and conjugates or fusion proteins comprising any of the above. Antibodies can be of Ig isotype (e.g., IgG, IgE, IgM, IgD, or IgA), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1or IgA2), or any subclass (e.g., IgG2aor IgG2b) of Ig). In certain embodiments, antibodies described herein are IgG antibodies, or a class (e.g., human IgG1 or IgG4) or subclass thereof. In certain embodiments, antibodies described herein are mIgG antibodies, or a class (e.g., mIgG1 or mIgG2a) or subclass thereof. In some embodiments, the antibody is a human, humanized, or chimeric IgG1 or IgG4 monoclonal antibody. In some embodiments, the term antibodies refers to a monoclonal or polyclonal antibody population. Antibodies described herein can be produced by any standard methods known in the art, e.g., recombinant production in host cells, see, e.g., § 5.6; or synthetic production.

[0103] As used herein, the term “antibody mimetic” refers to non-Ig based antigen binding domain. Various antibody-like scaffolds are known in the art. For example, 10th type III domain of fibronectin (e.g., AdNectins®) and designed ankyrin repeat proteins (e.g., DARPins®) have been used as alternative scaffolds for antigen-binding domains, see, e.g., Gebauer and Skerra, Engineered protein scaffolds as next-generation antibody therapeutics. Curr Opin Chem Biol 13:245-255 (2009) and Stumpp et al., Darpins: A new generation of protein therapeutics. Drug Discovery Today 13: 695-701 (2008), the full contents of each of which is incorporated by reference herein for all purposes. Exemplary antibody-like scaffolds include, but are not limited to, lipocalins (see, e.g., US7250297) (e.g., Anticalin®), protein A-derived molecules such as z- domains of protein a (see, e.g., US5831012) (e.g., Affibody®), A domains of membrane receptorsAttorney Docket No.62801.71WO01 stabilized by disulfide bonds and Ca2+ (see, e.g., US7803907) (e.g., Avimer / Maxibody®), a serum transferrin (see, e.g., US2004023334) (e.g., Transbody®); a designed ankyrin repeat protein (see, e.g., US7417130) (e.g., DARPin®), a fibronectin (see, e.g., US6818418) (e.g., AdNectin®), a C- type lectin domain (see, e.g., US2004132094) (e.g., Tetranectin®); a human gamma-crystallin or ubiquitin (see, e.g., US7838629) (e.g., Affilin®); a kunitz type domain of human protease inhibitors (see, e.g., US2004209243), C-Type Lectins (see, e.g., US2004132094) (e.g., Tetranectins®), cysteine knots or knottins (see, e.g., US7186524) (e.g., Microbodies®), nucleic acid aptamers (see, e.g., US5475096), thioredoxin A scaffold (see, e.g., US6004746) (peptide aptamers), and 10th type III domain of fibronectin (see, e.g., US6818418) (e.g., AdNectins®), and cystine-dense peptides (see, e.g., WO2023023031). Additional exemplary antibody-like scaffolds are known in the art and for example described in Storz U. Intellectual property protection: strategies for antibody inventions. MAbs. 2011;3(3):310-317. doi:10.4161 / mabs.3.3.15530. The entire contents of each of the foregoing references is incorporated herein by reference for all purposes. Antibody like scaffolds include e.g., naturally occurring antigen binders, variant (e.g., functional variants) of naturally occurring antigen binders, fragments (e.g., functional fragments) of naturally occurring antigen binders, and synthetic antigen binders (i.e., not naturally occurring antigen binders).

[0104] The terms “CH1” and “CH1 region” are used interchangeably herein and refer to the first constant region of an immunoglobulin heavy chain. The amino acid sequence of an exemplary reference hIgG1 CH1 region is set forth in SEQ ID NO: 90; and the amino acid sequence of an exemplary reference hIgG4 CH1 region is set forth in SEQ ID NO: 103.

[0105] The terms “CH2” and “CH2 region” are used interchangeably herein and refer to the second constant region of an immunoglobulin heavy chain. The amino acid sequence of an exemplary reference hIgG1 CH2 region is set forth in SEQ ID NO: 92; and the amino acid sequence of an exemplary reference hIgG4 CH2 region is set forth in SEQ ID NO: 106.

[0106] The terms “CH3” and “CH3 region” are used interchangeably herein and refer to the third constant region of an immunoglobulin heavy chain. The amino acid sequence of an exemplary reference hIgG1 CH3 region is set forth in SEQ ID NO: 93; and the amino acid sequence of an exemplary reference hIgG4 CH3 region is set forth in SEQ ID NO: 107.

[0107] As used herein, the term “chimeric antigen receptor” or “CAR” refers to a recombinant polypeptide construct comprising at least an extracellular antigen-binding domain (e.g.,Attorney Docket No.62801.71WO01 comprising a immunoreceptor inhibitory protein described herein), a transmembrane domain, and an intracellular signaling domain comprising one or more functional signaling domains derived from a stimulatory molecule. In some embodiments, the domains in the CAR polypeptide construct are in the same polypeptide chain. In some embodiments, the domains in the CAR polypeptide construct are not contiguous with each other, for example, are in different polypeptide chains.

[0108] As used herein, the term “circular RNA” refers to a translatable RNA molecule that forms a circular structure through covalent or non-covalent bonds. In some embodiments, the circular RNA is covalently closed.

[0109] As used herein, the term “conjugation” refers to chemical conjugation of a protein with a moiety (e.g., small molecule, polypeptide, nucleic acid molecule, carbohydrate, lipid, synthetic polymer (e.g., polymers of polyethylene glycol (PEG)), etc.). The moiety can be directly connected to the protein or indirectly connected through a linker, e.g., as described herein. Chemical conjugation methods are well known in the art, as are commercially available conjugation reagents and kits, with detailed instructions for their use readily available from the commercial suppliers.

[0110] As used herein, the term “derived from,” with reference to a nucleic acid molecule refers to a nucleic acid molecule that has at least 70% sequence identity to a reference nucleic acid molecule (e.g., a naturally occurring nucleic acid molecule) or a fragment thereof. The term “derived from,” with reference to a protein refers to a protein that comprises an amino acid sequence that has at least 70% sequence identity to the amino acid sequence of a reference protein (e.g., a naturally occurring protein). The term “derived from” as used herein does not denote any specific process or method for obtaining the nucleic acid molecule, polypeptide, or protein. For example, the nucleic acid molecule, polypeptide, or protein can be recombinantly produced or chemically synthesized.

[0111] As used herein, the term “diagnosing” or “diagnosis” refers to a determination of the presence, absence, severity, or course of treatment of a disease (e.g., an infection, e.g., a viral infection). The term “diagnosing” encompasses an initial determination as well as subsequent determinations (e.g., monitoring) after the initial determination.

[0112] As used herein, the term “disease” refers to any abnormal condition that impairs physiological function. The term is used broadly to encompass any disorder, illness, abnormality, pathology, sickness, condition, or syndrome in which physiological function is impaired, irrespective of the nature of the etiology.Attorney Docket No.62801.71WO01

[0113] The terms “DNA” and “polydeoxyribonucleotide” are used interchangeably herein and refer to macromolecules that include multiple deoxyribonucleotides that are polymerized via phosphodiester bonds. Deoxyribonucleotides are nucleotides in which the sugar is deoxyribose.

[0114] The term “effector function” when used in reference to an antibody refers to those biological activities attributable to the Fc region of an antibody, which therefore vary with the antibody isotype. Antibody effector functions include, but are not limited to, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement dependent cytotoxicity (CDC), Fc receptor binding (e.g., FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and / or FcγRIIIb (e.g., FcγRI, FcγIIa, and / or FcγIIIa)), and Clq binding.

[0115] As used herein, the term “Fc region” refers to the C-terminal region of an Ig heavy chain that comprises from N- to C-terminus at least a CH2 region operably connected to a CH3 region. In some embodiments, the Fc region comprises an Ig hinge region or at least a portion of an Ig hinge region operably connected to the N-terminus of the CH2 region. In some embodiments, the Fc region is engineered relative to a reference Fc region, see, e.g., §§ 5.4.5.3, 5.4.5.4(i), 5.4.5.4(ii). Additional examples of proteins with engineered Fc regions can be found in Saunders 2019 (K. O. Saunders, “Conceptual Approaches to Modulating Antibody Effector Functions and Circulation Half-Life,” 2019, Frontiers in Immunology, V. 10, Art. 1296, pp. 1-20, the entire contents of which is incorporated by reference herein for all purposes).

[0116] The term “functional variant” as used herein in reference to a protein refers to a protein that comprises at least one but no more than 15%, not more than 12%, no more than 10%, no more than 8% amino acid variation (e.g., substitution, deletion, addition) compared to the amino acid sequence of a reference protein, wherein the protein retains at least one particular function of the reference protein. Not all functions of the reference protein (e.g., wild type) need be retained by the functional variant of the protein. In some instances, one or more functions are selectively reduced or eliminated. In some embodiments, the reference protein is a wild type protein. For example, a functional variant of a TNF superfamily ligand binding protein can refer to a TNF superfamily ligand binding protein comprising one or more amino acid substitution as compared to a reference TNF superfamily ligand binding protein (e.g., a wild type protein) that retains the ability to specifically bind the TNF superfamily ligand.

[0117] The term “functional fragment” as used herein in reference to a protein refers to a fragment of a reference protein that retains at least one particular function. Not all functions of theAttorney Docket No.62801.71WO01 reference protein need be retained by a functional fragment of the protein. In some instances, one or more functions are selectively reduced or eliminated. In some embodiments, the reference protein is a wild type protein. For example, a functional fragment of a TNF superfamily ligand binding protein can refer to a fragment of a TNF superfamily ligand binding protein that retains the ability to specifically bind the TNF superfamily ligand.

[0118] As used herein, the term “fuse” and grammatical equivalents thereof refer to the operable connection of at least a first polypeptide to a second polypeptide, wherein the first and second polypeptides are not naturally found operably connected together. For example, the first and second polypeptides are derived from different proteins. The term fuse encompasses both a direct connection of the at least two polypeptides through a peptide bond, and the indirect connection through a linker (e.g., a peptide linker).

[0119] As used herein, the term “fusion protein” and grammatical equivalents thereof refers to a protein that comprises at least one polypeptide operably connected to another polypeptide, wherein the first and second polypeptides are not naturally found operably connected together. For example, the first and second polypeptides of the fusion protein are each derived from different proteins. The at least two polypeptides of the fusion protein can be directly operably connected through a peptide bond; or can be indirectly operably connected through a linker (e.g., a peptide linker). Therefore, for example, the term fusion polypeptide encompasses embodiments, wherein Polypeptide A is directly operably connected to Polypeptide B through a peptide bond (Polypeptide A – Polypeptide B), and embodiments, wherein Polypeptide A is operably connected to Polypeptide B through a peptide linker (Polypeptide A – peptide linker – Polypeptide B).

[0120] As used herein, the term “half-life extension moiety” refers to a moiety (e.g., small molecule, polypeptide, nucleic acid molecule, carbohydrate, lipid, synthetic polymer (e.g., polymers of PEG), etc.) that when conjugated or otherwise operably connected (e.g., fused) to a protein (the subject protein), increases the half-life of the subject protein in vivo when administered to a subject (e.g., a human subject). The pharmacokinetic properties of the protein can be evaluated utilizing in vivo models known in the art.

[0121] As used herein, the term “half-life extension polypeptide” or “half-life extension protein” refers to a protein that when operably connected to another protein (the subject protein), increases the half-life of the subject protein in vivo when administered to a subject (e.g., a human subject). The pharmacokinetic properties of the protein can be evaluated utilizing in vivo modelsAttorney Docket No.62801.71WO01 known in the art.

[0122] As used herein, the term “heterologous”, when used to describe a first element in reference to a second element means that the first element and second element do not exist in nature disposed as described. For example, a polypeptide comprising a “heterologous moiety” means a polypeptide that is joined to a moiety (e.g., small molecule, polypeptide, nucleic acid molecule, carbohydrate, lipid, synthetic polymer (e.g., polymers of PEG), etc.) that is not joined to the polypeptide in nature. In one embodiment, the heterologous moiety is not derived from a protein comprising or consisting of the amino acid sequence set forth in any one of SEQ ID NOS: 7-16. For example, a non-limiting example of a heterologous moiety is a heterologous polypeptide (as defined herein). In one embodiment, the heterologous polypeptide is a polypeptide derived from a protein other than a protein comprising or consisting of the amino acid sequence set forth in any one of SEQ ID NOS: 7-16. For example, a non-limiting example of a heterologous polypeptide, as described herein, is a human Ig Fc region.

[0123] As used, herein the term “heterologous signal peptide” refers to a signal peptide that is not operably connected to a subject protein in nature. For example, in reference to a polypeptide comprising a signal peptide from human IL-2 operably connected to human IL-12, the human IL- 2 signal peptide would constitute a heterologous signal peptide. The terms “signal peptide” and “signal sequence” are used interchangeably herein.

[0124] The terms “hinge” or “hinge region” are used interchangeably herein and refer to the hinge region of an immunoglobulin heavy chain. The amino acid sequence of an exemplary reference hIgG1 hinge region is set forth in SEQ ID NO: 91; and the amino acid sequence of an exemplary reference hIgG4 hinge region is set forth in SEQ ID NO: 104.

[0125] As used herein, the term “homologous signal peptide” refers to a signal peptide that is operably connected to a subject protein in nature. For example, in reference to a polypeptide comprising a signal peptide from human IL-2 operably connected to human IL-2, the human IL-2 signal peptide would constitute a homologous signal peptide.

[0126] As used herein, the term “immunogen” refers to a substance that is capable of inducing an immune response (e.g., an adaptive immune response) in a subject (e.g., a human subject). An immunogen may have one or more isoforms, sequence variants, or splice variants that have equivalent biological and immunological activity, and are thus also considered for the purposes of this disclosure to be immunogenic equivalents of the immunogen.Attorney Docket No.62801.71WO01

[0127] As used herein, the term “immunogenic peptide or protein” refers to a peptide or protein that comprises an immunogen.

[0128] As used herein, the term “immunoreceptor inhibitory protein” refers to a protein (e.g., a protein described herein) that inhibits (e.g., partially, fully) a function of one or more immune receptor (e.g., one or more of TNFSF receptor). Exemplary functions include binding to a cognate ligand (e.g., one or more cognate TNFSF ligand), signaling (e.g., signaling induced by binding to a cognate ligand), etc.

[0129] As used herein, the term “in combination with” means that two (or more) different agents or treatments are administered to a subject as part of a defined treatment regimen for a particular disease or condition. The treatment regimen defines the doses and periodicity of administration of each agent such that the effects of the separate agents on the subject overlap. In some embodiments, the delivery of the two or more agents is simultaneous or concurrent and the agents may be co-formulated. In other embodiments, the two or more agents are not co-formulated and are administered in a sequential manner as part of a prescribed. In some embodiments, administration of two or more agents or treatments in combination is such that the reduction in a symptom, or other parameter related to the condition is greater than what would be observed with one agent or treatment delivered alone or in the absence of the other. The effect of the two treatments can be partially additive, wholly additive, or greater than additive (e.g., synergistic). Sequential or substantially simultaneous administration of each therapeutic agent can be effected by any appropriate route including, but not limited to, oral routes, intravenous routes, and intramuscular routes. The therapeutic agents can be administered by the same route or by different routes.

[0130] As used herein, the term “isolated” with reference to a polypeptide, protein, or nucleic acid molecule refers to a polypeptide, protein, or nucleic acid molecule that is substantially free of other cellular components with which it is associated in the natural state.

[0131] As used herein the term “LTα” or “Lymphotoxin α” or “Tumor Necrosis Factor Ligand Superfamily Member 1” refers to the multifunctional immunomodulatory cytokine of the TNFSF. The amino acid sequence of an exemplary reference immature form of human LTα (hTNFα) protein is set forth in SEQ ID NO: 121 and the amino acid sequence of an exemplary mature form of hLTα protein is set forth in SEQ ID NO: 122.

[0132] As used herein, the term “moiety” is used generically to describe any macro or microAttorney Docket No.62801.71WO01 molecule that can be operably connected to a protein described herein. Exemplary moieties include, but are not limited to small molecules, polypeptides, nucleic acid molecules (e.g., DNA, RNA), carbohydrates, lipids, synthetic polymers (e.g., polymers of PEG).

[0133] As used herein, the term “modified nucleotide,” “nucleotide modification,” or use of the term “modification” and the like in reference to a nucleotide or nucleic acid sequence refers to a nucleotide comprising a chemical modification, e.g., a modified sugar moiety, a modified nucleobase, and / or a modified internucleoside linkage, or any combination thereof. Exemplary modifications are provided herein, see, e.g., § 5.5.4.2. In certain embodiments of the instant disclosure, inclusion of a deoxynucleotide - which is acknowledged as a naturally occurring form of nucleotide - if present within an RNA molecule is considered to constitute a modified nucleotide.

[0134] As used herein, the term “obtaining a sample” refers to the acquisition of a sample. The term includes the direct acquisition from a subject and the indirect acquisition through one or more third parties wherein one of the third parties directly acquired the sample from the subject.

[0135] As used herein, the term “operably connected” refers to the linkage of two moieties in a functional relationship. For example, a polypeptide is operably connected to another polypeptide when they are linked (either directly or indirectly via a peptide linker) in frame such that both polypeptides are functional (e.g., a fusion protein described herein). Or for example, a transcription regulatory nucleic acid molecule e.g., a promoter, enhancer, or other expression control element is operably linked to a nucleic acid molecule that encodes a protein if it affects the transcription of the nucleic acid molecule that encodes the protein. The term “operably connected” can also refer to the conjugation of a moiety to e.g., a nucleic acid molecule or polypeptide (e.g., the conjugation of a PEG polymer to a protein).

[0136] The determination of “percent identity” between two sequences (e.g., peptide or protein (amino acid sequences) or polynucleotide (nucleic acid sequences)) can be accomplished using a mathematical algorithm. A specific, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin S & Altschul SF (1990) PNAS 87: 2264-2268, modified as in Karlin S & Altschul SF (1993) PNAS 90: 5873-5877, each of which is herein incorporated by reference in its entirety. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul SF et al., (1990) J Mol Biol 215: 403, which is herein incorporated by reference in its entirety. BLAST nucleotide searches can be performed withAttorney Docket No.62801.71WO01 the NBLAST nucleotide program parameters set, e.g., for score=100, wordlength=12 to obtain nucleotide sequences homologous to a nucleic acid molecule described herein. BLAST protein searches can be performed with the XBLAST program parameters set, e.g., to score 50, wordlength=3 to obtain amino acid sequences homologous to a protein molecule described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul SF et al., (1997) Nuc Acids Res 25: 3389-3402, which is herein incorporated by reference in its entirety. Alternatively, PSI BLAST can be used to perform an iterated search which detects distant relationships between molecules (Id.). When utilizing BLAST, Gapped BLAST, and PSI Blast programs, the default parameters of the respective programs (e.g., of XBLAST and NBLAST) can be used (see, e.g., National Center for Biotechnology Information (NCBI) on the worldwide web, ncbi.nlm.nih.gov). Another specific, non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, 1988, CABIOS 4:11-17, which is herein incorporated by reference in its entirety. Such an algorithm is incorporated in the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted.

[0137] As used herein, the term “pharmaceutical composition” means a composition that is suitable for administration to an animal, e.g., a human subject, and comprises a therapeutic agent and a pharmaceutically acceptable carrier or diluent. A “pharmaceutically acceptable carrier or diluent” means a substance intended for use in contact with the tissues of human beings and / or non-human animals, and without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable therapeutic benefit / risk ratio.

[0138] As used herein, the term “plurality” means 2 or more (e.g., 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 9 or more, or 10 or more).

[0139] As used herein, the term “poly(A) sequence,” refers to a sequence of adenosine nucleotides, typically located at the 3'-end of a coding linear RNA, of up to about 1000 adenosine nucleotides. In some embodiments, the poly(A) sequence is essentially homopolymeric, e.g., a poly(A) sequence of e.g., 100 adenosine nucleotides having essentially the length of 100Attorney Docket No.62801.71WO01 nucleotides. In other embodiments, the poly(A) sequence may be interrupted by at least one nucleotide different from an adenosine nucleotide, e.g., a poly(A) sequence of e.g., 100 adenosine nucleotides may have a length of more than 100 nucleotides (comprising 100 adenosine nucleotides and in addition said at least one nucleotide - or a stretch of nucleotides - different from an adenosine nucleotide). It has to be understood that “poly(A) sequence” as defined herein typically relates to mRNA - however in the context of the invention, the term likewise relates to corresponding sequences in a DNA molecule (e.g., a “poly(T) sequence”).

[0140] The terms “polynucleotide” and “nucleic acid molecule” are used interchangeably herein and refer to a polymer of DNA or RNA. The nucleic acid molecule can be single-stranded or double-stranded; contain natural, non-natural, or altered nucleotides; and contain a natural, non- natural, or altered internucleotide linkage, such as a phosphoroamidate linkage or a phosphorothioate linkage, instead of the phosphodiester found between the nucleotides of an unmodified nucleic acid molecule. Nucleic acid molecules include, but are not limited to, all nucleic acid molecules which are obtained by any means available in the art, including, without limitation, recombinant means, e.g., the cloning of nucleic acid molecules from a recombinant library or a cell genome, using ordinary cloning technology and polymerase chain reaction, and the like, and by synthetic means. The skilled artisan will appreciate that, except where otherwise noted, nucleic acid sequences set forth in the instant application will recite thymidine (T) in a representative DNA sequence but where the sequence represents RNA (e.g., mRNA), the thymidines (Ts) would be substituted for uracils (Us). Thus, any of the RNA molecules encoded by a DNA identified by a particular sequence identification number may also comprise the corresponding RNA (e.g., mRNA) sequence encoded by the DNA, where each thymidine (T) of the DNA sequence is substituted with uracil (U).

[0141] As used herein, the terms “protein” and “polypeptide” refers to a polymer of at least 2 (e.g., at least 5) amino acids linked by a peptide bond. The term “polypeptide” does not denote a specific length of the polymer chain of amino acids. It is common in the art to refer to shorter polymers of amino acids (e.g., approximately 2-50 amino acids) as peptides; and to refer to longer polymers of amino acids (e.g., approximately over 50 amino acids) as polypeptides. However, the terms “peptide” and “polypeptide” and “protein” are used interchangeably herein. In some embodiments, the protein is folded into its three-dimensional structure. Where linear polypeptides are contemplated herein (i.e., primary structure (amino acid sequence)), it should be understoodAttorney Docket No.62801.71WO01 that proteins folded into their three-dimensional structure are also provided herein. Where proteins are contemplated herein (i.e., folded into their three-dimensional structure) polypeptides in their primary structure (i.e., the amino acid sequence) are also provided herein.

[0142] A “prophylactic” treatment is a treatment administered to a subject who does not exhibit signs of a disease or exhibits only early signs for the purpose of decreasing the risk of developing pathology.

[0143] The terms “RNA” and “polyribonucleotide” are used interchangeably herein and refer to macromolecules that include multiple ribonucleotides that are polymerized via phosphodiester bonds. Ribonucleotides are nucleotides in which the sugar is ribose. RNA may contain modified nucleotides; and contain natural, non-natural, or altered internucleotide linkages, such as a phosphoroamidate linkage or a phosphorothioate linkage, instead of the phosphodiester linkage found between the nucleotides of an unmodified nucleic acid molecule.

[0144] As used herein, the term “sample” encompass a variety of biological specimens obtained from a subject. Exemplary sample types include, e.g., blood and other liquid samples of biological origin (including, but not limited to, whole-blood, peripheral blood mononuclear cells (PBMCs), serum, plasma, urine, saliva, amniotic fluid, stool, synovial fluid, etc.), nasopharyngeal swabs, solid tissue samples such as biopsies (or cells derived therefrom and the progeny thereof), tissue cultures (or cells derived therefrom and the progeny thereof), and cell cultures (or cells derived therefrom and the progeny thereof). The term also includes samples that have been manipulated in any way after their procurement from a subject, such as by centrifugation, filtration, washing, precipitation, dialysis, chromatography, lysis, treatment with reagents, enriched for certain cell populations, refrigeration, freezing, staining, etc.

[0145] As used herein, the term “translatable RNA” refers to any RNA that encodes at least one polypeptide and can be translated to produce the encoded protein in vitro, in vivo, in situ or ex vivo. A translatable RNA may be an mRNA or a circular RNA encoding a polypeptide.

[0146] The term “(scFv)2” as used herein refers to an antibody that comprises a first and a second scFv operably connected (e.g., via a peptide linker). The first and second scFv can specifically bind the same or different antigens. In some embodiments, the first and second scFv are operably connected by a peptide linker.

[0147] The term “scFv-Fc” as used herein refers to an antibody that comprises a scFv operably linked (e.g., via a peptide linker) to an Fc domain or subunit of an Fc domain. In someAttorney Docket No.62801.71WO01 embodiments, a scFv is operably connected to only a first Fc domain of a first and a second Fc domain pair. In some embodiments, a first scFv is operably connected to a first Fc domain and a second scFv is operably connected to a second Fc domain of a first and second Fc domain pair.

[0148] The term “(scFv)2-Fc” as used herein refers to a (scFv)2operably linked (e.g., via a peptide linker) to an Fc domain or a subunit of an Fc domain. In some embodiments, a (scFv)2 is operably connected to only a first Fc domain of a first and a second Fc domain pair. In some embodiments, a first (scFv)2is operably connected to a first Fc domain and a second (scFv)2is operably connected to a second Fc domain of a first and second Fc domain pair.

[0149] As used herein, the term “single domain antibody” or “sdAb” refers to an antibody having a single monomeric variable antibody domain. A sdAb is able to specifically bind to a specific antigen. A VHH (as defined herein) is an example of a sdAb.

[0150] As used herein, the term “signal peptide” or “signal sequence” refers to a sequence (e.g., an amino acid sequence) that can direct the transport or localization of a protein to a certain organelle, cell compartment, or extracellular export. The term encompasses both the signal sequence peptide and the nucleic acid sequence encoding the signal peptide. Thus, references to a signal peptide in the context of a nucleic acid refers to the nucleic acid sequence encoding the signal peptide.

[0151] As used herein, the term “specifically binds” refers to preferential interaction, i.e., significantly higher binding affinity, between a first protein (e.g., a ligand) and a second protein (e.g., the ligand’s cognate receptor) relative to other amino acid sequences. Herein, when a first protein is said to “specifically bind” to a second protein, it is understood that the first protein specifically binds to an epitope of the second protein. The term “epitope” refers to the portion of the second protein that the first protein specifically recognizes. The term specifically binds includes molecules that are cross reactive with the same epitope of a different species. For example, an antibody that specifically binds human TNFα may be cross reactive with TNFα of another species (e.g., cynomolgus, murine, etc.), and still be considered herein to specifically bind human TNFα. A protein can specifically bind more than one different protein.

[0152] As used herein, the term “subject” includes any animal, such as a human or other animal. In some embodiments, the subject is a vertebrate animal (e.g., mammal, bird, fish, reptile, or amphibian). In some embodiments, the subject is a human. In some embodiments, the method subject is a non-human mammal. In some embodiments, the subject is a non-human mammal isAttorney Docket No.62801.71WO01 such as a non-human primate (e.g., monkeys, apes), ungulate (e.g., cattle, buffalo, sheep, goat, pig, camel, llama, alpaca, deer, horses, donkeys), carnivore (e.g., dog, cat), rodent (e.g., rat, mouse), or lagomorph (e.g., rabbit). In some embodiments, the subject is a bird, such as a member of the avian taxa Galliformes (e.g., chickens, turkeys, pheasants, quail), Anseriformes (e.g., ducks, geese), Paleaognathae (e.g., ostriches, emus), Columbiformes (e.g., pigeons, doves), or Psittaciformes (e.g., parrots).

[0153] As used herein, the term “therapeutically effective amount” of a therapeutic agent refers to any amount of the therapeutic agent that, when used alone or in combination with another therapeutic agent, improves a disease condition, e.g., protects a subject against the onset of a disease (or infection); improves a symptom of disease or infection, e.g., decreases severity of disease or infection symptoms, decreases frequency or duration of disease or infection symptoms, increases disease or infection symptom-free periods; prevents or reduces impairment or disability due to the disease or infection; or promotes disease (or infection) regression. The ability of a therapeutic agent to improve a disease condition can be evaluated using a variety of methods known to the skilled practitioner, such as in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by assaying the activity of the agent in in vitro assays.

[0154] As used herein, the terms “treat,” treating,” “treatment,” and the like refer to reducing or ameliorating a disease or infection and / or symptom(s) associated therewith or obtaining a desired pharmacologic and / or physiologic effect. It will be appreciated that, although not precluded, treating a disease or infection does not require that the disease or infection, or symptom(s) associated therewith be completely eliminated. In some embodiments, the effect is therapeutic, i.e., without limitation, the effect partially or completely reduces, diminishes, abrogates, abates, alleviates, decreases the intensity of, or cures a disease and / or adverse symptom attributable to the disease or infection. In some embodiments, the effect is preventative, i.e., the effect protects or prevents an occurrence or reoccurrence of a disease or infection. To this end, the presently disclosed methods comprise administering a therapeutically effective amount of a composition as described herein.

[0155] As used herein the term “TNFα” or “Tumor Necrosis Factor α” or “Tumor Necrosis Factor Ligand Superfamily Member 2” refers to the multifunctional immunomodulatory cytokine of the TNFSF. The amino acid sequence of an exemplary reference membrane human TNFα (hTNFα) protein is set forth in SEQ ID NO: 1 and the amino acid sequence of an exemplaryAttorney Docket No.62801.71WO01 reference soluble hTNFα protein is set forth in SEQ ID NO: 2.

[0156] As used herein the term “TNFR1” or “Tumor Necrosis Factor Receptor Superfamily Member 1A” refers to the receptor of the TNFSF that binds, e.g., TNFα. TNFR1 can be expressed both as a transmembrane protein and as a secreted protein through proteolytic processing. The amino acid sequence of an exemplary reference immature human TNFR1 (hTNFR1) protein is set forth in SEQ ID NO: 3 and the amino acid sequence of an exemplary reference mature hTNFR1 protein is set forth in SEQ ID NO: 4.

[0157] As used herein the term “TNFR2” or “Tumor Necrosis Factor Receptor Superfamily Member 1B” refers to the receptor of the TNFSF that binds, e.g., TNFα. The amino acid sequence of an exemplary reference immature human TNFR2 (hTNFR2) protein is set forth in SEQ ID NO: 5 and the amino acid sequence of an exemplary reference mature hTNFR2 protein is set forth in SEQ ID NO: 6.

[0158] As used herein, the term “variant” or “variation” with reference to a nucleic acid molecule, refers to a nucleic acid molecule that comprises at least one substitution, alteration, inversion, addition, or deletion of nucleotide compared to a reference nucleic acid molecule. As used herein, the term “variant” or “variation” with reference to a protein refers to a protein that comprises at least one substitution, alteration, inversion, addition, or deletion of an amino acid residue compared to a reference protein.

[0159] As used herein, the term “variant Ig Fc fusion protein” refers to a fusion protein comprising an immunoreceptor inhibitory protein described herein and an Ig Fc region, wherein the Ig Fc region comprises one or more variation (e.g., one or more amino acid substitution, deletion, or addition)) that decreases or abolishes one or more Fc effector function, relative to a reference Ig Fc fusion protein that does not comprise the one or more variation.

[0160] The terms “VL” and “VL domain” are used interchangeably to refer to the light chain variable region of an antibody.

[0161] The terms “VH” and “VH domain” are used interchangeably to refer to the heavy chain variable region of an antibody.

[0162] The term “VHH” as used herein refers to a type of single domain antibody (sdAb) that has a single monomeric heavy chain variable antibody domain (VH). Such antibodies can be found in or produced from camelid mammals (e.g., camels, llamas) which are naturally devoid of light chains or synthetically produced.Attorney Docket No.62801.71WO01

[0163] As used herein, the term “5'-untranslated region” or “5'-UTR” refers to a part of a nucleic acid molecule located 5' (i.e., “upstream”) of a coding sequence and which is not translated into protein. Typically, a 5'-UTR starts with the transcriptional start site and ends before the start codon of the coding sequence. A 5'-UTR may comprise elements for controlling gene expression, also called regulatory elements. Such regulatory elements may be, e.g., ribosomal binding sites, miRNA binding sites etc. The 5'-UTR may be post-transcriptionally modified, e.g., by enzymatic or post-transcriptional addition of a 5'-cap structure.

[0164] As used herein the term “3'-untranslated region” or “3'-UTR” refers to a part of a nucleic acid molecule located 3' (i.e., downstream) of a coding sequence and which is not translated into protein. A 3'-UTR may located between a coding sequence and an (optional) terminal poly(A) sequence of a nucleic acid sequence. A 3'-UTR may comprise elements for controlling gene expression, also called regulatory elements. Such regulatory elements may be, e.g., ribosomal binding sites, miRNA binding sites etc. 5.2 Immunoreceptor Inhibitory Proteins

[0165] The present disclosure provides, inter alia, immunoreceptor inhibitory proteins (IIPs) (and functional fragments and variants thereof) that specifically bind to TNF superfamily (TNFSF) ligand TNFα. The TNF superfamily of ligands and receptors regulates multiple cellular functions, including e.g., immune responses, cell proliferation, cell survival, cell differentiation, and programmed cell death. The TNF superfamily comprises 19 ligands and 29 receptors. Exemplary TNF superfamily ligands and their cognate receptors, include, e.g., TNFα and TNFR1 / TNFR2.

[0166] The amino acid sequence of the membrane and soluble form of hTNFα is set forth in SEQ ID NOS: 1 and 2, respectively. The amino acid sequence of the immature and mature form of hLTα is set forth in SEQ ID NOS: 121 and 122, respectively. The amino acid sequence of the immature and mature form of hTNFR1 is set forth in SEQ ID NOS: 3 and 4, respectively. The amino acid sequence of the immature and mature form of hTNFR2 is set forth in SEQ ID NOS: 5 and 6, respectively. See Table 1, herein. Table 1. The Amino Acid Sequence of human TNF Superfamily Reference Ligands and Receptors. D ri ti n Amin A id S n SEQ IDAttorney Docket No.62801.71WO01 Exemplary Ligands MSTESMIRDVELAEEALPKKTGGPQGSRRCLFLSLFSFLIVAGATTAttorney Docket No.62801.71WO01 DSSPGGHGTQVNVTCIVNVCSSSDHSSQCSSQASSTMGDTDSSPSE SPKDEQVPFSKEECAFRSQLETPETLLGSTEEKPLPLGVPDAGMKP Snd functional fragments and variants thereof) that specifically bind TNFα. The amino acid sequence of the immunoreceptor inhibitory proteins provided herein is set forth in Table 2. The amino acid sequence of the mature form of the immunoreceptor inhibitory proteins and polypeptides (i.e., lacking the native signal peptide) is set forth in SEQ ID NOS: 7-11. The amino acid sequence of the immature form of the immunoreceptor inhibitory proteins (i.e., containing the native signal peptide) is set forth in SEQ ID NOS: 12-16.

[0168] The signal peptides have been computationally predicted using standard methods (see, e.g., Teufel, F., Almagro Armenteros, J.J., Johansen, A.R. et al. SignalP 6.0 predicts all five types of signal peptides using protein language models. Nat Biotechnol (2022). https: / / doi.org / 10.1038 / s41587-021-01156-3, the entire contents of which is incorporated by reference herein for all purposes). A person of ordinary skill in the art would know how to experimentally identify and / or validate a computationally predicted signal peptide using standard methods known in the art, e.g., expression of the immunoreceptor inhibitory protein from a host cell and sequencing of the intracellular form and the extracellular form of the expressed protein (see, e.g., Zhang Z, Henzel WJ. Signal peptide prediction based on analysis of experimentally verified cleavage sites. Protein Sci. 2004;13(10):2819-2824. doi:10.1110 / ps.04682504, the entire contents of which is incorporated by reference herein for all purposes). Table 2. The Amino Acid Sequence of Immunoreceptor Inhibitory Proteins. Description Amino Acid Sequence SEQ IDAttorney Docket No.62801.71WO01 peptide IIP-2 SLQCKNNTYYNSQYVKCCKLCEPGTFYSKKCDEKNDTICEKCPDGS with t n tiv i n l YTSVYNHSPACVSCRGYCDYNQVETTSCTPTSNRICKCKLSSYCLV 8, ory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises an amino acid sequence at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a protein set forth in Table 2. In some embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises an amino acid sequence at least about 85% identical to the amino acid sequence of a protein set forth in Table 2. In some embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises an amino acid sequence at least about 90% identical to the amino acid sequence of a protein set forth in Table 2. In some embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises an amino acid sequence at least about 95%Attorney Docket No.62801.71WO01 identical to the amino acid sequence of a protein set forth in Table 2. In some embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises an amino acid sequence at least about 99% identical to the amino acid sequence of a protein set forth in Table 2.

[0170] In some embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises an amino acid sequence about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a protein set forth in Table 2. In some embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises an amino acid sequence about 85% identical to the amino acid sequence of a protein set forth in Table 2. In some embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises an amino acid sequence about 90% identical to the amino acid sequence of a protein set forth in Table 2. In some embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises an amino acid sequence about 95% identical to the amino acid sequence of a protein set forth in Table 2. In some embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises an amino acid sequence about 99% identical to the amino acid sequence of a protein set forth in Table 2. In some embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises an amino acid sequence about 100% identical to the amino acid sequence of a protein set forth in Table 2.

[0171] In some embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) consists of an amino acid sequence at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a protein set forth in Table 2. In some embodiments, the amino acid sequence of the immunoreceptor inhibitory protein consists of an amino acid sequence at least about 85% identical to the amino acid sequence of a protein set forth in Table 2. In some embodiments, the amino acid sequence ofAttorney Docket No.62801.71WO01 the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) consists of an amino acid sequence at least about 90% identical to the amino acid sequence of a protein set forth in Table 2. In some embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) consists of an amino acid sequence at least about 95% identical to the amino acid sequence of a protein set forth in Table 2. In some embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) consists of an amino acid sequence at least about 99% identical to the amino acid sequence of a protein set forth in Table 2.

[0172] In some embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) consists of an amino acid sequence about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a protein set forth in Table 2. In some embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) consists of an amino acid sequence about 85% identical to the amino acid sequence of a protein set forth in Table 2. In some embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) consists of an amino acid sequence about 90% identical to the amino acid sequence of a protein set forth in Table 2. In some embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) consists of an amino acid sequence about 95% identical to the amino acid sequence of a protein set forth in Table 2. In some embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) consists of an amino acid sequence about 99% identical to the amino acid sequence of a protein set forth in Table 2. In some embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) consists of an amino acid sequence about 100% identical to the amino acid sequence of a protein set forth in Table 2.

[0173] In some embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof)Attorney Docket No.62801.71WO01 comprises the amino acid sequence of a protein set forth in Table 2, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises the amino acid sequence of a protein set forth in Table 2, and further comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises the amino acid sequence of a protein set forth in Table 2, and further consists of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises the amino acid sequence of a protein set forth in Table 2, and further comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises the amino acid sequence of a protein set forth in Table 2, and further consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises the amino acid sequence of a protein set forth in Table 2, and further comprises or no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.).

[0174] In some embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) consists of the amino acid sequence of a protein set forth in Table 2, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) consists of the amino acid sequence of a protein set forth in Table 2, and further comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of theAttorney Docket No.62801.71WO01 immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) consists of the amino acid sequence of a protein set forth in Table 2, and further consists of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) consists of the amino acid sequence of a protein set forth in Table 2, and further comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) consists of the amino acid sequence of a protein set forth in Table 2, and further consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) consists of the amino acid sequence of a protein set forth in Table 2, and further comprises or no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.).

[0175] In some embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 7-16. For example, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) may comprise an amino acid sequence at least 85% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 7-16. The amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) may comprise an amino acid sequence at least 90% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 7-16. The amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) may comprise an amino acid sequence at least 95% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 7-16. In some embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) may comprise an amino acid sequence 100% identical to the amino acidAttorney Docket No.62801.71WO01 sequence set forth in any one of SEQ ID NOS: 7-16.

[0176] In some embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 7-16. For example, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) may consist of an amino acid sequence at least 85% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 7-16. The amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) may consist of an amino acid sequence at least 90% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 7-16. The amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) may consist of an amino acid sequence at least 95% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 7-16. In some embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) may consist of an amino acid sequence 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 7-16.

[0177] In embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises the amino acid sequence set forth in any one of SEQ ID NOS: 7-16, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises the amino acid sequence set forth in any one of SEQ ID NOS: 7-16, and further comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises the amino acid sequence set forth in any one of SEQ ID NOS: 7-16, and further comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of theAttorney Docket No.62801.71WO01 immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises the amino acid sequence set forth in any one of SEQ ID NOS: 7-16, and further consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises the amino acid sequence set forth in any one of SEQ ID NOS: 7-16, and further comprises or consists of no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.).

[0178] In embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) consists of the amino acid sequence set forth in any one of SEQ ID NOS: 7-16, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) consists of the amino acid sequence set forth in any one of SEQ ID NOS: 7-16, and further comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) consists of the amino acid sequence set forth in any one of SEQ ID NOS: 7-16, and further comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) consists of the amino acid sequence set forth in any one of SEQ ID NOS: 7-16, and further consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) consists of the amino acid sequence set forth in any one of SEQ ID NOS: 7-16, and further comprises or consists of no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.).

[0179] In some embodiments, the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises a homologousAttorney Docket No.62801.71WO01 signal peptide operably connected to the immunoreceptor inhibitory protein. In some embodiments, the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises a homologous signal peptide operably connected to the N-terminus of the immunoreceptor inhibitory protein. In some embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises the amino acid sequence set forth in any one of SEQ ID NOS: 7-16 and comprises a homologous signal peptide operably connected to the N- terminus of the immunoreceptor inhibitory protein. In some embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) consists of the amino acid sequence set forth in any one of SEQ ID NOS: 7-16 and comprises a homologous signal peptide operably connected to the N- terminus of the immunoreceptor inhibitory protein.

[0180] In some embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 7-11. For example, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) may comprise an amino acid sequence at least 85% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 7-11. The amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) may comprise an amino acid sequence at least 90% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 7-11. The amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) may comprise an amino acid sequence at least 95% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 7-11. In some embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) may comprise an amino acid sequence 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 7-11.

[0181] In some embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof)Attorney Docket No.62801.71WO01 consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 7-11. For example, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) may consist of an amino acid sequence at least 85% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 7-11. The amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) may consist of an amino acid sequence at least 90% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 7-11. The amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) may consist of an amino acid sequence at least 95% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 7-11. In some embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) may consist of an amino acid sequence 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 7-11.

[0182] In embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises the amino acid sequence set forth in any one of SEQ ID NOS: 7-11, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises the amino acid sequence set forth in any one of SEQ ID NOS: 7-11, and further comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises the amino acid sequence set forth in any one of SEQ ID NOS: 7-11, and further comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises the amino acid sequence set forth in any one of SEQ ID NOS: 7-11, and further consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g.,Attorney Docket No.62801.71WO01 substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises the amino acid sequence set forth in any one of SEQ ID NOS: 7-11, and further comprises or consists of no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.).

[0183] In embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) consists of the amino acid sequence set forth in any one of SEQ ID NOS: 7-11, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) consists of the amino acid sequence set forth in any one of SEQ ID NOS: 7-11, and further comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) consists of the amino acid sequence set forth in any one of SEQ ID NOS: 7-11, and further comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) consists of the amino acid sequence set forth in any one of SEQ ID NOS: 7-11, and further consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) consists of the amino acid sequence set forth in any one of SEQ ID NOS: 7-11, and further comprises or consists of no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.).

[0184] In some embodiments, the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises a homologous signal peptide operably connected to the immunoreceptor inhibitory protein. In some embodiments, the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises a homologous signal peptide operably connectedAttorney Docket No.62801.71WO01 to the N-terminus of the immunoreceptor inhibitory protein. In some embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises the amino acid sequence set forth in any one of SEQ ID NOS: 7-11 and comprises a homologous signal peptide operably connected to the N- terminus of the immunoreceptor inhibitory protein. In some embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) consists of the amino acid sequence set forth in any one of SEQ ID NOS: 7-11 and comprises a homologous signal peptide operably connected to the N- terminus of the immunoreceptor inhibitory protein.

[0185] In some embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 12-16. For example, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) may comprise an amino acid sequence at least 85% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 12-16. The amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) may comprise an amino acid sequence at least 90% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 12-16. The amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) may comprise an amino acid sequence at least 95% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 12-16. In some embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) may comprise an amino acid sequence 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 12-16.

[0186] In some embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 12-16. For example, the amino acid sequence of the immunoreceptorAttorney Docket No.62801.71WO01 inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) may consist of an amino acid sequence at least 85% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 12-16. The amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) may consist of an amino acid sequence at least 90% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 12-16. The amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) may consist of an amino acid sequence at least 95% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 12-16. In some embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) may consist of an amino acid sequence 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 12-16.

[0187] In embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises the amino acid sequence set forth in any one of SEQ ID NOS: 12-16, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises the amino acid sequence set forth in any one of SEQ ID NOS: 12-16, and further comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises the amino acid sequence set forth in any one of SEQ ID NOS: 12-16, and further comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises the amino acid sequence set forth in any one of SEQ ID NOS: 12-16, and further consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises the amino acid sequence set forth in any one of SEQ ID NOS:Attorney Docket No.62801.71WO01 12-16, and further comprises or consists of no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.).

[0188] In embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) consists of the amino acid sequence set forth in any one of SEQ ID NOS: 12-16, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) consists of the amino acid sequence set forth in any one of SEQ ID NOS: 12-16, and further comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) consists of the amino acid sequence set forth in any one of SEQ ID NOS: 12-16, and further comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) consists of the amino acid sequence set forth in any one of SEQ ID NOS: 12-16, and further consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) consists of the amino acid sequence set forth in any one of SEQ ID NOS: 12-16, and further comprises or consists of no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.).

[0189] In some embodiments, the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises a homologous signal peptide operably connected to the immunoreceptor inhibitory protein. In some embodiments, the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises a homologous signal peptide operably connected to the N-terminus of the immunoreceptor inhibitory protein. In some embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises the amino acid sequence set forth in any one ofAttorney Docket No.62801.71WO01 SEQ ID NOS: 12-16 and comprises a homologous signal peptide operably connected to the N- terminus of the immunoreceptor inhibitory protein. In some embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) consists of the amino acid sequence set forth in any one of SEQ ID NOS: 12-16 and comprises a homologous signal peptide operably connected to the N- terminus of the immunoreceptor inhibitory protein.

[0190] In specific embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 7. For example, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) may comprise an amino acid sequence at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 7. The amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) may comprise an amino acid sequence at least 90% identical to the amino acid sequence set forth in any one of in SEQ ID NO: 7. The amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) may comprise an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 7. In specific embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) may comprise an amino acid sequence 100% identical to the amino acid sequence set forth in SEQ ID NO: 7.

[0191] In specific embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 7. For example, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) may consist of an amino acid sequence at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 7. The amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) may consist of an amino acid sequenceAttorney Docket No.62801.71WO01 at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 7. The amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) may consist of an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 7. In specific embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) may consist of an amino acid sequence 100% identical to the amino acid sequence set forth in SEQ ID NO: 7.

[0192] In embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises the amino acid sequence set forth in SEQ ID NO: 7, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid variations (e.g., substitutions, additions, deletions, etc.). In specific embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises the amino acid sequence set forth in SEQ ID NO: 7, and further comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In specific embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises the amino acid sequence set forth in SEQ ID NO: 7, and further comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In specific embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises the amino acid sequence set forth in SEQ ID NO: 7, and further consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In specific embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises the amino acid sequence set forth in SEQ ID NO: 7, and further comprises or consists of no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.).

[0193] In embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) consists of the amino acid sequence set forth in SEQ ID NO: 7, and further comprises 1 or more but less thanAttorney Docket No.62801.71WO01 15% (less than 12%, less than 10%, less than 8%), amino acid variations (e.g., substitutions, additions, deletions, etc.). In specific embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) consists of the amino acid sequence set forth in SEQ ID NO: 7, and further comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In specific embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) consists of the amino acid sequence set forth in SEQ ID NO: 7, and further comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In specific embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) consists of the amino acid sequence set forth in SEQ ID NO: 7, and further consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In specific embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) consists of the amino acid sequence set forth in SEQ ID NO: 7, and further comprises or consists of no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.).

[0194] In specific embodiments, the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises a homologous signal peptide operably connected to the immunoreceptor inhibitory protein. In specific embodiments, the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises a homologous signal peptide operably connected to the N-terminus of the immunoreceptor inhibitory protein. In specific embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises the amino acid sequence set forth in SEQ ID NO: 7 and comprises a homologous signal peptide operably connected to the N-terminus of the immunoreceptor inhibitory protein. In specific embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) consists of the amino acid sequence set forth in SEQ ID NO: 7 and comprises a homologous signal peptide operably connected to the N-terminus of theAttorney Docket No.62801.71WO01 immunoreceptor inhibitory protein.

[0195] In specific embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 12. For example, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) may comprise an amino acid sequence at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 12. The amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) may comprise an amino acid sequence at least 90% identical to the amino acid sequence set forth in any one of in SEQ ID NO: 12. The amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) may comprise an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 12. In specific embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) may comprise an amino acid sequence 100% identical to the amino acid sequence set forth in SEQ ID NO: 12.

[0196] In specific embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 12. For example, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) may consist of an amino acid sequence at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 12. The amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) may consist of an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 12. The amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) may consist of an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 12. In specific embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment,Attorney Docket No.62801.71WO01 functional variant, or functional fragment / variant thereof) may consist of an amino acid sequence 100% identical to the amino acid sequence set forth in SEQ ID NO: 12.

[0197] In embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises the amino acid sequence set forth in SEQ ID NO: 12, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid variations (e.g., substitutions, additions, deletions, etc.). In specific embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises the amino acid sequence set forth in SEQ ID NO: 12, and further comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In specific embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises the amino acid sequence set forth in SEQ ID NO: 12, and further comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In specific embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises the amino acid sequence set forth in SEQ ID NO: 12, and further consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In specific embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises the amino acid sequence set forth in SEQ ID NO: 12, and further comprises or consists of no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.).

[0198] In embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) consists of the amino acid sequence set forth in SEQ ID NO: 12, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid variations (e.g., substitutions, additions, deletions, etc.). In specific embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) consists of the amino acid sequence set forth in SEQ ID NO: 12, and further comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g.,Attorney Docket No.62801.71WO01 substitutions, additions, deletions, etc.). In specific embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) consists of the amino acid sequence set forth in SEQ ID NO: 12, and further comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In specific embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) consists of the amino acid sequence set forth in SEQ ID NO: 12, and further consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In specific embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) consists of the amino acid sequence set forth in SEQ ID NO: 12, and further comprises or consists of no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.).

[0199] In specific embodiments, the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises a homologous signal peptide operably connected to the immunoreceptor inhibitory protein. In specific embodiments, the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises a homologous signal peptide operably connected to the N-terminus of the immunoreceptor inhibitory protein. In specific embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises the amino acid sequence set forth in SEQ ID NO: 12 and comprises a homologous signal peptide operably connected to the N-terminus of the immunoreceptor inhibitory protein. In specific embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) consists of the amino acid sequence set forth in SEQ ID NO: 12 and comprises a homologous signal peptide operably connected to the N-terminus of the immunoreceptor inhibitory protein.

[0200] In some embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) no more than 300, 290, 280, 270, 260, 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 115, 110, 100, 95, 90, 80, 70, 60, or 50 amino acids in length. In some embodiments, theAttorney Docket No.62801.71WO01 amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) is less than 120, 115, 110, 100, 95, 90, 80, 70, 60, or 50 amino acids in length. In some embodiments, the amino acid sequence of the immunoreceptor inhibitory protein (or a functional fragment, functional variant, or functional fragment / variant thereof) is from about 50-300, 50-250, 50-200, 50-150, 50-120, 50-110, 50-100, 50-90, 50-60, 60-120, 60-110, 60-100, 60-90, 60-80, 60-70, 70-120, 70-110, 70-100, 70-90, 70- 80, 80-120, 80-110, 80-100, 80-90, 90-120, 90-110, 90-100, 100-120, or 100-110. 5.3 Exemplary Properties of Immunoreceptor Inhibitory Proteins

[0201] In some embodiments, the immunoreceptor inhibitory proteins described herein are immunosuppressive (e.g., when administered to a subject). In some embodiments, the immunoreceptor inhibitory proteins described herein are anti-inflammatory (e.g., when administered to a subject). In some embodiments, the immunoreceptor inhibitory proteins described herein suppress pro-inflammatory response (e.g., when administered to a subject).

[0202] In some embodiments, the immunoreceptor inhibitory proteins described herein bind a subset (e.g., one or more) TNFSF ligand. In some embodiments, the immunoreceptor inhibitory protein specifically binds TNFα. In some embodiments, the immunoreceptor inhibitory protein specifically binds hTNFα. In some embodiments, the immunoreceptor inhibitory protein can act as a decoy receptor for a TNFSF ligand described herein. In some embodiments, the immunoreceptor inhibitory protein inhibits or reduces (e.g., prevents) binding of TNFα to TNFR1. In some embodiments, the immunoreceptor inhibitory protein specifically binds to TNFα and inhibits or reduces (e.g., prevents) binding of TNFα to TNFR1. In some embodiments, the immunoreceptor inhibitory protein inhibits binding of hTNFα to hTNFR1. In some embodiments, the immunoreceptor inhibitory protein specifically binds to hTNFα and inhibits or reduces (e.g., prevents) binding of hTNFα to hTNFR1. In some embodiments, the immunoreceptor inhibitory protein inhibits or reduces (e.g., prevents) binding of TNFα to TNFR2. In some embodiments, the immunoreceptor inhibitory protein specifically binds to TNFα and inhibits or reduces (e.g., prevents) binding of TNFα to TNFR2. In some embodiments, the immunoreceptor inhibitory protein inhibits or reduces (e.g., prevents) binding of hTNFα to hTNFR2. In some embodiments, the immunoreceptor inhibitory protein specifically binds to hTNFα and inhibits or reduces (e.g., prevents) binding of hTNFα to hTNFR2. In some embodiments, the immunoreceptor inhibitoryAttorney Docket No.62801.71WO01 protein inhibits or reduces (e.g., prevents) binding of TNFα to TNFR1 and TNFR2. In some embodiments, the immunoreceptor inhibitory protein specifically binds to TNFα and inhibits or reduces (e.g., prevents) binding of TNFα to TNFR1 and TNFR2. In some embodiments, the immunoreceptor inhibitory protein inhibits or reduces (e.g., prevents) binding of hTNFα to hTNFR1 and TNFR2. In some embodiments, the immunoreceptor inhibitory protein specifically binds to hTNFα and inhibits or reduces (e.g., prevents) binding of hTNFα to hTNFR1 and TNFR2. In some embodiments, the immunoreceptor inhibitory protein inhibits NF-κB signaling mediated by binding of hTNFα to hTNFR1 (e.g., as measured according to Example 4). In some embodiments, the immunoreceptor inhibitory protein inhibits NF-κB signaling mediated by binding of hTNFα to hTNFR2 (e.g., as measured according to Example 4).

[0203] In some embodiments, the immunoreceptor inhibitory protein specifically binds LTα. In some embodiments, the immunoreceptor inhibitory protein specifically binds hLTα. In some embodiments, the immunoreceptor inhibitory protein inhibits or reduces (e.g., prevents) binding of LTα to TNFR1. In some embodiments, the immunoreceptor inhibitory protein specifically binds to LTα and inhibits or reduces (e.g., prevents) binding of LTα to TNFR1. In some embodiments, the immunoreceptor inhibitory protein inhibits or reduces (e.g., prevents) binding of hLTα to hTNFR1. In some embodiments, the immunoreceptor inhibitory protein specifically binds to hLTα and inhibits or reduces (e.g., prevents) binding of hLTα to hTNFR1. In some embodiments, the immunoreceptor inhibitory protein inhibits or reduces (e.g., prevents) binding of LTα to TNFR2. In some embodiments, the immunoreceptor inhibitory protein specifically binds to LTα and inhibits or reduces (e.g., prevents) binding of LTα to TNFR2. In some embodiments, the immunoreceptor inhibitory protein inhibits or reduces (e.g., prevents) binding of hLTα to hTNFR2. In some embodiments, the immunoreceptor inhibitory protein specifically binds to hLTα and inhibits or reduces (e.g., prevents) binding of hLTα to hTNFR2. In some embodiments, the immunoreceptor inhibitory protein inhibits or reduces (e.g., prevents) binding of LTα to TNFR1 and TNFR2. In some embodiments, the immunoreceptor inhibitory protein specifically binds to LTα and inhibits or reduces (e.g., prevents) binding of LTα to TNFR1 and TNFR2. In some embodiments, the immunoreceptor inhibitory protein inhibits or reduces (e.g., prevents) binding of hLTα to hTNFR1 and TNFR2. In some embodiments, the immunoreceptor inhibitory protein specifically binds to hLTα and inhibits or reduces (e.g., prevents) binding of hLTα to hTNFR1 and TNFR2. In some embodiments, the immunoreceptor inhibitory protein inhibits or reduces (e.g.,Attorney Docket No.62801.71WO01 prevents) NF-κB signaling mediated by binding of hLTα to hTNFR1 (e.g., as measured according to Example 4). In some embodiments, the immunoreceptor inhibitory protein inhibits or reduces (e.g., prevents) NF-κB signaling mediated by binding of hLTα to hTNFR2 (e.g., as measured according to Example 4).

[0204] Binding affinity can be measured by standard assays known in the art. For example, binding affinity can be measured by surface plasmon resonance (SPR) (e.g., BIAcore®-based assay), a common method known in the art (see, e.g., Wilson, Science 295:2103, 2002; Wolff et al., Cancer Res. 55:2560, 1993; and U.S. Patent Nos. 5,283,173, 5,468,614, the full contents of each of which are incorporated by reference herein for all purposes). SPR measures changes in the concentration of molecules at a sensor surface as molecules bind to or dissociate from the surface. The change in the SPR signal is directly proportional to the change in mass concentration close to the surface, thereby allowing measurement of binding kinetics between two molecules (e.g., proteins). The dissociation constant for the complex can be determined by monitoring changes in the refractive index with respect to time as buffer is passed over the chip.

[0205] Other suitable assays for measuring the binding of one protein to another (e.g., binding of a protein described herein to a TNFSF ligand) include, for example, immunoassays such as enzyme linked immunosorbent assays (ELISA) and radioimmunoassays (RIA), or determination of binding by monitoring the change in the spectroscopic or optical properties of the proteins through fluorescence, UV absorption, circular dichroism, or nuclear magnetic resonance (NMR). Other exemplary assays include, but are not limited to, Western blot, analytical ultracentrifugation, spectroscopy, flow cytometry, sequencing and other methods for detection of binding of proteins. 5.4 Immunoreceptor Inhibitory Protein Fusions & Conjugates

[0206] In some embodiments, the immunoreceptor inhibitory protein (e.g., described herein) is operably connected to a heterologous moiety (e.g., a heterologous polypeptide) forming a fusion or conjugate protein, respectively. As such, further provided herein are, inter alia, fusion proteins comprising an immunoreceptor inhibitory protein (e.g., described herein) and one or more heterologous proteins (or a functional fragment, functional variant, or domain thereof). Further provided herein are, inter alia, conjugates comprising an immunoreceptor inhibitory protein (e.g., described herein) (or a nucleic acid molecule encoding an immunoreceptor inhibitory protein (e.g., described herein) and one or more heterologous moieties.Attorney Docket No.62801.71WO01

[0207] Heterologous moieties include, but are not limited to, proteins, peptides, small molecules, nucleic acid molecules (e.g., DNA, RNA, DNA / RNA hybrid molecules), carbohydrates, lipids, synthetic polymers (e.g., polymers of PEG), and any combination thereof. In some embodiments, the heterologous moiety is a detectable moiety (e.g., a protein, e.g., a fluorescent protein). In some embodiments, the heterologous moiety is an imaging agent. In some embodiments, the heterologous moiety comprises a radioligand. In some embodiments, the heterologous moiety is a diagnostic agent. In some embodiments, the heterologous moiety is a non-effector moiety, e.g., a protein sequence that acts as a “handle” or linker but has otherwise no independent biological effect. In some embodiments, the heterologous moiety is a therapeutic agent.

[0208] In some embodiments, the heterologous moiety (e.g., protein) comprises an antibody, an antibody mimetic, or one or more Ig constant region (Fc region). In some embodiments, the heterologous moiety comprises one or more Ig constant region (Fc region). In some embodiments, the heterologous moiety comprises an Fc region. The heterologous moiety can be any one or more of (any combination of) the foregoing. 5.4.1 Radioligands

[0209] In some embodiments, the heterologous moiety comprises a radioisotope. As such, provided herein are radioligands comprising an immunoreceptor inhibitory protein (e.g., described herein) operably connected (e.g., through a linker) to one more radioisotope. In some embodiments, the radioisotope acts as a therapeutic agent. In some embodiments, the radioisotope acts as an imaging agent. In some embodiments, the immunoreceptor inhibitory protein (e.g., described herein) acts as a targeting moiety for the radioisotope. In some embodiments, the radioisotope and the immunoreceptor inhibitory protein (e.g., described herein) are operably connected through a linker.

[0210] Radioisotopes are known in the art. See, e.g., Sgouros, G., Bodei, L., McDevitt, M.R. et al. Radiopharmaceutical therapy in cancer: clinical advances and challenges. Nat Rev Drug Discov 19, 589–608 (2020). https: / / doi.org / 10.1038 / s41573-020-0073-9; and Zhang, Longjiang et al. “Delivery of therapeutic radioisotopes using nanoparticle platforms: potential benefit in systemic radiation therapy.” Nanotechnology, science and applications vol.3159-70.3 Dec.2010, doi:10.2147 / NSA.S7462; the entire contents of each of which are incorporated herein by referenceAttorney Docket No.62801.71WO01 for all purposes.

[0211] Exemplary radioisotopes include, but are not limited to, Lutetium-177, Radium-223, Iodine-131, Iodine-125, Fluorine-18, Ir-192, Xenon-133, Yttrium-90, Carbon-11, Idium-111, Strontium-89, Copper-67, Copper-64, Rhenium-186, Actinium-225, Astatine-211, Bismuth-213, Bismuth-212, Samarium-153, Holmium-166, Thorium-227, and Lead-212.

[0212] Methods of operably connecting proteins to radionuclides (e.g., through one or more linkers) are known in the art. See, e.g., Gupta, Suprit et al. “Antibody labeling with radioiodine and radiometals.” Methods in molecular biology (Clifton, N.J.) vol. 1141 (2014): 147-57. doi:10.1007 / 978-1-4939-0363-4_9; Marion Chomet, State of the Art in Radiolabeling of Antibodies with Common and Uncommon Radiometals for Preclinical and Clinical Immuno-PET, Bioconjugate Chem.2021, 32, 7, 1315–1330; Martina Steiner, Dario Neri; Antibody-Radionuclide Conjugates for Cancer Therapy: Historical Considerations and New Trends. Clin Cancer Res 15 October 2011; 17 (20): 6406–6416. https: / / doi.org / 10.1158 / 1078-0432.CCR-11-0483; the entire contents of each of which are incorporated herein by reference for all purposes. 5.4.2 Chimeric Antigen Receptors

[0213] In some embodiments, an immunoreceptor inhibitory protein described herein is part of a chimeric antigen receptor (CAR). In some embodiments, an immunoreceptor inhibitory protein described herein is the extracellular antigen-binding domain of a CAR. Standard CAR domains are known in art, including, e.g., transmembrane domains and intracellular signaling domains. See, e.g., WO2024056809, WO2023240064A1, and WO2023205148A1, WO2023133092A1, the entire contents of each of which is incorporated herein by reference for all purposes.

[0214] Exemplary transmembrane domains include, e.g., the alpha, beta or zeta chain of T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8 (for example, CD8 alpha, CD8 beta), CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. In some embodiments, a transmembrane domain may include at least the transmembrane region(s) of a costimulatory molecule, for example, MHC class I molecule, TNF receptor proteins, Immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), activating NK cell receptors, BTLA, a Toll ligand receptor, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CDlla / CD18), 4-1BB (CD137), B7-H3, CDS,Attorney Docket No.62801.71WO01 ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD 19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDlld, ITGAE, CD103, ITGAL, CDlla, LFA-1, ITGAM, CDllb, ITGAX, CDllc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD 150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, LAT, GADS, SLP- 76, PAG / Cbp, CD 19a, and a ligand that specifically binds with CD83. In some instances, the transmembrane domain can be attached to the extracellular region of the CAR, for example, the antigen-binding domain of the CAR, via a hinge, for example, a hinge from a human protein. For example, in some embodiments, the hinge can be a human Ig (immunoglobulin) hinge, for example, an IgG4 hinge, or a CD8a hinge.

[0215] Exemplary intracellular signaling domains include, e.g., the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivative or variant of these sequences and any recombinant sequence that has the same functional capability. In some embodiments, the intracellular signaling domain comprises a primary signaling domain and one or more costimulatory signaling domain. Exemplary primary signaling domains, include, e.g., intracellular signaling domains of TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278 (also known as “ICOS”), FccRI, DAP10, DAP12, CD32, and CD66d. Exemplary of proteins with costimulatory domains suitable for use in CAR described herein include, e.g., MHC class I molecule, TNF receptor proteins, Immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), activating NK cell receptors, BTLA, a Toll ligand receptor, 0X40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD1 la / CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRFl), NKp44, NKp30, NKp46, CD 19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD lid, ITGAE, CD 103, ITGAL, CDlla, LFA-1, ITGAM, CDllb, ITGAX, CDllc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1Attorney Docket No.62801.71WO01 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD 160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, LAT, GADS, SLP- 76, PAG / Cbp, CD 19a, and a ligand that specifically binds with CD83, and the like. 5.4.3 Signal Peptides

[0216] In some embodiments, the heterologous polypeptide is a heterologous signal peptide. Heterologous signal peptides are known in the art. In some embodiments, the immunoreceptor inhibitory protein comprises a heterologous signal peptide operably connected to the immunoreceptor inhibitory protein. In some embodiments, the immunoreceptor inhibitory protein comprises a heterologous signal peptide operably connected to the N-terminus of the immunoreceptor inhibitory protein. In some embodiments, the amino acid sequence of the immunoreceptor inhibitory protein comprises the amino acid sequence set forth in any one of SEQ ID NOS: 7-16 and comprises a heterologous signal peptide operably connected to the N-terminus of the immunoreceptor inhibitory protein. In some embodiments, the amino acid sequence of the immunoreceptor inhibitory protein consists of the amino acid sequence set forth in any one of SEQ ID NOS: 7-16 and comprises a heterologous signal peptide operably connected to the N-terminus of the immunoreceptor inhibitory protein. In some embodiments, the amino acid sequence of the immunoreceptor inhibitory protein comprises the amino acid sequence set forth in any one of SEQ ID NOS: 7-11 and comprises a heterologous signal peptide operably connected to the N-terminus of the immunoreceptor inhibitory protein. In some embodiments, the amino acid sequence of the immunoreceptor inhibitory protein consists of the amino acid sequence set forth in any one of SEQ ID NOS: 7-11 and comprises a heterologous signal peptide operably connected to the N-terminus of the immunoreceptor inhibitory protein.

[0217] Commonly used heterologous signal peptides are known in the art, for example, the native signal peptide of human interleukin 2 (hIL-2), human oncostatin M (hOSM), human chymotrypsinogen (hCTRB1), human trypsinogen 2 (hTRY2), and human insulin (hINS). A person of ordinary skill can determine the appropriate signal peptide using standard methodology known in the art. The amino acid sequence of exemplary signal peptides is provided in Table 3. Table 3. The Amino Acid Sequence of Exemplary Signal Peptides. Description Amino Acid Sequence SEQ ID NOAttorney Docket No.62801.71WO01 hIL-2MYRMQLLSCIALSLALVTNS17hOSMMGVLLTQRTLLSLVLALLFPSMASM18the amino acid sequence of any one of the signal peptides set forth in Table 3. In some embodiments, the amino acid sequence of the signal peptide comprises the amino acid sequence of any one of the signal peptides set forth in Table 3, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the signal peptide comprises the amino acid sequence of any one of the signal peptides set forth in Table 3, comprising 1, 2, or 3 amino acid variations (e.g., substitutions, deletions, additions). In some embodiments, the amino acid sequence of the signal peptide comprises the amino acid sequence of any one of the signal peptides set forth in Table 3, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid substitutions. In some embodiments, the amino acid sequence of the signal peptide comprises the amino acid sequence of any one of the signal peptides set forth in Table 3, comprising 1, 2, or 3 amino acid substitutions.

[0219] In some embodiments, the amino acid sequence of the signal peptide consists of the amino acid sequence of any one of the signal peptides set forth in Table 3. In some embodiments, the amino acid sequence of the signal peptide consists of the amino acid sequence of any one of the signal peptides set forth in Table 3, and further consists of 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the signal peptide consists of the amino acid sequence of any one of the signal peptides set forth in Table 3, comprising 1, 2, or 3 amino acid variations (e.g., substitutions, deletions, additions). In some embodiments, the amino acid sequence of the signal peptide consists of the amino acid sequence of any one of the signal peptides set forth in Table 3, and further consists of 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid substitutions. In some embodiments, the amino acid sequence of the signal peptide consists of the amino acid sequence of any one of the signal peptides set forth in Table 3, comprising 1, 2, or 3 amino acid substitutions.Attorney Docket No.62801.71WO01

[0220] In some embodiments, the amino acid sequence of the signal peptide comprises the amino acid sequence set forth in any one of SEQ ID NOS: 17-21. In some embodiments, the amino acid sequence of the signal peptide comprises the amino acid sequence set forth in any one of SEQ ID NOS: 17-21, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the signal peptide comprises the amino acid sequence set forth in any one of SEQ ID NOS: 17-21, comprising 1, 2, or 3 amino acid variations (e.g., substitutions, deletions, additions). In some embodiments, the amino acid sequence of the signal peptide comprises the amino acid sequence set forth in any one of SEQ ID NOS: 17-21, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid substitutions. In some embodiments, the amino acid sequence of the signal peptide comprises the amino acid sequence set forth in any one of SEQ ID NOS: 17-21, comprising 1, 2, or 3 amino acid substitutions.

[0221] In some embodiments, the amino acid sequence of the signal peptide consists of the amino acid sequence set forth in any one of SEQ ID NOS: 17-21. In some embodiments, the amino acid sequence of the signal peptide consists of the amino acid sequence set forth in any one of SEQ ID NOS: 17-21, and further consists of 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the signal peptide consists of the amino acid sequence set forth in any one of SEQ ID NOS: 17-21, comprising 1, 2, or 3 amino acid variations (e.g., substitutions, deletions, additions). In some embodiments, the amino acid sequence of the signal peptide consists of the amino acid sequence set forth in any one of SEQ ID NOS: 17-21, and further consists of 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid substitutions. In some embodiments, the amino acid sequence of the signal peptide consists of the amino acid sequence set forth in any one of SEQ ID NOS: 17-21, comprising 1, 2, or 3 amino acid substitutions. 5.4.4 Half-Life Extension Moieties

[0222] In some embodiments, the heterologous moiety (e.g., protein) is a half-life extension moiety (e.g., protein). Various half-life extension moieties are known in the art. See, e.g., Ko S, Jo M, Jung ST. Recent Achievements and Challenges in Prolonging the Serum Half-Lives ofAttorney Docket No.62801.71WO01 Therapeutic IgG Antibodies Through Fc Engineering. BioDrugs. 2021;35(2):147-157. doi:10.1007 / s40259-021-00471-0 (hereinafter “Ko 2021”); Bech, E. M., Pedersen, S. L., & Jensen, K. J. (2018). Chemical Strategies for Half-Life Extension of Biopharmaceuticals: Lipidation and Its Alternatives. ACS medicinal chemistry letters, 9(7), 577–580. https: / / doi.org / 10.1021 / acsmedchemlett.8b00226 (hereinafter “Bech 2018”); Mester S, Evers M, Meyer S, et al. Extended plasma half-life of albumin-binding domain fused human IgA upon pH- dependent albumin engagement of human FcRn in vitro and in vivo. MAbs. 2021;13(1):1893888. doi:10.1080 / 19420862.2021.1893888 (hereinafter “Mester 2021”); Kontermann RE. Strategies for extended serum half-life of protein therapeutics. Curr Opin Biotechnol. 2011;22(6):868-876. doi:10.1016 / j.copbio.2011.06.012 (hereinafter “Kontermann 2011”); Strohl W. R. (2015). Fusion Proteins for Half-Life Extension of Biologics as a Strategy to Make Biobetters. BioDrugs : clinical immunotherapeutics, biopharmaceuticals and gene therapy, 29(4), 215–239. https: / / doi.org / 10.1007 / s40259-015-0133-6; Zaman R, Islam RA, Ibnat N, et al. Current strategies in extending half-lives of therapeutic proteins. J Control Release. 2019;301:176-189. doi:10.1016 / j.jconrel.2019.02.016; Chen C, Constantinou A, Chester KA, et al. Glycoengineering approach to half-life extension of recombinant biotherapeutics. Bioconjug Chem. 2012;23(8):1524-1533. doi:10.1021 / bc200624a; Gupta, Vijayalaxmi et al. “Protein PEGylation for cancer therapy: bench to bedside.” Journal of cell communication and signaling vol. 13,3 (2019): 319-330. doi:10.1007 / s12079-018-0492-0; Martin Schlapschy, et al, PASylation: a biological alternative to PEGylation for extending the plasma half-life of pharmaceutically active proteins, Protein Engineering, Design and Selection, Volume 26, Issue 8, August 2013, Pages 489–501, https: / / doi.org / 10.1093 / protein / gzt023; Strohl, William R. “Fusion Proteins for Half- Life Extension of Biologics as a Strategy to Make Biobetters.” BioDrugs : clinical immunotherapeutics, biopharmaceuticals and gene therapy vol. 29,4 (2015): 215-39. doi:10.1007 / s40259-015-0133-6; the entire contents of each of which are incorporated by reference herein for all purposes.

[0223] Exemplary half-life extension moieties include, but are not limited to, an immunoglobulin (e.g., human Ig (hIg), murine Ig (mIg)), a fragment of an Ig (e.g., hIg, mIg), an Ig (e.g., hIg, mIg) constant region, a fragment of an Ig (e.g., hIg, mIg) constant region, an Ig (e.g., hIg, mIg) Fc region, human transferrin, a human transferrin binding moiety (e.g., small molecule, lipid, protein, peptide, etc.), human serum albumin (HSA), a fragment of HSA, an HSA bindingAttorney Docket No.62801.71WO01 moiety (e.g., small molecule, lipid, protein, peptide, etc.) (e.g., an antibody, a Streptococcal protein G (see, e.g., Mester 2021), polyethylene glycol (PEG) (and polymers thereof) (e.g., pegylation), lipids, small molecules, carbohydrates (e.g., glycosylation, polysialic acid (polysialylation), hydroxyethyl starch (HES) (HESylation), heparosan (HEPylation)).

[0224] In some embodiments, the heterologous polypeptide is a half-life extension polypeptide. Exemplary half-life extension polypeptides include, but are not limited to, an Ig, a fragment of an Ig, one or more Ig heavy chain constant region, a fragment of an Ig constant region, an Ig Fc region, a hIg, a fragment of a hIg, one or more hIg heavy chain constant region, a fragment of a hIg constant region, a hIg Fc region, a mIg, a fragment of a mIg, one or more mIg heavy chain constant region, a fragment of a mIg constant region, a mIg Fc region, human transferrin, a fragment of human transferrin, a human transferrin binding protein (e.g., an antibody) HSA, and HSA binding proteins (e.g., an antibody, a Streptococcal protein G). In some embodiments, the half-life extension polypeptide comprises an Ig Fc region (e.g., hIg Fc region). In some embodiments, the Ig (e.g., hIg, mIg) Fc region of a fusion protein described herein comprises one or more amino acid variation (e.g., relative to a reference Ig (e.g., hIg, mIg) Fc region (e.g., a wild- type Ig (e.g., hIg, mIg) Fc region)) that enhances serum half-life of the fusion protein (e.g., relative to a reference Ig (e.g., hIg, mIg) Fc region (e.g., a wild-type Ig (e.g., hIg, mIg) Fc region)). See, e.g., § 5.4.5.2.

[0225] In some embodiments, half-life extension is mediated through one or more of lipidation, glycosylation, polysialylation, HESylation, HEPylation, and / or pegylation. In some embodiments, half-life extension is mediated through one or more of lipidation, HESylation, HEPylation, and / or pegylation. In some embodiments, half-life extension is mediated through glycosylation. In some embodiments, half-life extension is mediated through polysialylation.

[0226] In some embodiments, the half-life extension moiety comprises one or more lipids. See, e.g., Bech 2018. In some embodiments, the half-life extension moiety comprises one or more post translational modifications (e.g., glycosylation, polysialylation, etc.).

[0227] In some embodiments, the half-life extension moiety (e.g., protein) is altered (e.g., compared to a reference half-life extension moiety (e.g., protein)) to further enhance half-life of the fusion protein or conjugate. Various alterations to known half-life extension moieties (e.g., proteins) are known in the art. See, e.g., Ko 2021, Bech 2018, Mester 2021, and Kontermann 2011. Modifications include, e.g., amino acid variations (e.g., substitutions, additions, deletions) and postAttorney Docket No.62801.71WO01 translational modifications (e.g., altered lipidation, glycosylation, polysialylation, HESylation, HEPylation, pegylation, etc.).

[0228] The immunoreceptor inhibitory protein described herein fused or conjugated to a half- life extending moiety or a half-life extending moiety can be evaluated for their pharmacokinetic properties utilizing standard in vivo methods known in the art. See, e.g., Avery, Lindsay B et al. “Utility of a human FcRn transgenic mouse model in drug discovery for early assessment and prediction of human pharmacokinetics of monoclonal antibodies.” mAbs vol.8,6 (2016): 1064-78. doi:10.1080 / 19420862.2016.1193660; Conner, Christopher M et al. “A precisely humanized FCRN transgenic mouse for preclinical pharmacokinetics studies.” Biochemical pharmacology vol. 210 (2023): 115470. doi:10.1016 / j.bcp.2023.115470; and Kathryn Ball et al., PK and Biodistribution of Therapeutic Proteins, Drug Metabolism and Disposition June 1, 2022, 50 (6) 858-866; DOI: https: / / doi.org / 10.1124 / dmd.121.000463 (hereinafter “Ball 2022”), the entire contents of each of which are incorporated herein by reference for all purposes. 5.4.5 Ig Fusion Proteins 5.4.5.1 Antibody Fusion Proteins

[0229] In some embodiments, the heterologous protein comprises an antibody. In some embodiments, the antibody can act to further target the immunoreceptor inhibitory protein e.g., to a specified cell or tissue type expressing a specific protein (e.g., cell surface protein). Exemplary antibodies include, full-length antibodies, scFvs, Fabs, single domain antibodies (e.g., VHHs), scFv-Fcs, Fab-Fcs, and single domain antibody-Fcs (e.g., VHH-Fcs). In some embodiments, the antibody comprises a full-length antibody. In some embodiments, the antibody comprises a scFv. In some embodiments, the antibody comprises a Fab. In some embodiments, the antibody comprises a single domain antibody. In some embodiments, the antibody comprises a VHH. In some embodiments, the antibody comprises an Fc region.

[0230] In specific embodiments, the heterologous protein comprises an antibody that specifically binds a cytokine (e.g., an interleukin). In specific embodiments, the heterologous protein comprises an antibody that specifically binds an interleukin (e.g., a human interleukin). In specific embodiments, the heterologous protein comprises an antibody that specifically binds interleukin 23 (IL-23). In specific embodiments, the heterologous protein comprises an antibody that specifically binds human IL-23.Attorney Docket No.62801.71WO01 5.4.5.2 Ig Fusion Proteins

[0231] In some embodiments, the heterologous protein comprises one or more Ig heavy chain constant regions (e.g., a CH2 region, a CH3 region, a hinge region, an Fc region (e.g., in some embodiments, preferably an Fc region) (or any combination of the foregoing). In some embodiments, the Ig is an IgG. In some embodiments, the IgG is IgG1, IgG2, IgG3, or IgG4 (e.g., in some embodiments preferably an IgG1 or IgG4).

[0232] In some embodiments, the heterologous protein comprises an IgG CH2 region and an IgG CH3 region. In some embodiments, the heterologous protein comprises a partial IgG hinge region, IgG CH2 region, and IgG CH3 region. In some embodiments, the heterologous protein comprises an IgG hinge region, IgG CH2 region, and IgG CH3 region. In some embodiments, the heterologous protein comprises an IgG1 CH2 region and an IgG1 CH3 region. In some embodiments, the heterologous protein comprises a partial IgG1 hinge region, IgG1 CH2 region, and IgG1 CH3 region. In some embodiments, the heterologous protein comprises an IgG1 hinge region, IgG1 CH2 region, and IgG1 CH3 region. In some embodiments, the heterologous protein comprises an IgG4 CH2 region and an IgG4 CH3 region. In some embodiments, the heterologous protein comprises a partial IgG4 hinge region, IgG4 CH2 region, and IgG4 CH3 region. In some embodiments, the heterologous protein comprises an IgG4 hinge region, IgG4 CH2 region, and IgG4 CH3 region.

[0233] In some embodiments, the heterologous protein consists of an IgG CH2 region and an IgG CH3 region. In some embodiments, the heterologous protein consists of a partial IgG hinge region, IgG CH2 region, and IgG CH3 region. In some embodiments, the heterologous protein consists of an IgG hinge region, IgG CH2 region, and IgG CH3 region. In some embodiments, the heterologous protein consists of an IgG1 CH2 region and an IgG1 CH3 region. In some embodiments, the heterologous protein consists of a partial IgG1 hinge region, IgG1 CH2 region, and IgG1 CH3 region. In some embodiments, the heterologous protein consists of an IgG1 hinge region, IgG1 CH2 region, and IgG1 CH3 region. In some embodiments, the heterologous protein consists of an IgG4 CH2 region and an IgG4 CH3 region. In some embodiments, the heterologous protein consists of a partial IgG4 hinge region, IgG4 CH2 region, and IgG4 CH3 region. In some embodiments, the heterologous protein consists of an IgG4 hinge region, IgG4 CH2 region, and IgG4 CH3 region.

[0234] In some embodiments, the heterologous protein comprises an Ig Fc region. In someAttorney Docket No.62801.71WO01 embodiments, the Ig Fc region comprises at least a portion of a hinge region, a CH2 region, and a CH3 region. In some embodiments, the Ig Fc region comprises a hinge region, a CH2 region, and a CH3 region. In some embodiments, the Ig Fc region comprises at least a portion of an IgG hinge region, an IgG CH2 region, and an IgG CH3 region. In some embodiments, the Ig Fc region comprises an IgG hinge region, an IgG CH2 region, and an IgG CH3 region. In some embodiments, the Ig Fc region comprises at least a portion of an IgG1 hinge region, an IgG1 CH2 region, and an IgG1 CH3 region. In some embodiments, the Ig Fc region comprises an IgG1 hinge region, an IgG1 CH2 region, and an IgG1 CH3 region. In some embodiments, the Ig Fc region comprises at least a portion of an IgG4 hinge region, an IgG4 CH2 region, and an IgG4 CH3 region. In some embodiments, the Ig Fc region comprises an IgG4 hinge region, an IgG4 CH2 region, and an IgG4 CH3 region.

[0235] In some embodiments, the heterologous protein consists of an Ig Fc region. In some embodiments, the Ig Fc region consists of at least a portion of a hinge region, a CH2 region, and a CH3 region. In some embodiments, the Ig Fc region consists of a hinge region, a CH2 region, and a CH3 region. In some embodiments, the Ig Fc region consists of at least a portion of an IgG hinge region, an IgG CH2 region, and an IgG CH3 region. In some embodiments, the Ig Fc region consists of an IgG hinge region, an IgG CH2 region, and an IgG CH3 region. In some embodiments, the Ig Fc region consists of at least a portion of an IgG1 hinge region, an IgG1 CH2 region, and an IgG1 CH3 region. In some embodiments, the Ig Fc region consists of an IgG1 hinge region, an IgG1 CH2 region, and an IgG1 CH3 region. In some embodiments, the Ig Fc region consists of at least a portion of an IgG4 hinge region, an IgG4 CH2 region, and an IgG4 CH3 region. In some embodiments, the Ig Fc region consists of an IgG4 hinge region, an IgG4 CH2 region, and an IgG4 CH3 region.

[0236] In some embodiments, the heterologous protein comprises one or more hIg heavy chain constant regions (e.g., a CH2 region, a CH3 region, a hinge region, an Fc region). In some embodiments, the hIg is a human IgG (hIgG). In some embodiments, the hIgG is hIgG1, IgG2, IgG3, or IgG4. In some embodiments, the hIgG is IgG1 or IgG4. In some embodiments, the hIgG is hIgG1. In some embodiments, the hIgG is hIgG4.

[0237] In some embodiments, the heterologous protein comprises a hIgG CH2 region and a hIgG CH3 region. In some embodiments, the heterologous protein comprises a partial hIgG hinge region, hIgG CH2 region, and hIgG CH3 region. In some embodiments, the heterologous proteinAttorney Docket No.62801.71WO01 comprises a hIgG hinge region, hIgG CH2 region, and hIgG CH3 region. In some embodiments, the heterologous protein comprises a hIgG1 CH2 region and a hIgG1 CH3 region. In some embodiments, the heterologous protein comprises a partial hIgG1 hinge region, hIgG1 CH2 region, and hIgG1 CH3 region. In some embodiments, the heterologous protein comprises a hIgG1 hinge region, hIgG1 CH2 region, and hIgG1 CH3 region. In some embodiments, the heterologous protein comprises a hIgG4 CH2 region and a hIgG4 CH3 region. In some embodiments, the heterologous protein comprises a partial hIgG4 hinge region, hIgG4 CH2 region, and hIgG4 CH3 region. In some embodiments, the heterologous protein comprises a hIgG4 hinge region, hIgG4 CH2 region, and hIgG4 CH3 region.

[0238] In some embodiments, the heterologous protein consists of a hIgG CH2 region and a hIgG CH3 region. In some embodiments, the heterologous protein consists of a partial hIgG hinge region, hIgG CH2 region, and hIgG CH3 region. In some embodiments, the heterologous protein consists of a hIgG hinge region, hIgG CH2 region, and hIgG CH3 region. In some embodiments, the heterologous protein consists of a hIgG1 CH2 region and a hIgG1 CH3 region. In some embodiments, the heterologous protein consists of a partial hIgG1 hinge region, hIgG1 CH2 region, and hIgG1 CH3 region. In some embodiments, the heterologous protein consists of a hIgG1 hinge region, hIgG1 CH2 region, and hIgG1 CH3 region. In some embodiments, the heterologous protein consists of a hIgG4 CH2 region and a hIgG4 CH3 region. In some embodiments, the heterologous protein consists of a partial hIgG4 hinge region, hIgG4 CH2 region, and hIgG4 CH3 region. In some embodiments, the heterologous protein consists of a hIgG4 hinge region, hIgG4 CH2 region, and hIgG4 CH3 region.

[0239] In some embodiments, the heterologous protein comprises a hIg Fc region. In some embodiments, the hIg Fc region comprises at least a portion of a hinge region, a CH2 region, and a CH3 region. In some embodiments, the hIg Fc region comprises a hinge region, a CH2 region, and a CH3 region. In some embodiments, the hIg Fc region comprises at least a portion of a hIgG hinge region, a hIgG CH2 region, and a hIgG CH3 region. In some embodiments, the hIg Fc region comprises a hIgG hinge region, a hIgG CH2 region, and a hIgG CH3 region. In some embodiments, the hIg Fc region comprises at least a portion of a hIgG1 hinge region, a hIgG1 CH2 region, and a hIgG1 CH3 region. In some embodiments, the hIg Fc region comprises a hIgG1 hinge region, a hIgG1 CH2 region, and a hIgG1 CH3 region. In some embodiments, the hIg Fc region comprises at least a portion of a hIgG4 hinge region, a hIgG4 CH2 region, and a hIgG4 CH3 region. In someAttorney Docket No.62801.71WO01 embodiments, the hIg Fc region comprises a hIgG4 hinge region, a hIgG4 CH2 region, and a hIgG4 CH3 region.

[0240] In some embodiments, the heterologous protein consists of a hIg Fc region. In some embodiments, the hIg Fc region consists of at least a portion of a hinge region, a CH2 region, and a CH3 region. In some embodiments, the hIg Fc region consists of a hinge region, a CH2 region, and a CH3 region. In some embodiments, the hIg Fc region consists of at least a portion of a hIgG hinge region, a hIgG CH2 region, and a hIgG CH3 region. In some embodiments, the hIg Fc region consists of a hIgG hinge region, a hIgG CH2 region, and a hIgG CH3 region. In some embodiments, the hIg Fc region consists of at least a portion of a hIgG1 hinge region, a hIgG1 CH2 region, and a hIgG1 CH3 region. In some embodiments, the hIg Fc region consists of a hIgG1 hinge region, a hIgG1 CH2 region, and a hIgG1 CH3 region. In some embodiments, the hIg Fc region consists of at least a portion of a hIgG4 hinge region, a hIgG4 CH2 region, and a hIgG4 CH3 region. In some embodiments, the hIg Fc region consists of a hIgG4 hinge region, a hIgG4 CH2 region, and a hIgG4 CH3 region.

[0241] The amino acid sequence of exemplary reference hIgG1 and hIgG4 heavy chain constant regions and hIg light chain constant regions, which can be incorporated in one or more of the embodiments described herein (e.g., fusion proteins and polypeptide), is provided in Table 4. Table 4. The Amino Acid Sequence of Exemplary hIg heavy chain constant region components and hIg light chain constant regions. Description Amino Acid Sequence SEQ ID NAttorney Docket No.62801.71WO01 PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH hIgG1 CH2 Region + EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL CH3 Re ion HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTAttorney Docket No.62801.71WO01 FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG With C-terminal Lysine VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVAttorney Docket No.62801.71WO01 hIgG4 Partial Hinge PCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD Region + CH2 Region + VSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVL CH3 Re ion TVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQAttorney Docket No.62801.71WO01 GQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVA Ig light chain kappa WKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSH constant region (λCL) 52 RSYSCQVTHEGSTVEKTVAPTECS sesan amino acid sequence set forth in Table 4. In some embodiments, the amino acid sequence of the heterologous protein comprises an amino acid sequence set forth in Table 4, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the heterologous protein comprises an amino acid sequence set forth in Table 4, comprising at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the heterologous protein comprises an amino acid sequence set forth in Table 4, comprising about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the heterologous protein comprises an amino acid sequence set forth in Table 4, comprising no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid variations (e.g., amino acid substitutions, deletions, or additions).

[0243] In some embodiments, the amino acid sequence of the heterologous protein consists of an amino acid sequence set forth in Table 4. In some embodiments, the amino acid sequence of the heterologous protein consists of an amino acid sequence set forth in Table 4, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the heterologous protein consists of an amino acid sequence set forth in Table 4, comprising at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the heterologous protein consists of an amino acid sequence set forth in Table 4, comprising about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the heterologous protein consists of an amino acid sequence set forth in Table 4, comprising no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid variations (e.g., amino acid substitutions, deletions, or additions).

[0244] In some embodiments, the amino acid sequence of the heterologous protein comprises the amino acid sequence set forth in any one of SEQ ID NOS: 22-52 or 123-124. In some embodiments, the amino acid sequence of the heterologous protein comprises the amino acidAttorney Docket No.62801.71WO01 sequence set forth in any one of SEQ ID NOS: 22-52 or 123-124, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the heterologous protein comprises the amino acid sequence set forth in any one of SEQ ID NOS: 22- 52 or 123-124, comprising at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the heterologous protein comprises the amino acid sequence set forth in any one of SEQ ID NOS: 22-52 or 123-124, comprising about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the heterologous protein comprises the amino acid sequence set forth in any one of SEQ ID NOS: 22-52 or 123-124, comprising no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., amino acid substitutions, deletions, or additions).

[0245] In some embodiments, the amino acid sequence of the heterologous protein consists of the amino acid sequence set forth in any one of SEQ ID NOS: 22-52 or 123-124. In some embodiments, the amino acid sequence of the heterologous protein consists of the amino acid sequence set forth in any one of SEQ ID NOS: 22-52 or 123-124, and further comprising 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the heterologous protein consists of the amino acid sequence set forth in any one of SEQ ID NOS: 22- 52 or 123-124, comprising at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the heterologous protein consists of the amino acid sequence set forth in any one of SEQ ID NOS: 22-52 or 123-124, comprising about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the heterologous protein consists of the amino acid sequence set forth in any one of SEQ ID NOS: 90-12, comprising no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., amino acid substitutions, deletions, or additions).

[0246] In some embodiments, wherein the heterologous protein comprises a CH3 region (e.g., comprises an Fc region; a hinge region, CH2 region, and CH3 region, etc.), the CH3 region lacks the C-terminal lysine (e.g., residue 232 of SEQ ID NO: 99, numbering according to SEQ ID NO: 99; or e.g., residue 229 of SEQ ID NO: 113, numbering according to SEQ ID NO: 113). In someAttorney Docket No.62801.71WO01 embodiments, the CH3 region further lacks the C-terminal glycine (e.g., residue 231 of SEQ ID NO: 99, numbering according to SEQ ID NO: 99; or e.g., residue 228 of SEQ ID NO: 113, numbering according to SEQ ID NO: 113).

[0247] In some embodiments, the heterologous protein comprises one or more mIg heavy chain constant regions (e.g., a CH2 region, a CH3 region, a hinge region, an Fc region). In some embodiments, the mIg is mIgG (mIgG). In some embodiments, the mIgG is mIgG1, mIgG2a, mIgG2c, mIgG2b, or mIgG3. In some embodiments, the mIgG is mIgG1 or mIgG2a. In some embodiments, the mIgG is mIgG1. In some embodiments, the mIgG is mIgG2a.

[0248] In some embodiments, the heterologous protein comprises a mIgG CH2 region and a mIgG CH3 region. In some embodiments, the heterologous protein comprises a partial mIgG hinge region, mIgG CH2 region, and mIgG CH3 region. In some embodiments, the heterologous protein comprises a mIgG hinge region, mIgG CH2 region, and mIgG CH3 region. In some embodiments, the heterologous protein comprises a mIgG1 CH2 region and a mIgG1 CH3 region. In some embodiments, the heterologous protein comprises a partial mIgG1 hinge region, mIgG1 CH2 region, and mIgG1 CH3 region. In some embodiments, the heterologous protein comprises a mIgG1 hinge region, mIgG1 CH2 region, and mIgG1 CH3 region. In some embodiments, the heterologous protein comprises a mIgG2a CH2 region and a mIgG2a CH3 region. In some embodiments, the heterologous protein comprises a partial mIgG2a hinge region, mIg2a CH2 region, and mIgG2a CH3 region. In some embodiments, the heterologous protein comprises a mIgG2a hinge region, mIgG2a CH2 region, and mIgG2a CH3 region.

[0249] In some embodiments, the heterologous protein comprises a mIg Fc region. In some embodiments, the mIg Fc region comprises at least a portion of a hinge region, a CH2 region, and a CH3 region. In some embodiments, the mIg Fc region comprises a hinge region, a CH2 region, and a CH3 region. In some embodiments, the mIg Fc region comprises at least a portion of a mIgG hinge region, a mIgG CH2 region, and a mIgG CH3 region. In some embodiments, the mIg Fc region comprises a mIgG hinge region, a mIgG CH2 region, and a mIgG CH3 region. In some embodiments, the mIg Fc region comprises at least a portion of a mIgG1 hinge region, a mIgG1 CH2 region, and a mIgG1 CH3 region. In some embodiments, the mIg Fc region comprises a mIgG1 hinge region, a mIgG1 CH2 region, and a mIgG1 CH3 region. In some embodiments, the mIg Fc region comprises at least a portion of a mIgG2a hinge region, a mIgG2a CH2 region, and a mIgG2a CH3 region. In some embodiments, the mIg Fc region comprises a mIgG2a hinge region,Attorney Docket No.62801.71WO01 a mIgG2a CH2 region, and a mIgG2a CH3 region.

[0250] In some embodiments, the heterologous protein consists of a mIgG CH2 region and a mIgG CH3 region. In some embodiments, the heterologous protein consists of a partial mIgG hinge region, mIgG CH2 region, and mIgG CH3 region. In some embodiments, the heterologous protein consists of a mIgG hinge region, mIgG CH2 region, and mIgG CH3 region. In some embodiments, the heterologous protein consists of a mIgG1 CH2 region and a mIgG1 CH3 region. In some embodiments, the heterologous protein consists of a partial mIgG1 hinge region, mIgG1 CH2 region, and mIgG1 CH3 region. In some embodiments, the heterologous protein consists of a mIgG1 hinge region, mIgG1 CH2 region, and mIgG1 CH3 region. In some embodiments, the heterologous protein consists of a mIgG2a CH2 region and a mIgG2a CH3 region. In some embodiments, the heterologous protein consists of a partial mIgG2a hinge region, mIg2a CH2 region, and mIgG2a CH3 region. In some embodiments, the heterologous protein consists of a mIgG2a hinge region, mIgG2a CH2 region, and mIgG2a CH3 region.

[0251] In some embodiments, the heterologous protein consists of a mIg Fc region. In some embodiments, the mIg Fc region consists of at least a portion of a hinge region, a CH2 region, and a CH3 region. In some embodiments, the mIg Fc region consists of a hinge region, a CH2 region, and a CH3 region. In some embodiments, the mIg Fc region consists of at least a portion of a mIgG hinge region, a mIgG CH2 region, and a mIgG CH3 region. In some embodiments, the mIg Fc region consists of a mIgG hinge region, a mIgG CH2 region, and a mIgG CH3 region. In some embodiments, the mIg Fc region consists of at least a portion of a mIgG1 hinge region, a mIgG1 CH2 region, and a mIgG1 CH3 region. In some embodiments, the mIg Fc region consists of a mIgG1 hinge region, a mIgG1 CH2 region, and a mIgG1 CH3 region. In some embodiments, the mIg Fc region consists of at least a portion of a mIgG2a hinge region, a mIgG2a CH2 region, and a mIgG2a CH3 region. In some embodiments, the mIg Fc region consists of a mIgG2a hinge region, a mIgG2a CH2 region, and a mIgG2a CH3 region.

[0252] The amino acid sequence of exemplary reference mIgG1 and mIgG2a heavy chain constant regions, which can be incorporated in one or more of the embodiments described herein (e.g., fusion proteins and polypeptide), is provided in Table 5. Table 5. The Amino Acid Sequence of Exemplary mIg heavy chain constant region components. Description Amino Acid SequenceSEQAttorney Docket No.62801.71WO01 ID NO AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGAttorney Docket No.62801.71WO01 mIgG2a CH3 Region LTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYS KLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPG Without C-terminal 65

[0253] In some embodiments, the amino acid sequence of the heterologous protein comprisesAttorney Docket No.62801.71WO01 an amino acid sequence set forth in Table 5. In some embodiments, the amino acid sequence of the heterologous protein comprises an amino acid sequence set forth in Table 5, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the heterologous protein comprises an amino acid sequence set forth in Table 5, comprising at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the heterologous protein comprises an amino acid sequence set forth in Table 5, comprising about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the heterologous protein comprises an amino acid sequence set forth in Table 5, comprising no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid variations (e.g., amino acid substitutions, deletions, or additions).

[0254] In some embodiments, the amino acid sequence of the heterologous protein consists of an amino acid sequence set forth in Table 5. In some embodiments, the amino acid sequence of the heterologous protein consists of an amino acid sequence set forth in Table 5, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the heterologous protein consists of an amino acid sequence set forth in Table 5, comprising at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the heterologous protein consists of an amino acid sequence set forth in Table 5, comprising about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the heterologous protein consists of an amino acid sequence set forth in Table 5, comprising no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid variations (e.g., amino acid substitutions, deletions, or additions).

[0255] In some embodiments, the amino acid sequence of the heterologous protein comprises the amino acid sequence set forth in any one of SEQ ID NOS: 53-73. In some embodiments, the amino acid sequence of the heterologous protein comprises the amino acid sequence set forth in any one of SEQ ID NOS: 53-73, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the heterologous proteinAttorney Docket No.62801.71WO01 comprises the amino acid sequence set forth in any one of SEQ ID NOS: 53-73, comprising at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the heterologous protein comprises the amino acid sequence set forth in any one of SEQ ID NOS: 53-73, comprising about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the heterologous protein comprises the amino acid sequence set forth in any one of SEQ ID NOS: 53-73, comprising no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., amino acid substitutions, deletions, or additions).

[0256] In some embodiments, the amino acid sequence of the heterologous protein consists of the amino acid sequence set forth in any one of SEQ ID NOS: 53-73. In some embodiments, the amino acid sequence of the heterologous protein consists of the amino acid sequence set forth in any one of SEQ ID NOS: 53-73, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the heterologous protein consists of the amino acid sequence set forth in any one of SEQ ID NOS: 53-73, comprising at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the heterologous protein consists of the amino acid sequence set forth in any one of SEQ ID NOS: 53-73, comprising about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the heterologous protein consists of the amino acid sequence set forth in any one of SEQ ID NOS: 53-73, comprising no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., amino acid substitutions, deletions, or additions).

[0257] In some embodiments, wherein the heterologous protein comprises a CH3 region (e.g., comprises an Fc region; a hinge region, CH2 region, and CH3 region, etc.), the CH3 region lacks the C-terminal lysine (e.g., residue 227 of SEQ ID NO: 129, numbering according to SEQ ID NO: 129; or e.g., residue 223 of SEQ ID NO: 136, numbering according to SEQ ID NO: 136). In some embodiments, the CH3 region further lacks the C-terminal glycine (e.g., residue 226 of SEQ ID NO: 129, numbering according to SEQ ID NO: 129; or e.g., residue 222 of SEQ ID NO: 136, numbering according to SEQ ID NO: 136). 5.4.5.3 Half-Life ExtensionAttorney Docket No.62801.71WO01

[0258] In some embodiments, the Ig (e.g., hIg, mIg) Fc region of a fusion protein described herein (an Fc region, an antibody, etc.) exhibits enhanced serum half-life, e.g., relative to a reference Ig (e.g., hIg, mIg) Fc region (e.g., a wild-type Ig (e.g., hIg, mIg) Fc region).

[0259] Standard in vitro and / or in vivo assays known in the art can be conducted to evaluate serum half-life. See, e.g., Ko S, Jo M, Jung ST. Recent Achievements and Challenges in Prolonging the Serum Half-Lives of Therapeutic IgG Antibodies Through Fc Engineering. BioDrugs. 2021;35(2):147-157. doi:10.1007 / s40259-021-00471-0, the entire contents of which are incorporated herein by reference for all purposes.

[0260] In some embodiments, the Ig (e.g., hIg, mIg) Fc region of a fusion protein described herein (an Fc region, an antibody, etc.) exhibits enhanced serum half-life (e.g., relative to a reference Ig (e.g., hIg, mIg) Fc region (e.g., a wild-type Ig (e.g., hIg, mIg) Fc region)) through enhanced binding affinity for the FcRn receptor (e.g., the human FcRn receptor) (e.g., relative to a reference Ig (e.g., hIg, mIg) Fc region (e.g., a wild-type Ig (e.g., hIg, mIg) Fc region)).

[0261] In some embodiments, the Ig (e.g., hIg, mIg) Fc region of a fusion protein described herein (an Fc region, an antibody, etc.) exhibits enhanced serum half-life (e.g., relative to a reference Ig (e.g., hIg, mIg) Fc region (e.g., a wild-type Ig (e.g., hIg, mIg) Fc region)) through enhanced binding affinity for the FcRn receptor (e.g., the human FcRn receptor) at a pH of from about 5.5-6.5 (e.g., relative to a reference Ig (e.g., hIg, mIg) Fc region (e.g., a wild-type Ig (e.g., hIg, mIg) Fc region)). In some embodiments, the Ig (e.g., hIg, mIg) Fc region of a fusion protein described herein (an Fc region, an antibody, etc.) exhibits enhanced serum half-life (e.g., relative to a reference Ig (e.g., hIg, mIg) Fc region (e.g., a wild-type Ig (e.g., hIg, mIg) Fc region)) through enhanced binding affinity for the FcRn receptor (e.g., the human FcRn receptor) at a pH of from about 5.5-6.5 and no substantial change in binding affinity for the FcRn receptor (e.g., the human FcRn receptor) at a pH of from about 7.0-7.5 (e.g., relative to a reference Ig (e.g., hIg, mIg) Fc region (e.g., a wild-type Ig (e.g., hIg, mIg) Fc region)). In some embodiments, the Ig (e.g., hIg, mIg) Fc region of a fusion protein described herein (an Fc region, an antibody, etc.) exhibits enhanced serum half-life (e.g., relative to a reference Ig (e.g., hIg, mIg) Fc region (e.g., a wild- type Ig (e.g., hIg, mIg) Fc region)) through enhanced binding affinity for the FcRn receptor (e.g., the human FcRn receptor) at a pH of from about 6.0-6.5 (e.g., relative to a reference Ig (e.g., hIg, mIg) Fc region (e.g., a wild-type Ig (e.g., hIg, mIg) Fc region)) and a decrease in binding affinity for the FcRn receptor (e.g., the human FcRn receptor) at a pH of from about 7.0-7.5 (e.g., relativeAttorney Docket No.62801.71WO01 to a reference Ig (e.g., hIg, mIg) Fc region (e.g., a wild-type Ig (e.g., hIg, mIg) Fc region)).

[0262] In some embodiments, the Ig (e.g., hIg, mIg) Fc region of a fusion protein described herein (an Fc region, an antibody, etc.) exhibits enhanced serum half-life (e.g., relative to a reference Ig (e.g., hIg, mIg) Fc region (e.g., a wild-type Ig (e.g., hIg, mIg) Fc region)) through enhanced binding affinity for the FcRn receptor (e.g., the human FcRn receptor) at a pH of about 6 (e.g., relative to a reference Ig (e.g., hIg, mIg) Fc region (e.g., a wild-type Ig (e.g., hIg, mIg) Fc region)). In some embodiments, the Ig (e.g., hIg, mIg) Fc region of a fusion protein described herein (an Fc region, an antibody, etc.) exhibits enhanced serum half-life (e.g., relative to a reference Ig (e.g., hIg, mIg) Fc region (e.g., a wild-type Ig (e.g., hIg, mIg) Fc region)) through enhanced binding affinity for the FcRn receptor (e.g., the human FcRn receptor) at a pH of about 6 and no substantial change in binding affinity for the FcRn receptor (e.g., the human FcRn receptor) at a pH of about 7.4 (e.g., relative to a reference Ig (e.g., hIg, mIg) Fc region (e.g., a wild- type Ig (e.g., hIg, mIg) Fc region)). In some embodiments, the Ig (e.g., hIg, mIg) Fc region of a fusion protein described herein (an Fc region, an antibody, etc.) exhibits enhanced serum half-life (e.g., relative to a reference Ig (e.g., hIg, mIg) Fc region (e.g., a wild-type Ig (e.g., hIg, mIg) Fc region)) through enhanced binding affinity for the FcRn receptor (e.g., the human FcRn receptor) at a pH of about 6 (e.g., relative to a reference Ig (e.g., hIg, mIg) Fc region (e.g., a wild-type Ig (e.g., hIg, mIg) Fc region)) and a decrease in binding affinity for the FcRn receptor (e.g., the human FcRn receptor) at a pH of about 7.4 (e.g., relative to a reference Ig (e.g., hIg, mIg) Fc region (e.g., a wild-type Ig (e.g., hIg, mIg) Fc region)).

[0263] In some embodiments, the Ig (e.g., hIg, mIg) Fc region of a fusion protein described herein comprises one or more amino acid variation (e.g., relative to a reference Ig (e.g., hIg, mIg) Fc region (e.g., a wild-type Ig (e.g., hIg, mIg) Fc region)) that enhances serum half-life of the fusion protein (e.g., relative to a reference Ig (e.g., hIg, mIg) Fc region (e.g., a wild-type Ig (e.g., hIg, mIg) Fc region)).

[0264] In some embodiments, the Ig (e.g., hIg, mIg) Fc region of a fusion protein described herein comprises one or more amino acid variation (e.g., relative to a reference Ig (e.g., hIg, mIg) Fc region (e.g., a wild-type Ig (e.g., hIg, mIg) Fc region)) that enhances serum half-life of the fusion protein (e.g., relative to a reference Ig (e.g., hIg, mIg) Fc region (e.g., a wild-type Ig (e.g., hIg, mIg) Fc region)) through altered binding to the FcRn receptor (e.g., as described herein) (e.g., an FcRn binding profile described herein).Attorney Docket No.62801.71WO01

[0265] Exemplary amino acid variations of an Ig (e.g., hIg, mIg) Fc region that enhance serum half-life of the Ig Fc region (or a protein comprising the same) are known in the art. See, e.g., Ko 2021 (and references cited therein) (including e.g., Table 1 of Ko 2021); Xinhua Wang, Mary Mathieu, Randall J Brezski, IgG Fc engineering to modulate antibody effector functions, Protein & Cell, Volume 9, Issue 1, January 2018, Pages 63–73, https: / / doi.org / 10.1007 / s13238-017-0473- 8; US8546543B2; WO2024059652A1; US11591368 (e.g., H433K / N434F); Ko, S., Park, S., Sohn, M.H. et al. An Fc variant with two mutations confers prolonged serum half-life and enhanced effector functions on IgG antibodies. Exp Mol Med 54, 1850–1861 (2022). https: / / doi.org / 10.1038 / s12276-022-00870-5; the entire contents of each of which is incorporated herein by reference for all purposes.

[0266] Table 10 below, provides exemplary amino acid substitutions (and combinations thereof) and glycoengineering that can be utilized to extend half-life of proteins (e.g., fusion proteins described herein) comprising an Ig Fc region (or fragment thereof). Amino acids in Table 10 are numbered according to the EU numbering scheme. The amino acid substitutions set forth in Table 10 are with reference to an IgG1 Fc region (except where noted). However, a person of ordinary skill in the could identify the corresponding amino acid in a non-IgG1 Fc region, for example in an IgG2 or IgG4 Fc region, should the base amino acid be different between the IgG1 and non-IgG1 Fc region. Table 10. Exemplary hIg Fc Variations to Extend Half-Life. Variation / Glycoengineering Exemplary Effects on Effector FunctionAttorney Docket No.62801.71WO01 H433K / N434F Extended Half-Life H433K / N434F (IgG4) Extended Half-Life hehIg Fc (e.g., IgG1 Fc) region comprises any one or more of the amino acid substitutions set forth in Table 10 (i.e., any one or more amino acid substitution set forth in any set of amino acid substitutions set forth in Table 10). In some embodiments, the hIg Fc (e.g., IgG1 Fc) comprises any one or more of the sets of amino acid substitutions set forth in Table 10. In some embodiments, the hIg Fc (e.g., IgG1 Fc) region comprises any one or more of the glycosylation changes set forth in Table 10.

[0268] For example, amino acid variations include, but are not limited to, M428L / N434S, EU numbering according to Kabat; M252Y / S254T / T256E, EU numbering according to Kabat; N434A, EU numbering according to Kabat; N434W, EU numbering according to Kabat; T256D / T307Q, EU numbering according to Kabat; T256D / T307W, EU numbering according to Kabat; M252Y / T256D, EU numbering according to Kabat; T307Q / Q311V / A378V, EU numbering according to Kabat; T256D / H286D / T307R / Q311V / A378V, EU numbering according to Kabat; and L309D / Q311H / N434S, EU numbering according to Kabat. Further amino acid modifications include, H433K / N434F (of IgG1) or H433K / N434F (of IgG4), EU numbering according to Kabat.

[0269] In some embodiments, the Ig (e.g., hIg, mIg) Fc region of a fusion protein described herein comprises one or more alteration (including various post-translational modifications e.g., glycosylation, sialylation) that mediates enhanced serum half-life, e.g., relative to a reference (e.g., wild type) Ig (e.g., hIg, mIg) Fc region. In some embodiments, the Ig (e.g., hIg, mIg) Fc region of a fusion protein described herein comprises one or more post-translational modification (e.g., glycosylation, sialylation) that mediates enhanced serum half-life, e.g., relative to a reference (e.g., wild type) Ig (e.g., hIg, mIg) Fc region. In some embodiments, the Ig (e.g., hIg, mIg) Fc region of a fusion protein described herein comprises altered glycosylation that mediates enhanced serum half-life, e.g., relative to a reference (e.g., wild type) Ig (e.g., hIg, mIg) Fc region. In some embodiments, the Ig (e.g., hIg, mIg) Fc region of a fusion protein described herein comprises altered lipidation that mediates enhanced serum half-life, e.g., relative to a reference (e.g., wild type) Ig (e.g., hIg, mIg) Fc region. In some embodiments, the Ig (e.g., hIg, mIg) Fc region of aAttorney Docket No.62801.71WO01 fusion protein described herein comprises altered sialylation that mediates enhanced serum half- life, e.g., relative to a reference (e.g., wild type) Ig (e.g., hIg, mIg) Fc region. In some embodiments, the Ig (e.g., hIg, mIg) Fc region of a fusion protein described herein is pegylated, which mediates enhanced serum half-life, e.g., relative to a reference (e.g., wild type) Ig (e.g., hIg, mIg) Fc region. 5.4.5.4 Ig Fc Effector Function

[0270] In some embodiments, the Ig (e.g., hIg, mIg) Fc region of a fusion protein described herein exhibits modulation (e.g., a decrease or increase) of one or more Fc effector function, e.g., relative to a reference (e.g., wild type) Ig (e.g., hIg, mIg) Fc region. Exemplary Ig (e.g., hIg, mIg) Fc effector functions include, but are not limited to, antibody dependent cellular cytotoxicity (ADCC), antibody dependent cellular phagocytosis (ADCP), complement dependent cytotoxicity (CDC), and binding affinity to one or more human Fc receptor (e.g., an Fcγ receptor (e.g., FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and / or FcγRIIIb (e.g., FcγRI, FcγIIa, and / or FcγIIIa))).

[0271] Standard in vitro and / or in vivo assays known in the art can be conducted to evaluate Fc effector function, including, any one or more of ADCC, CDC, ADCP, Fc receptor (e.g., Fcγ receptor) binding affinity, and C1q binding affinity.

[0272] For example, ADCC activity can be assessed utilizing standard (radioactive and non- radioactive) methods known in the art (see, e.g., WO2006 / 082515, WO2012 / 130831), the entire contents of each of which is incorporated by reference herein for all purposes). For example, ADCC activity can be assessed using a chromium-5 (51Cr) assay. Briefly,51Cr is pre-loaded into target cells expressing CD20, NK cells are added to the culture, and radioactivity in the cell culture supernatant is assessed (indicative of lysis of the target cells by the NK cells). Similar non- radioactive assays can also be utilized that employ a similar method, but the target cells are pre- loaded with fluorescent dyes, such as calcein-AM, CFSE, BCECF, or lanthanide flurophore (Europium). See, e.g., Parekh, Bhavin S et al. “Development and validation of an antibody- dependent cell-mediated cytotoxicity-reporter gene assay.” mAbs vol. 4,3 (2012): 310-8. Doi:10.4161 / mabs.19873, the entire contents of which is incorporated by reference herein for all purposes. Exemplary commercially available non-radioactive assays include, for example, ACTI™ non-radioactive cytotoxicity assay for flow cytometry (Cell Technology, Inc. Mountain View, Calif.; and CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, Wis.). Additional non-limiting examples of in vitro assays that can be used to assess ADCC activity ofAttorney Docket No.62801.71WO01 a fusion protein described herein include those described in US5500362; US5821337; Hellstrom, I., et al., Proc. Nat’l Acad. Sci. USA 83 (1986) 7059-7063; Hellstrom, I., et al., Proc. Nat’l Acad. Sci. USA 82 (1985) 1499-1502; and Bruggemann, M., et al., J. Exp. Med. 166 (1987) 1351-1361, the entire contents of each of which is incorporated by reference herein. Alternatively, or additionally, ADCC activity of a fusion protein described herein may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes, et al., Proc. Nat’l Acad. Sci. USA 95 (1998) 652-656, the entire contents of which is incorporated by reference herein for all purposes.

[0273] C1q binding assays can be utilized to assess the ability of a hIg fusion protein described herein to bind C1q (or bind with less affinity than a reference fusion protein) and hence lack (or have decreased) CDC activity. The binding of a hIg fusion protein described herein to C1q can be determined by a variety of in vitro assays (e.g., biochemical or immunological based assays) known in the art for determining Fc-C1q interactions, including e.g., equilibrium methods (e.g., enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA)), or kinetic methods (e.g., surface plasmon resonance (SPR) analysis), and other methods such as indirect binding assays, competitive inhibition assays, fluorescence resonance energy transfer (FRET), gel electrophoresis, and chromatography (e.g., gel filtration). These and other methods may utilize a label on one or more of the components being examined and / or employ a variety of detection methods including but not limited to chromogenic, fluorescent, luminescent, or isotopic labels. A detailed description of binding affinities and kinetics can be found in e.g., Paul, W. E., ed., Fundamental Immunology, 4thEd., Lippincott-Raven, Philadelphia (1999), the entire contents of which is incorporated by reference herein. For example, see, e.g., C1q and C3c binding ELISAs described in WO2006 / 029879 and WO2005 / 100402, the entire contents of each of which is incorporated by reference herein for all purposes. Additional CDC activity assays include those described in e.g., Gazzano-Santoro, et al., J. Immunol. Methods 202 (1996) 163; Cragg, M. S., et al., Blood 101 (2003) 1045-1052; and Cragg, M. S., and Glennie, M. J., Blood 103 (2004) 2738- 2743), the entire contents of each of which is incorporated by reference herein for all purposes.

[0274] ADCP activity can be measured by in vitro or in vivo methods known in the art and also commercially available assays (see, e.g., van de Donk NW, Moreau P, Plesner T, et al. “Clinical efficacy and management of monoclonal antibodies targeting CD38 and SLAMF7 in multiple myeloma,” Blood, 127(6):681‐695 (2016), the entire contents of each of which is incorporated by reference herein for all purposes). For example, a primary cell based ADCP assayAttorney Docket No.62801.71WO01 can be used in which fresh human peripheral blood mononuclear cells (PBMCs) are isolated, monocytes isolated and differentiated in culture to macrophages using standard procedures. The macrophages are fluorescently labeled added to cultures containing fluorescently labeled target cells expressing CD20 and a fusion protein described herein. Phagocytosis events can be analyzed using FACS screening and / or microscopy. A modified reporter version of the above described assay can also be used that employs an engineered cell line that stably expresses FcγRIIa (CD32a) as the effector cell line (e.g., an engineered T cell line, e.g., THP-1), removing the requirement for primary cells. Exemplary ADCP assays are described in e.g., Ackerman, M. E. et al. A robust, high-throughput assay to determine the phagocytic activity of clinical antibody samples. J. Immunol. Methods 366, 8–19 (2011); and Mcandrew, E. G. et al. Determining the phagocytic activity of clinical antibody samples. J. Vis. Exp. 3588 (2011). Doi:10.3791 / 3588; the entire contents of each of which is incorporated by reference herein.

[0275] Binding of a hIg fusion protein described herein to an Ig (e.g., hIg, mIg) Fc receptor can be determined by a variety of in vitro assays (e.g., biochemical or immunological based assays) known in the art for determining Fc-Fc receptor interactions, i.e., specific binding of an Fc region to an Fc receptor. Common assays include equilibrium methods (e.g., enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA)), or kinetic methods (e.g., surface plasmon resonance (SPR) analysis), and other methods such as indirect binding assays, competitive inhibition assays, fluorescence resonance energy transfer (FRET), gel electrophoresis, and chromatography (e.g., gel filtration). These and other methods may utilize a label on one or more of the components being examined and / or employ a variety of detection methods including but not limited to chromogenic, fluorescent, luminescent, or isotopic labels. A detailed description of binding affinities and kinetics can be found in e.g., Paul, W. E., ed., Fundamental Immunology, 4” Ed., Lippincott-Raven, Philadelphia (1999), the entire contents of which is incorporated by reference herein for all purposes. (i) Reduced Ig Effector Function

[0276] In some embodiments, the Ig Fc region exhibits a decrease in or no detectable activity of one or more Fc effector. As described above, exemplary Ig Fc effector functions include, but are not limited to, ADCC, ADCP, CDC, binding affinity to C1q, and binding affinity to one or more human Fc receptor (e.g., an Fcγ receptor (e.g., FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and / or FcγRIIIb)).Attorney Docket No.62801.71WO01

[0277] In some embodiments, the hIg Fc region is modified (e.g., comprises one or more variation (e.g., one or more amino acid substitution, deletion, addition, etc.); altered glycosylation)) (referred to herein as a “modified hIg Fc”). In some embodiments, the modification (e.g., the variation (e.g., one or more amino acid substitution, deletion, addition, etc.); altered glycosylation decreases or abolishes one or more Fc effector function, relative to a reference hIg Fc that does not comprise the modification (e.g., the one or more variation (e.g., the one or more amino acid substitution, deletion, addition, etc.; the altered glycosylation)).

[0278] In some embodiments, the modified Ig (e.g., hIg, mIg) Fc fusion protein exhibits no detectable or decreased ADCC compared to a reference fusion protein that does not comprise the Ig (e.g., hIg, mIg) Fc modification (e.g., the one or more variation (e.g., one or more amino acid substitution, deletion, or addition)). In some embodiments, the modified Ig (e.g., hIg, mIg) Fc fusion protein exhibits no detectable or decreased CDC compared to a reference fusion protein that does not comprise the Ig (e.g., hIg, mIg) Fc modification (e.g., the one or more variation (e.g., one or more amino acid substitution, deletion, or addition)). In some embodiments, the modified Ig (e.g., hIg, mIg) Fc fusion protein exhibits no detectable or decreased ADCP compared to a reference fusion protein that does not comprise the Ig (e.g., hIg, mIg) Fc modification (e.g., the one or more variation (e.g., one or more amino acid substitution, deletion, or addition)).

[0279] In some embodiments, the modified Ig (e.g., hIg, mIg) Fc fusion protein exhibits decreased or no binding affinity to one or more Fc receptor (e.g., human Fc receptor) (e.g., an Fcγ receptor (e.g., an Fcγ receptor (e.g., FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and / or FcγRIIIb)) compared to a reference fusion protein that does not comprise the Ig (e.g., hIg, mIg) Fc modification (e.g., the one or more variation (e.g., one or more amino acid substitution, deletion, or addition)).

[0280] In some embodiments, the modified Ig (e.g., hIg, mIg) Fc fusion protein exhibits decreased or no binding affinity to FcγRI, FcγRIIa, FcγRIIIa, and / or FcγRIIIb compared to a reference fusion protein that does not comprise the hIg Fc modification (e.g., the one or more variation (e.g., one or more amino acid substitution, deletion, or addition)). In some embodiments, the modified Ig (e.g., hIg, mIg) Fc fusion protein exhibits decreased or no binding affinity to FcγRI compared to a reference fusion protein that does not comprise the Ig (e.g., hIg, mIg) Fc modification (e.g., the one or more variation (e.g., one or more amino acid substitution, deletion, or addition)). In some embodiments, the modified Ig (e.g., hIg, mIg) Fc fusion protein exhibitsAttorney Docket No.62801.71WO01 decreased or no binding affinity to FcγRIIa compared to a reference fusion protein that does not comprise the Ig (e.g., hIg, mIg) Fc modification (e.g., the one or more variation (e.g., one or more amino acid substitution, deletion, or addition)). In some embodiments, the modified Ig (e.g., hIg, mIg) Fc fusion protein exhibits decreased or no binding affinity to FcγRIIIa compared to a reference fusion protein that does not comprise the Ig (e.g., hIg, mIg) Fc modification (e.g., the one or more variation (e.g., one or more amino acid substitution, deletion, or addition)). In some embodiments, the modified Ig (e.g., hIg, mIg) Fc fusion protein exhibits decreased or no binding affinity to FcγRIIIb compared to a reference fusion protein that does not comprise the Ig (e.g., hIg, mIg) Fc modification (e.g., the one or more variation (e.g., one or more amino acid substitution, deletion, or addition)).

[0281] In some embodiments, the modified Ig (e.g., hIg, mIg) Fc fusion protein exhibits increased binding affinity to one or more Fc receptor (e.g., human Fc receptor) (e.g., an Fcγ receptor (e.g., FcγRIIb)) compared to a reference fusion protein that does not comprise the hIg Fc modification (e.g., the one or more variation (e.g., one or more amino acid substitution, deletion, or addition)). In some embodiments, the modified Ig (e.g., hIg, mIg) Fc fusion protein exhibits increased binding affinity to FcγRIIb compared to a reference fusion protein that does not comprise the Ig (e.g., hIg, mIg) Fc modification (e.g., the one or more variation (e.g., one or more amino acid substitution, deletion, or addition)).

[0282] In some embodiments, the modified Ig (e.g., hIg, mIg) Fc fusion protein exhibits decreased or no binding affinity to C1q compared to a reference fusion protein that does not comprise the Ig (e.g., hIg, mIg) Fc modification (e.g., the one or more variation (e.g., one or more amino acid substitution, deletion, or addition)).

[0283] Amino acid substitutions that decrease or abolish one or more Ig (e.g., hIg, mIg) Fc effector function are known in the art. See for example, Saunders Kevin, “Conceptual Approaches to Modulating Antibody Effector Functions and Circulation Half-Life,” Frontiers in Immunology, v10 (June 7, 2019) DOI=10.3389 / fimmu.2019.01296, the full contents of which is incorporated by reference herein for all purposes, see more particularly for example, e.g., Table 3 of Saunders.

[0284] Table 11 below, provides exemplary amino acid substitutions (and combinations thereof) and glycoengineering that can be utilized to decrease one or more hIg Fc effector function. Amino acids in Table 11 are numbered according to the EU numbering scheme. The effects on effector function set forth in Table 11 are exemplary only and not intended to be limiting. TheAttorney Docket No.62801.71WO01 amino acid substitutions set forth in Table 11 are with reference to an IgG1 Fc region (except where noted). However, a person of ordinary skill in the could identify the corresponding amino acid in a non-IgG1 Fc region, for example in an IgG2 or IgG4 Fc region, should the base amino acid be different between the IgG1 and non-IgG1 Fc region. Table 11. Exemplary hIg Fc Variations and Glycoengineering to Decreases Effector Function. Exemplary Effects on Effector Function Variation / Glycoengineering (Non-Limiting) I, I, I,Attorney Docket No.62801.71WO01 Decreased CDC V264A Decreased C1q binding D r d CDC, , he hIg Fc (e.g., IgG1 Fc) region comprises any one or more of the amino acid substitutions set forth in Table 11 (i.e., any one or more amino acid substitution set forth in any set of amino acid substitutions set forth in Table 11). In some embodiments, the hIg Fc (e.g., IgG1 Fc) comprises any one or more of the sets of amino acid substitutions set forth in Table 11. In some embodiments, the hIg Fc (e.g., IgG1 Fc) region comprises any one or more of the glycosylation changes set forth in Table 11.

[0286] In some embodiments, the modified Ig Fc fusion protein comprises a hIg Fc region comprising one or more amino acid variation. In some embodiments, the modified hIg Fc fusion protein comprises a hIg4 Fc region comprising one or more amino acid variation. In some embodiments, the hIgG4 Fc region comprises an amino acid substitution at amino acid positions S228, F234, and / or L235, EU numbering according to Kabat. In some embodiments, the hIgG4 Fc region comprises the following amino acid substitutions S228P, F234A, and / or L235A, EUAttorney Docket No.62801.71WO01 numbering according to Kabat. In some embodiments, the hIgG4 Fc region comprises the following amino acid substitutions S228P, F234A, and / or L235E, EU numbering according to Kabat. In some embodiments, the hIgG4 Fc comprises the following amino acid substitutions S228P and / or L235E, EU numbering according to Kabat.

[0287] In some embodiments, the S228P variation stabilized the hinge region. See, e.g., Silva, John-Paul et al. “The S228P mutation prevents in vivo and in vitro IgG4 Fab-arm exchange as demonstrated using a combination of novel quantitative immunoassays and physiological matrix preparation.” The Journal of biological chemistry vol. 290,9 (2015): 5462-9. doi:10.1074 / jbc.M114.600973, the entire contents of which is incorporated herein by reference for all purposes.

[0288] In some embodiments, the modified hIg Fc fusion protein comprises a hIgG1 Fc region comprising one or more amino acid variations. In some embodiments, the hIgG1 Fc region comprises an amino acid substitution at amino acid positions L234, L235, and / or P329, EU numbering according to Kabat. In some embodiments, the hIgG1 Fc region comprises the following amino acid substitutions L234A and / or L235A, EU numbering according to Kabat. In some embodiments, the hIgG1 Fc region comprises the following amino acid substitutions L234A, L235A, and P329G, EU numbering according to Kabat. In some embodiments, the hIgG1 Fc region comprises the following amino acid substitutions L234A, L235A, and P329A, EU numbering according to Kabat.

[0289] In some embodiments, the modified hIg Fc fusion protein comprises a hIgG1 Fc region comprising one or more amino acid variations. In some embodiments, the hIgG1 Fc region comprises an amino acid substitution at amino acid positions L235, G237, and / or P329, EU numbering according to Kabat. In some embodiments, the hIgG1 Fc region comprises the following amino acid substitutions L235A and / or G237A, EU numbering according to Kabat. In some embodiments, the hIgG1 Fc region comprises the following amino acid substitutions L235A, G237A, and P329G, EU numbering according to Kabat. In some embodiments, the hIgG1 Fc region comprises the following amino acid substitutions L235A, G237A, and P329A, EU numbering according to Kabat.

[0290] The amino acid sequence of exemplary variant hIg Fc regions that are known in the art to exhibit a decrease in one more effector function is provided in Table 6.Attorney Docket No.62801.71WO01 Table 6. The amino acid sequence of exemplary variant hIg Fc Regions. Description Amino Acid Sequence SEQ ID NOAttorney Docket No.62801.71WO01 S228P / F234A / L235A EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKS With C-terminal L sine LSLSLGKAttorney Docket No.62801.71WO01 (Variant) REPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSV S228P / F234A / L235A MHEALHNHYTQKSLSLSLG ioncomprising an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a polypeptide set forth in Table 6. For example, the variant hIg Fc fusion protein may comprise a hIg Fc region comprising an amino acid sequence at least 85% identical to the amino acid sequence of a polypeptide set forth in Table 6. The variant hIg Fc fusion protein may comprise a hIg Fc region comprising an amino acid sequence at least 90% identical to the amino acid sequence of a polypeptide set forth in Table 6. The variant hIg Fc fusion protein may comprise a hIg Fc region comprising an amino acid sequence at least 95% identical to the amino acid sequence of a polypeptide set forth in Table 6. In some embodiments, the variant hIg Fc fusion protein preferably may comprise a hIg Fc region comprising an amino acid sequence 100% identical to the amino acid sequence of a polypeptide set forth in Table 6.

[0292] In some embodiments, the variant hIg Fc fusion protein comprises a hIg Fc region consisting of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a polypeptide set forth in Table 6. For example, the variant hIg Fc fusion protein may comprise a hIg Fc region consisting of an amino acid sequence at least 85% identical to the amino acid sequence of a polypeptide set forth in Table 6. The variant hIg Fc fusion protein may comprise a hIg Fc region consisting of an amino acid sequence at least 90% identical to the amino acid sequence of a polypeptide set forth in Table 6. The variant hIg Fc fusion protein may comprise a hIg Fc region consisting of an amino acid sequence at least 95% identical to the amino acid sequence of a polypeptide set forth in Table 6. In some embodiments, the variant hIg Fc fusion protein preferably may comprise a hIg Fc region consisting of an amino acid sequence 100% identical to the amino acid sequence of a polypeptide set forth in Table 6.

[0293] In some embodiments, the amino acid sequence of the variant hIg Fc fusion protein comprises a hIg Fc region that comprises the amino acid sequence of a polypeptide set forth in Table 6, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, lessAttorney Docket No.62801.71WO01 than 8%), amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the variant hIg Fc fusion protein comprises a hIg Fc region that comprises the amino acid sequence of a polypeptide set forth in Table 6, and further comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the variant hIg Fc fusion protein comprises a hIg Fc region that comprises the amino acid sequence of a polypeptide set forth in Table 6, and further comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the variant hIg Fc fusion protein comprises a hIg Fc region that comprises the amino acid sequence of a polypeptide set forth in Table 6, and further consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the variant hIg Fc fusion protein comprises a hIg Fc region that comprises the amino acid sequence of a polypeptide set forth in Table 6, and further comprises no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.).

[0294] In some embodiments, the amino acid sequence of the variant hIg Fc fusion protein comprises a hIg Fc region that consists of the amino acid sequence of a polypeptide set forth in Table 6, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the variant hIg Fc fusion protein comprises a hIg Fc region that consists of the amino acid sequence of a polypeptide set forth in Table 6, and further comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the variant hIg Fc fusion protein comprises a hIg Fc region that consists of the amino acid sequence of a polypeptide set forth in Table 6, and further comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the variant hIg Fc fusion protein comprises a hIg Fc region that consists of the amino acid sequence of a polypeptide set forth in Table 6, and further consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the variant hIg Fc fusion protein comprises a hIg Fc region that consists of the amino acid sequence of a polypeptide set forth in Table 6, and further comprises no moreAttorney Docket No.62801.71WO01 than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.).

[0295] In some embodiments, the amino acid sequence of the variant hIg Fc fusion protein comprises a hIg Fc region that comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 74-87. For example, the amino acid sequence of the variant hIg Fc fusion protein may comprise a hIg Fc region that comprises an amino acid sequence at least 85% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 74-87. The amino acid sequence of the variant hIg Fc fusion protein may comprise a hIg Fc region that comprises an amino acid sequence at least 90% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 74-87. The amino acid sequence of the variant hIg Fc fusion protein may comprise a hIg Fc region that comprises an amino acid sequence at least 95% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 74-87. In some embodiments, the amino acid sequence of the variant hIg Fc fusion protein preferably may comprise a hIg Fc region that comprises an amino acid sequence 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 74-87.

[0296] In some embodiments, the amino acid sequence of the variant hIg Fc fusion protein comprises a hIg Fc region that consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 74-87. For example, the amino acid sequence of the variant hIg Fc fusion protein may comprise a hIg Fc region that consists of an amino acid sequence at least 85% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 74-87. The amino acid sequence of the variant hIg Fc fusion protein may comprise a hIg Fc region that consists of an amino acid sequence at least 90% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 74-87. The amino acid sequence of the variant hIg Fc fusion protein may comprise a hIg Fc region that consists of an amino acid sequence at least 95% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 74-87. In some embodiments, the amino acid sequence of the variant hIg Fc fusion protein preferably may comprise a hIg Fc region that consists of an amino acid sequence 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 74-87.

[0297] In some embodiments, the amino acid sequence of the variant hIg Fc fusion proteinAttorney Docket No.62801.71WO01 comprises a hIg Fc region that comprises the amino acid sequence set forth in any one of SEQ ID NOS: 74-87, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the variant hIg Fc fusion protein comprises a hIg Fc region that comprises the amino acid sequence set forth in any one of SEQ ID NOS: 74-87, and further comprises or consists of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the variant hIg Fc fusion protein comprises a hIg Fc region that comprises the amino acid sequence set forth in any one of SEQ ID NOS: 74-87, and further comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the variant hIg Fc fusion protein comprises a hIg Fc region that comprises the amino acid sequence set forth in any one of SEQ ID NOS: 74-87, and further consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the variant hIg Fc fusion protein comprises a hIg Fc region that comprises the amino acid sequence set forth in any one of SEQ ID NOS: 74-87, and further comprises no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.).

[0298] In some embodiments, the amino acid sequence of the variant hIg Fc fusion protein comprises a hIg Fc region that consists of the amino acid sequence set forth in any one of SEQ ID NOS: 74-87, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the variant hIg Fc fusion protein comprises a hIg Fc region that consists of the amino acid sequence set forth in any one of SEQ ID NOS: 74-87, and further comprises or consists of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the variant hIg Fc fusion protein comprises a hIg Fc region that consists of the amino acid sequence set forth in any one of SEQ ID NOS: 74-87, and further comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the variant hIg Fc fusion protein comprises a hIg Fc region that consists of the amino acid sequence set forth in any one of SEQ ID NOS: 74-87, and further consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions,Attorney Docket No.62801.71WO01 etc.). In some embodiments, the amino acid sequence of the variant hIg Fc fusion protein comprises a hIg Fc region that consists of the amino acid sequence set forth in any one of SEQ ID NOS: 74- 87, and further comprises no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.).

[0299] In some embodiments, the variant mIg Fc fusion protein comprises a mIgG2a Fc region comprising one or more amino acid variations. In some embodiments, the mIgG2a Fc region comprises an amino acid substitution at amino acid positions L234, L235, and / or P329, EU numbering according to Kabat. In some embodiments, the mIgG2a Fc region comprises the following amino acid substitutions L234P and / or L235P, EU numbering according to Kabat. In some embodiments, the mIgG2a Fc region comprises the following amino acid substitutions L234P, L235P, and P329G, EU numbering according to Kabat. In some embodiments, the mIgG2a Fc region comprises the following amino acid substitutions L234P, L235P, and P329A, EU numbering according to Kabat.

[0300] In some embodiments, the variant mIg Fc fusion protein comprises a mIgG2a Fc region comprising one or more amino acid variations. In some embodiments, the mIgG2a Fc region comprises an amino acid substitution at amino acid positions L234, L235, and / or P329, EU numbering according to Kabat. In some embodiments, the mIgG2a Fc region comprises the following amino acid substitutions L234A and / or L235A, EU numbering according to Kabat. In some embodiments, the mIgG2a Fc region comprises the following amino acid substitutions L234A, L235A, and P329G, EU numbering according to Kabat. In some embodiments, the mIgG2a Fc region comprises the following amino acid substitutions L234A, L235A, and P329A, EU numbering according to Kabat.

[0301] The amino acid sequence of exemplary variant hIg Fc regions that are known in the art to exhibit a decrease in one more effector function is provided in Table 7. Table 7. The amino acid sequence of exemplary variant mIg Fc Regions. Description Amino Acid Sequence SEQ OAttorney Docket No.62801.71WO01 mIgG2a CH2 Region + APNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDV CH3 Region QISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSG KEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTAttorney Docket No.62801.71WO01

[0302] In some embodiments, the variant mIg Fc fusion protein comprises a mIg Fc region comprising an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a polypeptide set forth in Table 7. For example, the variant mIg Fc fusion protein may comprise a mIg Fc region comprising an amino acid sequence at least 85% identical to the amino acid sequence of a polypeptide set forth in Table 7. The variant mIg Fc fusion protein may comprise a mIg Fc region comprising an amino acid sequence at least 90% identical to the amino acid sequence of a polypeptide set forth in Table 7. The variant mIg Fc fusion protein may comprise a mIg Fc region comprising an amino acid sequence at least 95% identical to the amino acid sequence of a polypeptide set forth in Table 7. In some embodiments, the variant mIg Fc fusion protein preferably may comprise a mIg Fc region comprising an amino acid sequence 100% identical to the amino acid sequence of a polypeptide set forth in Table 7.

[0303] In some embodiments, the amino acid sequence of the variant mIg Fc fusion protein comprises a mIg Fc region that comprises or consists of the amino acid sequence of a polypeptide set forth in Table 7, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the variant mIg Fc fusion protein comprises a mIg Fc region that comprises or consists of the amino acid sequence of a polypeptide set forth in Table 7, and further comprises or consists of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the variant mIg Fc fusion protein comprises a mIg Fc region that comprises or consists of the amino acid sequence of a polypeptide set forth in Table 7, and further comprises or consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the variant mIg Fc fusion protein comprises a mIg Fc region that comprises or consists of the amino acid sequence of a polypeptide set forth in Table 7, and further comprises or consists of no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.).

[0304] In some embodiments, the amino acid sequence of the variant mIg Fc fusion protein comprises a mIg Fc region that comprises or consists of the amino acid sequence of a polypeptide set forth in Table 7, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid substitutions. In some embodiments, the amino acid sequence ofAttorney Docket No.62801.71WO01 the variant mIg Fc fusion protein comprises a mIg Fc region that comprises or consists of the amino acid sequence of a polypeptide set forth in Table 7, and further comprises or consists of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions. In some embodiments, the amino acid sequence of the variant mIg Fc fusion protein comprises a mIg Fc region that comprises or consists of the amino acid sequence of a polypeptide set forth in Table 7, and further comprises or consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions. In some embodiments, the amino acid sequence of the variant mIg Fc fusion protein comprises a mIg Fc region that comprises or consists of the amino acid sequence of a polypeptide set forth in Table 7, and further comprises or consists of no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions.

[0305] In some embodiments, the amino acid sequence of the variant mIg Fc fusion protein comprises a mIg Fc region that comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 88-95. For example, the amino acid sequence of the variant mIg Fc fusion protein may comprise a mIg Fc region that comprises an amino acid sequence at least 85% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 88-95. The amino acid sequence of the variant mIg Fc fusion protein may comprise a mIg Fc region that comprises an amino acid sequence at least 90% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 88-95. The amino acid sequence of the variant mIg Fc fusion protein may comprise a mIg Fc region that comprises an amino acid sequence at least 95% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 88-95. In some embodiments, the amino acid sequence of the variant mIg Fc fusion protein preferably may comprise a mIg Fc region that comprises an amino acid sequence 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 88-95. In some embodiments, the amino acid sequence of the variant mIg Fc fusion protein comprises a mIg Fc region that consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 88-95. For example, the amino acid sequence of the variant mIg Fc fusion protein may comprise a mIg Fc region that consists of an amino acid sequence at least 85% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 88-95. The amino acid sequence of the variant mIg Fc fusion protein may comprise a hIg Fc region that consists of an amino acid sequence at least 90% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 88-95. The amino acid sequence of the variant mIg Fc fusionAttorney Docket No.62801.71WO01 protein may comprise a mIg Fc region that consists of an amino acid sequence at least 95% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 88-95. In some embodiments, the amino acid sequence of the variant mIg Fc fusion protein preferably may comprise a mIg Fc region that consists of an amino acid sequence 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 88-95.

[0306] In some embodiments, the amino acid sequence of the variant mIg Fc fusion protein comprises a mIg Fc region that comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOS: 88-95, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the variant mIg Fc fusion protein comprises a mIg Fc region that comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOS: 88-95, and further comprises or consists of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the variant mIg Fc fusion protein comprises a mIg Fc region that comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOS: 88-95, and further comprises or consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the variant mIg Fc fusion protein comprises a mIg Fc region that comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOS: 88-95, and further comprises or consists of no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.).

[0307] In some embodiments, the amino acid sequence of the variant mIg Fc fusion protein comprises a mIg Fc region that comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOS: 88-95, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid substitutions. In some embodiments, the amino acid sequence of the variant mIg Fc fusion protein comprises a mIg Fc region that comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOS: 88-95, and further comprises or consists of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions. In some embodiments, the amino acid sequence of the variant mIg Fc fusion protein comprises a mIg Fc region that comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOS: 88-95, and further comprises or consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acidAttorney Docket No.62801.71WO01 substitutions. In some embodiments, the amino acid sequence of the variant mIg Fc fusion protein comprises a mIg Fc region that comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOS: 88-95, and further comprises or consists of no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions. (ii) Enhanced Ig Effector Function

[0308] In some embodiments, the Ig Fc region exhibits an enhancement (e.g., an increase) in one or more Fc effector function relative to a reference (e.g., wild type) Ig Fc region. Exemplary Ig Fc effector functions include, but are not limited to, ADCC, ADCP, CDC, binding affinity to C1q, and binding affinity to one or more human Fc receptor (e.g., an Fcγ receptor (e.g., (e.g., FcγRI, FcγRIIa, FcγRIIIa, and / or FcγRIIIb). In some embodiments, the Fc region exhibits one or more enhanced Fc effector function, relative to a reference Ig (e.g., hIg, mIg).

[0309] In some embodiments, the hIg Fc region is modified (e.g., comprises one or more variation (e.g., one or more amino acid substitution, deletion, addition, etc.); altered glycosylation (e.g., afucosylation))) (referred to herein as a “modified hIg Fc”). In some embodiments, the modification (e.g., the variation (e.g., one or more amino acid substitution, deletion, addition, etc.); altered glycosylation (e.g., afucosylation))) enhances (e.g., increases) one or more Fc effector function, relative to a reference hIg Fc that does not comprise the modification (e.g., the one or more variation (e.g., the one or more amino acid substitution, deletion, addition, etc.; the altered glycosylation (e.g., afucosylation))).

[0310] In some embodiments, the modified Ig (e.g., hIg, mIg) Fc fusion protein exhibits enhanced ADCC compared to a reference fusion protein that does not comprise the Ig (e.g., hIg, mIg) Fc modification (e.g., the one or more variation (e.g., one or more amino acid substitution, deletion, or addition)). In some embodiments, the modified Ig (e.g., hIg, mIg) Fc fusion protein exhibits enhanced CDC compared to a reference fusion protein that does not comprise the Ig (e.g., hIg, mIg) Fc modification (e.g., the one or more variation (e.g., one or more amino acid substitution, deletion, or addition)). In some embodiments, the modified Ig (e.g., hIg, mIg) Fc fusion protein exhibits enhanced ADCP compared to a reference fusion protein that does not comprise the Ig (e.g., hIg, mIg) Fc modification (e.g., the one or more variation (e.g., one or more amino acid substitution, deletion, or addition)).

[0311] In some embodiments, the modified Ig (e.g., hIg, mIg) Fc fusion protein exhibits enhanced binding affinity to one or more Fc receptor (e.g., human Fc receptor) (e.g., an FcγAttorney Docket No.62801.71WO01 receptor (e.g., FcγRI, FcγRIIa, FcγRIIIa, and / or FcγRIIIb) compared to a reference fusion protein that does not comprise the Ig (e.g., hIg, mIg) Fc modification (e.g., the one or more variation (e.g., one or more amino acid substitution, deletion, or addition)). In some embodiments, the modified Ig (e.g., hIg, mIg) Fc fusion protein exhibits enhanced binding affinity to FcγRI, FcγRIIa, FcγRIIIa, and / or FcγRIIIb compared to a reference fusion protein that does not comprise the hIg Fc modification (e.g., the one or more variation (e.g., one or more amino acid substitution, deletion, or addition)). In some embodiments, the modified Ig (e.g., hIg, mIg) Fc fusion protein exhibits enhanced binding affinity to FcγRI compared to a reference fusion protein that does not comprise the Ig (e.g., hIg, mIg) Fc modification (e.g., the one or more variation (e.g., one or more amino acid substitution, deletion, or addition)). In some embodiments, the modified Ig (e.g., hIg, mIg) Fc fusion protein exhibits enhanced binding affinity to FcγRIIa compared to a reference fusion protein that does not comprise the Ig (e.g., hIg, mIg) Fc modification (e.g., the one or more variation (e.g., one or more amino acid substitution, deletion, or addition)). In some embodiments, the modified Ig (e.g., hIg, mIg) Fc fusion protein exhibits enhanced binding affinity to FcγRIIIa compared to a reference fusion protein that does not comprise the Ig (e.g., hIg, mIg) Fc modification (e.g., the one or more variation (e.g., one or more amino acid substitution, deletion, or addition)). In some embodiments, the modified Ig (e.g., hIg, mIg) Fc fusion protein exhibits enhanced binding affinity to FcγRIIIb compared to a reference fusion protein that does not comprise the Ig (e.g., hIg, mIg) Fc modification (e.g., the one or more variation (e.g., one or more amino acid substitution, deletion, or addition)).

[0312] In some embodiments, the modified Ig (e.g., hIg, mIg) Fc fusion protein exhibits reduced binding affinity to one or more Fc receptor (e.g., human Fc receptor) ((e.g., an Fcγ receptor (e.g., FcγRIIb)) compared to a reference fusion protein that does not comprise the Ig (e.g., hIg, mIg) Fc modification (e.g., the one or more variation (e.g., one or more amino acid substitution, deletion, or addition)). In some embodiments, the modified Ig (e.g., hIg, mIg) Fc fusion protein exhibits reduced binding affinity to FcγRIIb compared to a reference fusion protein that does not comprise the Ig (e.g., hIg, mIg) Fc modification (e.g., the one or more variation (e.g., one or more amino acid substitution, deletion, or addition)).

[0313] In some embodiments, the modified Ig (e.g., hIg, mIg) Fc fusion protein exhibits enhanced binding affinity to C1q compared to a reference fusion protein that does not comprise the Ig (e.g., hIg, mIg) Fc modification (e.g., the one or more variation (e.g., one or more aminoAttorney Docket No.62801.71WO01 acid substitution, deletion, or addition)).

[0314] Amino acid substitutions and glycoengineering that enhance (e.g., increase) one or more hIg Fc effector function are known in the art. See for example, Liu R, Oldham RJ, Teal E, Beers SA, Cragg MS. Fc-Engineering for Modulated Effector Functions-Improving Antibodies for Cancer Treatment. Antibodies (Basel). 2020;9(4):64. Published 2020 Nov 17. doi:10.3390 / antib9040064; van der Horst HJ, Nijhof IS, Mutis T, Chamuleau MED. Fc- Engineered Antibodies with Enhanced Fc-Effector Function for the Treatment of B-Cell Malignancies. Cancers (Basel). 2020;12(10):3041. Published 2020 Oct 19. Doi:10.3390 / cancers12103041; and Saunders Kevin, “Conceptual Approaches to Modulating Antibody Effector Functions and Circulation Half-Life,” Frontiers in Immunology, v10 (June 7, 2019) DOI=10.3389 / fimmu.2019.01296, the full contents of each of which is incorporated by reference herein for all purposes.

[0315] Table 12 below, provides exemplary amino acid substitutions (and combinations thereof) and glycoengineering that can be utilized to increase one or more hIg Fc effector function. Amino acids in Table 12 are numbered according to the EU numbering scheme. The effects on effector function set forth in Table 12 are exemplary only and not intended to be limiting. The amino acid substitutions set forth in Table 12 are with reference to an IgG1 Fc region (except where noted). However, a person of ordinary skill in the could identify the corresponding amino acid in a non-IgG1 Fc region, for example in an IgG2 or IgG4 Fc region, should the base amino acid be different between the IgG1 and non-IgG1 Fc region. Table 12. Exemplary hIg Fc Variations and Glycoengineering to Increase Effector Function. Variation / Glycoengineering Exemplary Effects on Effector FunctionAttorney Docket No.62801.71WO01 L234Y / L235Q / G D270E / K326D / A3 236W / S239M / H 30M / K334E ghe hIg Fc (e.g., IgG1 Fc) region comprises any one or more of the amino acid substitutions set forth in Table 12 (i.e., any one or more amino acid substitution set forth in any set of amino acid substitutions set forth in Table 12). In some embodiments, the hIg Fc (e.g., IgG1 Fc) comprises any one or more of the sets of amino acid substitutions set forth in Table 12. In some embodiments, the hIg Fc (e.g., IgG1 Fc) region comprises any one or more of the glycosylation changes set forth in Table 12.

[0317] In some embodiments, the hIg Fc (e.g., IgG1 Fc) region comprises an amino acid substitution at any one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or more) of amino acid positions S298, E333, K334, S239, I332, P247, A339, A330, G236, F243, R292, Y300, V305, P396, L235, F243, R292, Y300, P396, F243, R292, Y300, V305, P396, K326, E333, S267E, H268, S324, S298, E333, K334, L234, L235, G236, S239, H268, D270, S298 D270, K326, A330, and / or K334. In some embodiments, the hIg Fc (e.g., IgG1 Fc) region comprises an amino acid substitution at from about 1-10 (e.g., 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2) of the following amino acid positions S298, E333, K334, S239, I332, P247, A339, A330, G236, F243, R292, Y300, V305, P396, L235, F243, R292, Y300, P396, F243, R292, Y300, V305, P396, K326, E333, S267E, H268, S324, S298,Attorney Docket No.62801.71WO01 E333, K334, L234, L235, G236, S239, H268, D270, S298 D270, K326, A330, and / or K334.

[0318] In some embodiments, the hIg Fc (e.g., IgG1 Fc) region comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or more) of the following amino acid substitutions S298A, E333A, K334A, S239D, I332E, P247I, A339Q, A330L, G236A, F243L, R292P, Y300L, V305I, P396L, L235V, F243L, R292P, Y300L, P396L, F243L, R292P, Y300L, V305I, P396L, K326W, E333S, S267E, H268E, S324T, S298A, E333A, K334A, L234Y, L235Q, G236W, S239M, H268D, D270E, S298A D270E, K326D, A330M, and / or K334E.

[0319] In some embodiments, the hIg Fc (e.g., IgG1 Fc) region comprises from about 1-10 (e.g., 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2) of the following amino acid substitutions S298A, E333A, K334A, S239D, I332E, P247I, A339Q, A330L, G236A, F243L, R292P, Y300L, V305I, P396L, L235V, F243L, R292P, Y300L, P396L, F243L, R292P, Y300L, V305I, P396L, K326W, E333S, S267E, H268E, S324T, S298A, E333A, K334A, L234Y, L235Q, G236W, S239M, H268D, D270E, S298A D270E, K326D, A330M, and / or K334E.

[0320] In some embodiments, the hIg Fc region comprises a hIgG1 Fc region comprising one or more amino acid variation relative to a reference hIgG1 Fc region.

[0321] In some embodiments, the hIgG1 Fc region comprises an amino acid substitution at one or more (e.g., 1, 2, or 3) of amino acid positions S298, E333, K334, EU numbering according to Kabat. In some embodiments, the hIgG1 Fc region comprises one or more (e.g., 1, 2, or 3) of the following amino acid substitutions S298A, E333A, and / or K334A, EU numbering according to Kabat.

[0322] In some embodiments, the hIgG1 Fc region comprises an amino acid substitution at one or more (e.g., 1 or 2) of amino acid positions S239 and / or I332, EU numbering according to Kabat. In some embodiments, the hIgG1 Fc region comprises one or more (e.g., 1 or 2) of the following amino acid substitutions S239D and / or I332E, EU numbering according to Kabat.

[0323] In some embodiments, the hIgG1 Fc region comprises an amino acid substitution at one or more (e.g., 1 or 2) of amino acid positions P247 and / or A339, EU numbering according to Kabat. In some embodiments, the hIgG1 Fc region comprises one or more (e.g., 1 or 2) of the following amino acid substitutions P247I and / or A339Q, EU numbering according to Kabat.

[0324] In some embodiments, the hIgG1 Fc region comprises an amino acid substitution at one or more (e.g., 1, 2, or 3) of amino acid positions S239, A330, and / or I332, EU numbering according to Kabat. In some embodiments, the hIgG1 Fc region comprises one or more (e.g., 1, 2,Attorney Docket No.62801.71WO01 or 3) of the following amino acid substitutions S239D, A330L, and / or I332E, EU numbering according to Kabat.

[0325] In some embodiments, the hIgG1 Fc region comprises an amino acid substitution at one or more (e.g., 1, 2, or 3) of amino acid positions G236, S239, and / or I332, EU numbering according to Kabat. In some embodiments, the hIgG1 Fc region comprises one or more (e.g., 1, 2, or 3) of the following amino acid substitutions G236A, S239D, and / or I332E, EU numbering according to Kabat.

[0326] In some embodiments, the hIgG1 Fc region comprises an amino acid substitution at one or more (e.g., 1, 2, 3, 4, or 5) of amino acid positions F243, R292, Y300, V305, and / or P396, EU numbering according to Kabat. In some embodiments, the hIgG1 Fc region comprises one or more (e.g., 1, 2, 3, 4, or 5) of the following amino acid substitutions F243L, R292P, Y300L, V305I, and / or P396L, EU numbering according to Kabat.

[0327] In some embodiments, the hIgG1 Fc region comprises an amino acid substitution at one or more (e.g., 1, 2, 3, 4, or 5) of amino acid positions L235, F243, R292, Y300, and P396, EU numbering according to Kabat. In some embodiments, the hIgG1 Fc region comprises one or more (e.g., 1, 2, 3, 4, or 5) of the following amino acid substitutions L235V, F243L, R292P, Y300L, and / or P396L, EU numbering according to Kabat.

[0328] In some embodiments, the hIgG1 Fc region comprises an amino acid substitution at one or more (e.g., 1, 2, 3, 4, 5, 6, or 7) of amino acid positions L234, L235, G236, S239, H268, D270, and / or S298, EU numbering according to Kabat. In some embodiments, the hIgG1 Fc region comprises one or more (e.g., 1, 2, 3, 4, 5, 6, or 7) of the following amino acid substitutions L234Y, L235Q, G236W, S239M, H268D, D270E, and / or S298A, EU numbering according to Kabat.

[0329] In some embodiments, the hIgG1 Fc region comprises an amino acid substitution at one or more (e.g., 1, 2, 3, or 4) of amino acid positions D270, K326, A330, and / or K334, EU numbering according to Kabat. In some embodiments, the hIgG1 Fc region comprises one or more (e.g., 1, 2, 3, or 4) of the following amino acid substitutions D270E, K326D, A330M, and / or K334E, EU numbering according to Kabat.

[0330] In some embodiments, the hIgG1 Fc region comprises an amino acid substitution at one or more (e.g., 1, 2, 3, 4, or 5) of amino acid positions F243, R292, Y300, V305, and / or P396, EU numbering according to Kabat. In some embodiments, the hIgG1 Fc region comprises one or more (e.g., 1, 2, 3, 4, or 5) of the following amino acid substitutions F243L, R292P, Y300L, V305I,Attorney Docket No.62801.71WO01 and / or P396L, EU numbering according to Kabat.

[0331] In some embodiments, the hIgG1 Fc region comprises an amino acid substitution at one or more (e.g., 1, 2, or 3) of amino acid positions S239, I332, and / or A330, EU numbering according to Kabat. In some embodiments, the hIgG1 Fc region comprises one or more (e.g., 1, 2, or 3) of the following amino acid substitutions S239D, I332E, and / or A330L, EU numbering according to Kabat.

[0332] In some embodiments, the hIgG1 Fc region comprises an amino acid substitution at one or more (e.g., 1, 2, 3, or 4) of amino acid positions S239, I332, A330, and / or G236, EU numbering according to Kabat. In some embodiments, the hIgG1 Fc region comprises one or more (e.g., 1, 2, 3, or 4) of the following amino acid substitutions S239D, I332E, A330L and / or G236A, EU numbering according to Kabat.

[0333] In some embodiments, the hIgG1 Fc region comprises an amino acid substitution at one or more (e.g., 1, 2, or 3) of amino acid positions S239, I332, and / or G326, EU numbering according to Kabat. In some embodiments, the hIgG1 Fc region comprises one or more (e.g., 1, 2, or 3) of the following amino acid substitutions S239D, I332E, and / or G326A, EU numbering according to Kabat.

[0334] In some embodiments, the hIgG1 Fc region comprises an amino acid substitution at amino acid position G326, EU numbering according to Kabat. In some embodiments, the hIgG1 Fc region comprises a G326A amino acid substitution, EU numbering according to Kabat.

[0335] In some embodiments, the hIgG1 Fc region comprises an amino acid substitution at one or more (e.g., 1, 2, or 3) of amino acid positions G236, S239, and / or I332, EU numbering according to Kabat. In some embodiments, the hIgG1 Fc region comprises one or more (e.g., 1, 2, or 3) of the following amino acid substitutions G236A, S239D, and / or I332E, EU numbering according to Kabat.

[0336] In some embodiments, the hIgG1 Fc region comprises an amino acid substitution at one or more (e.g., 1 or 2) of amino acid positions S239 and / or I332, EU numbering according to Kabat. In some embodiments, the hIgG1 Fc region comprises one or more (e.g., 1 or 2) of the following amino acid substitutions S239D and / or I332E, EU numbering according to Kabat.

[0337] In some embodiments, the hIgG1 Fc region comprises an amino acid substitution at one or more (e.g., 1 or 2) of amino acid positions K326 and / or E333, EU numbering according to Kabat. In some embodiments, the hIgG1 Fc region comprises one or more (e.g., 1 or 2) of theAttorney Docket No.62801.71WO01 following amino acid substitutions K326W and / or E333S, EU numbering according to Kabat.

[0338] In some embodiments, the hIgG1 Fc region comprises an amino acid substitution at one or more (e.g., 1, 2, or 3) of amino acid positions S267, H268, and / or S324, EU numbering according to Kabat. In some embodiments, the hIgG1 Fc region comprises one or more (e.g., 1, 2, or 3) of the following amino acid substitutions S267E, H268E, and / or S324T, EU numbering according to Kabat.

[0339] In some embodiments, the hIgG1 Fc region comprises an amino acid substitution at one or more (e.g., 1, 2, or 3) of amino acid positions S298, E333, and / or K334, EU numbering according to Kabat. In some embodiments, the hIgG1 Fc region comprises one or more (e.g., 1, 2, or 3) of the following amino acid substitutions S298A, E333A, and / or K334A, EU numbering according to Kabat.

[0340] In some embodiments, the hIgG1 Fc region comprises an amino acid substitution at one or more (e.g., 1 or 2) of amino acid positions S239 and / or I332, EU numbering according to Kabat. In some embodiments, the hIgG1 Fc region comprises one or more (e.g., 1 or 2) of the following amino acid substitutions S239D and / or I332E, EU numbering according to Kabat.

[0341] In some embodiments, the hIgG1 Fc region comprises an amino acid substitution at one or more (e.g., 1 or 2) of amino acid positions P247 and / or A339, EU numbering according to Kabat. In some embodiments, the hIgG1 Fc region comprises one or more (e.g., 1 or 2) of the following amino acid substitutions P247I and / or A339Q, EU numbering according to Kabat.

[0342] In some embodiments, the hIg Fc region comprises one or more changes to the glycosylation. In some embodiments, the hIg Fc region is afucosylated. In some embodiments, the hIg Fc region is afucosylated and exhibits enhanced (e.g., increased) ADCC compared to a reference hIg Fc region that is not afucosylated. Afucosylated antibodies can be made, e.g., through modifying the amino acid sequence, utilizing an engineered cell line (e.g., CHOK1-FUT8), and utilizing specific cell culture media. See, e.g., Pereira, Natasha A et al. “The "less-is-more" in therapeutic antibodies: Afucosylated anti-cancer antibodies with enhanced antibody-dependent cellular cytotoxicity.” mAbs vol.10,5 (2018): 693-711. doi:10.1080 / 19420862.2018.1466767, the entire contents of which are incorporated herein by reference for all purposes. 5.4.6 Linkers

[0343] As described herein, the heterologous moiety (e.g., heterologous protein) can beAttorney Docket No.62801.71WO01 directly operably connected or indirectly operably connected to the immunoreceptor inhibitory protein (e.g., described herein). In some embodiments, the heterologous protein is directly operably connected to the immunoreceptor inhibitory protein (e.g., described herein) via a peptide bond. In some embodiment, the heterologous protein is indirectly operably connected to the immunoreceptor inhibitory protein (e.g., described herein) via a peptide linker.

[0344] In some embodiments, the peptide linker is one or any combination of a cleavable linker, a non-cleavable linker, a flexible linker, a rigid linker, a helical linker, and / or a non-helical linker.

[0345] In some embodiments, the amino acid sequence of the peptide linker comprises from or from about 2-30, 5-30, 10-30, 15-30, 20-30, 25-30, 2-25, 5-25, 10-25, 15-25, 20-25, 2-20, 5-20, 10-20, 15-20, 2-15, 5-15, 10-15, 2-10, or 5-10 amino acid residues. In some embodiments, the amino acid sequence of the peptide linker comprises at least about 2, 3, 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 amino acid residues. In some embodiments, the amino acid sequence of the peptide linker comprises about 2, 3, 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 amino acid residues. In some embodiments, the amino acid sequence of the peptide linker consists of about 2, 3, 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 amino acid residues. In some embodiments, the amino acid sequence of the peptide linker comprises no more than about 2, 3, 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 amino acid residues. In some embodiments, the amino acid sequence of the peptide linker consists of no more than about 2, 3, 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 amino acid residues.

[0346] In some embodiments, the amino acid sequence of the peptide linker comprises glycine, serine, or both glycine and serine amino acid residues. In some embodiments, the amino acid sequence of the peptide linker comprises glycine, serine, and proline amino acid residues. In some embodiments, the amino acid sequence of the peptide linker consists of glycine, serine, or both glycine and serine amino acid residues. In some embodiments, the amino acid sequence of the peptide linker consists of glycine, serine, and proline amino acid residues.

[0347] The amino acid sequence of exemplary peptide linkers, which can be incorporated in one or more of the embodiments described herein (e.g., fusion proteins), is set provided in Table 8.Attorney Docket No.62801.71WO01 Table 8. The Amino Acid Sequence of Exemplary Peptide Linkers. Description Amino Acid Sequence SEQ ID NO A96the amino acid sequence of any one of the linkers set forth in Table 8. In some embodiments, the amino acid sequence of the peptide linker consists of the amino acid sequence of any one of the linkers set forth in Table 8. In some embodiments, the amino acid sequence of the peptide linker comprises the amino acid sequence of any one of the linkers set forth in Table 8, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the peptide linker consists of the amino acid sequence of any one of the linkers set forth in Table 8, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the peptide linker comprises the amino acid sequence of any one of the linkers set forth in Table 8, comprising 1, 2, or 3 amino acid variations (e.g., substitutions, deletions, additions). In some embodiments, the amino acid sequence of the peptide linker consists of the amino acid sequence of any one of the linkers set forth in Table 8, comprising 1, 2, or 3 amino acid variations (e.g., substitutions, deletions, additions). In some embodiments, the amino acid sequence of the peptide linker comprises the amino acid sequence of any one of the linkers set forth in Table 8, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid substitutions. In some embodiments, the amino acid sequence of the peptide linker consists of the amino acid sequence of any one of the linkers set forth in Table 8, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid substitutions. In some embodiments, the amino acid sequence of the peptide linker comprises the amino acid sequence of any one of the linkers set forth in TableAttorney Docket No.62801.71WO01 8, comprising 1, 2, or 3 amino acid substitutions. In some embodiments, the amino acid sequence of the peptide linker consists of the amino acid sequence of any one of the linkers set forth in Table 8, comprising 1, 2, or 3 amino acid substitutions.

[0349] In some embodiments, the amino acid sequence of the peptide linker comprises the amino acid sequence set forth in any one of SEQ ID NOS: 96-105. In some embodiments, the amino acid sequence of the peptide linker consists of the amino acid sequence set forth in any one of SEQ ID NOS: 96-105. In some embodiments, the amino acid sequence of the peptide linker comprises the amino acid sequence set forth in any one of SEQ ID NOS: 96-105, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the peptide linker consists of the amino acid sequence set forth in any one of SEQ ID NOS: 96-105, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the peptide linker comprises the amino acid sequence set forth in any one of SEQ ID NOS: 96-105, comprising 1, 2, or 3 amino acid variations (e.g., substitutions, deletions, additions). In some embodiments, the amino acid sequence of the peptide linker consists of the amino acid sequence set forth in any one of SEQ ID NOS: 96-105, comprising 1, 2, or 3 amino acid variations (e.g., substitutions, deletions, additions). In some embodiments, the amino acid sequence of the peptide linker comprises the amino acid sequence set forth in any one of SEQ ID NOS: 96-105, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid substitutions. In some embodiments, the amino acid sequence of the peptide linker consists of the amino acid sequence set forth in any one of SEQ ID NOS: 96-105, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid substitutions. In some embodiments, the amino acid sequence of the peptide linker comprises the amino acid sequence set forth in any one of SEQ ID NOS: 96- 105, comprising 1, 2, or 3 amino acid substitutions. In some embodiments, the amino acid sequence of the peptide linker consists of the amino acid sequence set forth in any one of SEQ ID NOS: 96- 105, comprising 1, 2, or 3 amino acid substitutions.

[0350] In some embodiments, the amino acid sequence of the peptide linker comprises the amino acid sequence set forth in SEQ ID NO: 105. In some embodiments, the amino acid sequence of the peptide linker consists of the amino acid sequence set forth in SEQ ID NO: 105. In someAttorney Docket No.62801.71WO01 embodiments, the amino acid sequence of the peptide linker comprises the amino acid sequence set forth in SEQ ID NO: 105, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the peptide linker consists of the amino acid sequence set forth in SEQ ID NO: 105, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the peptide linker comprises the amino acid sequence set forth in SEQ ID NO: 105, comprising 1, 2, or 3 amino acid variations (e.g., substitutions, deletions, additions). In some embodiments, the amino acid sequence of the peptide linker consists of the amino acid sequence set forth in SEQ ID NO: 105, comprising 1, 2, or 3 amino acid variations (e.g., substitutions, deletions, additions). In some embodiments, the amino acid sequence of the peptide linker comprises the amino acid sequence set forth in SEQ ID NO: 105, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid substitutions. In some embodiments, the amino acid sequence of the peptide linker consists of the amino acid sequence set forth in SEQ ID NO: 105, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid substitutions. In some embodiments, the amino acid sequence of the peptide linker comprises the amino acid sequence set forth in SEQ ID NO: 105, comprising 1, 2, or 3 amino acid substitutions. In some embodiments, the amino acid sequence of the peptide linker consists of the amino acid sequence set forth in SEQ ID NO: 105, comprising 1, 2, or 3 amino acid substitutions. 5.4.7 Orientation

[0351] The heterologous moiety (e.g., heterologous protein) and the immunoreceptor inhibitory protein (e.g., described herein) can be arranged in any configuration or order as long as the immunoreceptor inhibitory protein (e.g., described herein) maintains the ability to mediate its function (e.g., bind to its cognate partner) and in the embodiments wherein the heterologous moiety (e.g., heterologous protein) has a specific function, the heterologous moiety (e.g., heterologous protein) can mediate its function.

[0352] In some embodiments, the heterologous moiety is a heterologous protein (e.g., an Ig (e.g., hIg, mIg) Fc region (e.g., an Ig (e.g., hIg, mIg) Fc region described herein)) forming a fusion protein. In some embodiments, the fusion protein comprises from N- to C- terminus: anAttorney Docket No.62801.71WO01 immunoreceptor inhibitory protein (e.g., described herein) and a heterologous protein (e.g., an Ig (e.g., hIg, mIg) Fc region (e.g., an Ig (e.g., hIg, mIg) Fc region described herein)). In some embodiments, the fusion protein comprises from N- to C- terminus: an immunoreceptor inhibitory protein (e.g., described herein), a peptide linker (e.g., described herein), and a heterologous protein (e.g., an Ig (e.g., hIg, mIg) Fc region (e.g., an Ig (e.g., hIg, mIg) Fc region described herein)). In this specific orientation, the N-terminus of the immunoreceptor inhibitory protein (e.g., described herein) is operably connected to the C-terminus of the heterologous protein (e.g., an Ig (e.g., hIg, mIg) Fc region (e.g., an Ig (e.g., hIg, mIg) Fc region described herein)) either directly or indirectly through the peptide linker (e.g., described herein).

[0353] In some embodiments, the fusion protein comprises from N- to C- terminus: a heterologous protein (e.g., an Ig (e.g., hIg, mIg) Fc region (e.g., an Ig (e.g., hIg, mIg) Fc region described herein)) and an immunoreceptor inhibitory protein (e.g., described herein). In some embodiments, the fusion protein comprises from N- to C- terminus: a heterologous protein (e.g., an Ig (e.g., hIg, mIg) Fc region (e.g., an Ig (e.g., hIg, mIg) Fc region described herein)), a peptide linker (e.g., described herein), and an immunoreceptor inhibitory protein (e.g., described herein). In this specific orientation, the C-terminus of the immunoreceptor inhibitory protein (e.g., described herein) is operably connected to the N-terminus of the heterologous protein (e.g., an Ig (e.g., hIg, mIg) Fc region (e.g., an Ig (e.g., hIg, mIg) Fc region described herein)) either directly or indirectly through the peptide linker (e.g., described herein). 5.4.8 Multimeric Fusion Proteins

[0354] Provided herein are multimeric (e.g., dimeric) proteins comprising at least two fusion proteins or conjugates described herein (e.g., Ig (e.g., hIg, mIg) Fc fusion proteins described herein). In some embodiments, the protein is dimeric. In some embodiments, the protein is homodimeric. In some embodiments, the protein is heterodimeric. In some embodiments, the at least two fusion proteins described herein (e.g., Ig (e.g., hIg, mIg) Fc fusion proteins described herein) or conjugates associate via covalent or non-covalent interactions. In some embodiments, the at least two fusion proteins described herein (e.g., Ig (e.g., hIg, mIg) Fc fusion proteins described herein) or conjugates associate via at least one covalent interaction. In some embodiments, the at least two fusion proteins (e.g., Ig (e.g., hIg, mIg) Fc fusion proteins) or conjugates associate via one or more disulfide bond. In some embodiments, the at least two fusionAttorney Docket No.62801.71WO01 proteins (e.g., Ig (e.g., hIg, mIg) Fc fusion proteins) or conjugates associate via 1, 2, 3, 4, or more disulfide bonds.

[0355] In some embodiments, the protein is dimeric comprising a first fusion protein (e.g., a hIg Fc fusion protein) or conjugate described herein and a second fusion protein (e.g., an Ig (e.g., hIg, mIg) Fc fusion protein) or conjugate described herein, wherein the amino acid sequence of the first protein comprises an amino acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of the second protein. For example, the amino acid sequence of the first protein may comprise an amino acid sequence at least about 85% identical to the amino acid sequence of the second protein. For example, the amino acid sequence of the first protein may comprise an amino acid sequence at least about 90% identical to the amino acid sequence of the second protein. For example, the amino acid sequence of the first protein may comprise an amino acid sequence at least about 95% identical to the amino acid sequence of the second protein. In some embodiments, the amino acid sequence of the first protein may preferably comprise an amino acid sequence 100% identical to the amino acid sequence of the second protein.

[0356] In some embodiments, the protein is dimeric comprising a first Ig (e.g., hIg, mIg) Fc fusion protein and a second Ig (e.g., hIg, mIg) Fc fusion protein. In some embodiments, the dimeric protein is homodimeric. In some embodiments, the dimeric protein is heterodimeric. In some embodiments, the amino acid sequence of the first Ig (e.g., hIg, mIg) Fc fusion protein comprises an amino acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of the second Ig (e.g., hIg, mIg) Fc fusion protein.

[0357] An exemplary dimeric Ig (e.g., hIg, mIg) Fc fusion protein includes, for example, a protein comprising (i) a first Ig (e.g., hIg, mIg) Fc fusion protein comprising from N- to C- terminus: a first Ig (e.g., hIg, mIg) Fc region (e.g., described herein), a first peptide linker (e.g., described herein), and a first immunoreceptor inhibitory protein (e.g., described herein); and (ii) a second Ig (e.g., hIg, mIg) Fc fusion protein comprising from N- to C- terminus: a second Ig (e.g., hIg, mIg) Fc region (e.g., described herein), a second peptide linker (e.g., described herein), and a second immunoreceptor inhibitory protein (e.g., described herein). In some embodiments, the amino acid sequence of the first Ig (e.g., hIg, mIg) Fc fusion protein is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,Attorney Docket No.62801.71WO01 99%, or 100% identical to the amino acid sequence of the second Ig (e.g., hIg, mIg) Fc fusion protein. In this specific embodiment, the N-terminus of the immunoreceptor inhibitory protein (e.g., described herein) is operably connected to the C-terminus of the Ig (e.g., hIg, mIg) Fc region through the peptide linker (e.g., described herein).

[0358] Another exemplary dimeric Ig (e.g., hIg, mIg) Fc fusion protein includes, for example, a protein comprising (i) a first Ig (e.g., hIg, mIg) Fc fusion protein comprising from N- to C- terminus: a first immunoreceptor inhibitory protein (e.g., described herein), a first peptide linker (e.g., described herein), and a first Ig (e.g., hIg, mIg) Fc region (e.g., described herein); and (ii) a second immunoreceptor inhibitory protein (e.g., described herein), a second peptide linker (e.g., described herein), and a second Ig (e.g., hIg, mIg) Fc region (e.g., described herein). In some embodiments, the amino acid sequence of the first Ig (e.g., hIg, mIg) Fc fusion protein is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of the second Ig (e.g., hIg, mIg) Fc fusion protein. In this specific embodiment, the C-terminus of the immunoreceptor inhibitory protein (e.g., described herein) is operably connected to the N-terminus of the Ig (e.g., hIg, mIg) Fc region either directly or indirectly through the peptide linker (e.g., described herein). 5.4.9 Exemplary Ig Fusion Proteins

[0359] The amino acid sequence of exemplary immunoreceptor inhibitory fusion proteins (IIFPs) described herein is provided in Table 9. Each of the IIFPs 1-5 comprising the amnio acid sequence set forth in any one of SEQ ID NOS: 106, 109, 112, 115, or 118comprises from N- to C- terminus the hIL-2 signal sequence (hIL-2ss), an effector function reduced hIgG4 Fc region, a peptide linker, and an immunoreceptor inhibitory protein identified herein (IIPs 1-5) (e.g., see Table 2, SEQ ID NOS: 7-11). Each of the IIFPs 1-5 comprising the amnio acid sequence set forth in any one of SEQ ID NOS: 107-108, 110-111, 113-114, 116-117, or 119-120 comprises from N- to C-terminus an effector function reduced hIgG4 Fc region, a peptide linker, and an immunoreceptor inhibitory protein identified herein (IIPs 1-4) (e.g., see Table 2, SEQ ID NOS: 7- 11). The fusion proteins provided in Table 9 are exemplary only, and not intended to be limiting. Similar fusion proteins can be made utilizing the additional IIPs listed in Table 2, e.g., any one of IIPs 1-5.Attorney Docket No.62801.71WO01 Table 9. The Amino Acid Sequence of Exemplary Ig Fusion Proteins. Description Amino Acid Sequence SEQ ID NOAttorney Docket No.62801.71WO01 SPACVSCRGYCDYNQVETTSCTPTSNRICKCKLSSYCLVKGYNENCRVCV RKKMNAttorney Docket No.62801.71WO01 without hIL-2 EDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKE signal peptide YKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQ, ory fusion protein comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a polypeptide set forth in Table 9. In some embodiments, the amino acid sequence of the immunoreceptor inhibitory fusion protein comprises an amino acid sequence at least 85% identical to the amino acid sequence of a polypeptide set forth in Table 9. In some embodiments, the amino acid sequence of the immunoreceptor inhibitory fusion protein comprises an amino acid sequence at least 90% identical to the amino acid sequence of a polypeptide set forth in Table 9. In some embodiments, the amino acid sequence of the immunoreceptor inhibitory fusion protein comprises an amino acid sequence at least 95% identical to the amino acid sequence of a polypeptide set forth in Table 9. In some embodiments, the amino acid sequence of the immunoreceptor inhibitory fusion protein comprises an amino acid sequence at least 100% identical to the amino acid sequence of a polypeptide set forth in Table 9.Attorney Docket No.62801.71WO01

[0361] In some embodiments, the amino acid sequence of the immunoreceptor inhibitory fusion protein consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a polypeptide set forth in Table 9. In some embodiments, the amino acid sequence of the immunoreceptor inhibitory fusion protein consists of an amino acid sequence at least 85% identical to the amino acid sequence of a polypeptide set forth in Table 9. In some embodiments, the amino acid sequence of the immunoreceptor inhibitory fusion protein consists of an amino acid sequence at least 90% identical to the amino acid sequence of a polypeptide set forth in Table 9. In some embodiments, the amino acid sequence of the immunoreceptor inhibitory fusion protein consists of an amino acid sequence at least 95% identical to the amino acid sequence of a polypeptide set forth in Table 9. In some embodiments, the amino acid sequence of the immunoreceptor inhibitory fusion protein consists of an amino acid sequence at least 100% identical to the amino acid sequence of a polypeptide set forth in Table 9.

[0362] In some embodiments, the amino acid sequence of the immunoreceptor inhibitory fusion protein comprises the amino acid sequence of a polypeptide set forth in Table 9, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the immunoreceptor inhibitory fusion protein comprises the amino acid sequen...

Claims

Attorney Docket No.62801.71WO01 CLAIMS What is claimed is:

1. An isolated protein comprising an amino acid sequence at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any protein set forth in Table 2 or set forth in any one of SEQ ID NOS: 7-16.

2. The isolated protein of claim 1, comprising an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any protein set forth in Table 2 or set forth in any one of SEQ ID NOS: 7-16.

3. The isolated protein of claim 1 or 2, comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any protein set forth in Table 2 or set forth in any one of SEQ ID NOS: 7-16.

4. The isolated protein of any one of the preceding claims, comprising an amino acid sequence at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 7 or 12.

5. The isolated protein of any one of the preceding claims, comprising an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 7 or 12.

6. The isolated protein of any one of the preceding claims, comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 7 or 12.

7. The isolated protein of any one of the preceding claims, comprising the amino acid sequence set forth in SEQ ID NO: 7 or 12.

8. The isolated protein of any one of the preceding claims, wherein the protein exhibits anti- inflammatory properties (e.g., upon administration to a subject).

9. The isolated protein of any one of the preceding claims, wherein the protein specifically binds human TNFα (hTNFα); specifically binds human LTα (hLTα); or specifically binds hTNFα and hLTα.

10. The isolated protein of any one of the preceding claims, wherein the protein inhibits binding of hTNFα to human TNFR1 (hTNFR1) and / or human TNFR2 (hTNFR2).Attorney Docket No.62801.71WO01 11. The isolated protein of any one of the preceding claims, wherein the protein inhibits binding of hLTα to human TNFR1 (hTNFR1) and / or human TNFR2 (hTNFR2).

12. The isolated protein of any one of the preceding claims, wherein the protein comprises a homologous or heterologous signal peptide (e.g., operably connected to the N-terminus of the protein).

13. The protein of any one of the preceding claims operably connected to a heterologous moiety (e.g., described herein).

14. The protein of claim 13, wherein the heterologous moiety is a protein, peptide, small molecule, nucleic acid molecule (e.g., DNA, RNA, DNA / RNA hybrid molecule), lipid, or synthetic polymer.

15. The protein of claim 14, wherein the heterologous moiety is a protein.

16. A conjugate comprising the protein of any one of claims 1-15 operably connected to a heterologous moiety (e.g., described herein).

17. A radioligand comprising the protein of any one of claims 1-15 operably connected to a radionuclide.

18. A fusion protein comprising the protein of any one of claims 1-15 operably connected to a heterologous protein.

19. The fusion protein of claim 18, wherein the heterologous protein comprises an antibody.

20. The fusion protein of claim 19, wherein the antibody specifically binds a cytokine.

21. The fusion protein of claim 20, wherein the cytokine is an interleukin.

22. The fusion protein of claim 21, wherein the interleukin is interleukin 23 (IL-23) (e.g., human IL-23).

23. The fusion protein of any one of claims 18-22, wherein the heterologous protein comprises a half-life extension protein.

24. The fusion protein of any one of claims 18-23, wherein the heterologous protein comprises an immunoglobulin (Ig) (e.g., a human Ig (hIg)) Fc region.

25. The fusion protein of claim 24, wherein the Ig (e.g., hIg) Fc region comprises at least a portion of a hinge region, a CH2 region, and a CH3 region.

26. The fusion protein of claim 24 or 25, wherein the Ig (e.g., hIg) Fc region comprises a hinge region, a CH2 region, and a CH3 region.

27. The fusion protein of any one of claims 24-26, wherein the Ig is a hIg.Attorney Docket No.62801.71WO01 28. The fusion protein of claim 27, wherein the hIg is a human IgG (hIgG).

29. The fusion protein of any one of claims 27-28, wherein the hIgG is hIgG1 or hIgG4.

30. The fusion protein of any one of claims 24-29, wherein the Ig (e.g., hIg) Fc region comprises one or more amino acid substitutions relative to a reference Ig (e.g., hIg) Fc region that reduces or abolishes one or more of the following effector functions relative to the reference Ig (e.g., hIg) Fc region: antibody dependent cell mediated cytotoxicity (ADCC), complement dependent cytotoxicity (CDC), and / or affinity to one or more human Fc receptor (e.g., an Fcγ receptor (e.g., FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and / or FcγRIIIb (e.g., FcγRI, FcγIIa, and / or FcγIIIa))).

31. The fusion protein of any one of claims 24-30, wherein the Ig (e.g., hIg) Fc region does not substantially mediate ADCC, does not substantially mediate CDC, and / or does not bind to one or more human Fc receptor (e.g., an Fcγ receptor (e.g., FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and / or FcγRIIIb (e.g., FcγRI, FcγIIa, and / or FcγIIIa))).

32. The fusion protein of any one of claims 24-31, wherein the Ig is an hIgG4 and the amino acid sequence of the Fc region comprises an amino acid substitution at amino acid position S228, an amino acid substitution at amino acid position F234, and / or an amino acid substitution at amino acid position L235, numbering according to the EU index of Kabat.

33. The fusion protein of any one of claims 24-32, wherein the Ig is hIgG4 and the amino acid sequence of the Fc region comprises a proline at amino acid position S228, an alanine at amino acid position F234, and / or an alanine at amino acid position L235, numbering according to EU index of Kabat.

34. The fusion protein of any one of claims 24-33, wherein the Ig is hIgG1 and the amino acid sequence of the Fc region comprises an amino acid substitution at amino acid position L234, and / or an amino acid substitution at amino acid position L235, numbering according to the EU index of Kabat.

35. The fusion protein of any one of claims 24-34, wherein the Ig is hIgG1 and the amino acid sequence of the Fc region comprises an alanine at amino acid position L234 and / or an alanine at amino acid position L235, numbering according to the EU index of Kabat.

36. The fusion protein of any one of claims 24-35, wherein the Ig is hIgG1 and the amino acid sequence of the Fc region comprises an alanine at amino acid position L234, an alanine atAttorney Docket No.62801.71WO01 amino acid position L235, and / or a glycine at position P329 numbering according to the EU index of Kabat.

37. The fusion protein of any one of claims 24-36, wherein the Ig (e.g., hIg) Fc region comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any protein set forth in any one of Tables 4-7 or set forth in any one of SEQ ID NOS: 22-95 or 123-124.

38. The fusion protein of any one of claims 24-37, wherein the Ig (e.g., hIg) Fc region comprises an amino acid sequence comprising a set of amino acid variations set forth in any one of Tables 10-12.

39. The fusion protein of any one of claims 24-38, wherein the protein of any one of claims 1-15 is directly operably connected to the heterologous protein through a peptide bond.

40. The fusion protein of any one of claims 24-38, wherein the protein of any one of claims 1-15 is indirectly operably connected to the heterologous protein through a peptide linker.

41. The fusion protein of claim 40, wherein the amino acid sequence of the peptide linker comprises or consists of glycine or glycine and serine amino acid residues.

42. The fusion protein of any one of claims 40-41, wherein the amino acid of the peptide linker comprises or consists of (a) the amino acid sequence set forth in any one of SEQ ID NOS: 96-105; or (b) the amino acid sequence set forth in any one of SEQ ID NOS: 96-105 comprising or consisting of 1, 2, or 3 amino acid substitutions.

43. The fusion protein of any one of claims 24-42, wherein the fusion protein comprises from N- to C-terminus: the protein of any one of claims 1-15 and the heterologous protein.

44. The fusion protein of any one of claims 24-43, wherein the fusion protein comprises from N- to C-terminus: the protein of any one of claims 1-15, a peptide linker, and the heterologous protein.

45. The fusion protein of any one of claims 24-44, wherein the fusion protein comprises from N- to C-terminus: a signal peptide, the protein of any one of claims 1-15, a peptide linker, and the heterologous protein.

46. The fusion protein of any one of claims 24-45, wherein the fusion protein comprises from N- to C-terminus: the heterologous protein and the protein of any one of claims 1-15.Attorney Docket No.62801.71WO01 47. The fusion protein of any one of claims 24-46, wherein the fusion protein comprises from N- to C-terminus: the heterologous protein, a peptide linker, and the protein of any one of claims 1-15.

48. The fusion protein of any one of claims 24-47, wherein the fusion protein comprises from N- to C-terminus: a signal peptide, the heterologous protein, a peptide linker, and the protein of any one of claims 1-15.

49. The fusion protein of any one of claims 24-48, wherein the amino acid sequence of the fusion protein is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 106-120 or set forth in Table 9.

50. The fusion protein of any one of claims 24-49, wherein the amino acid sequence of the fusion protein is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 106-120.

51. The fusion protein of any one of claims 18-50, wherein the amino acid sequence of the fusion protein is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 106-108.

52. The fusion protein of any one of claims 18-51, wherein the amino acid sequence of the fusion protein is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 106-108.

53. The fusion protein of any one of claims 18-52, wherein the amino acid sequence of the fusion protein is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 106-108.

54. The fusion protein of any one of claims 18-53, wherein the amino acid sequence of the fusion protein comprises the amino acid sequence set forth in any one of SEQ ID NOS: 106-108.

55. A fusion protein comprising a first protein and a second protein, wherein the first protein comprises a first Ig (e.g., hIg) Fc region operably connected to a first protein of any one of claims 1-15; and wherein the second protein comprises a second Ig (e.g., hIg) Fc region operably connected to a second protein of any one of claims 1-15.Attorney Docket No.62801.71WO01 56. The fusion protein of claim 55, wherein the first Fc region and the second Fc region associate to form a dimer.

57. The fusion protein of any one of claims 55-56, wherein the first protein comprises an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of the second protein.

58. The fusion protein of claim 55-57, wherein the first Ig (e.g., hIg, mIg) Fc region comprises at least a portion of a hinge region, a CH2 region, and a CH3 region; and the second Ig (e.g., hIg) Fc region comprises at least a portion of a hinge region, a CH2 region, and a CH3 region.

59. The fusion protein of any one of claims 55-58, wherein the first Ig (e.g., hIg) Fc region comprises a hinge region, a CH2 region, and a CH3 region; and the second Ig (e.g., hIg) Fc region comprises a hinge region, a CH2 region, and a CH3 region.

60. The fusion protein of any one of claims 55-59, wherein the Ig of the first Ig Fc region is a hIg and the Ig of the second Ig Fc region is a hIg.

61. The fusion protein of claim 60, wherein the hIg of the first hIg Fc region is a hIgG and the hIg of the second hIg Fc region is a hIgG.

62. The fusion protein of claim 61, wherein the hIgG of the first hIg Fc region is hIgG4 and the hIgG of the first hIg Fc region is hIgG4.

63. The fusion protein of claim 61, wherein the hIgG of the first hIg Fc region is hIgG1 and the hIgG of the first hIg Fc region is hIgG1.

64. The fusion protein of any one of claims 55-63, wherein the first Ig (e.g., hIg) Fc region and the second Ig (e.g., hIg) Fc region each comprises one or more amino acid substitutions relative to a reference Ig (e.g., hIg) Fc region that reduces or abolishes one or more of the following effector functions relative to the reference Ig (e.g., hIg) Fc region: ADCC, CDC, and / or binding affinity to one or more Fc receptor (e.g., an Fcγ receptor (e.g., FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and / or FcγRIIIb (e.g., FcγRI, FcγIIa, and / or FcγIIIa))).

65. The fusion protein of any one of claims 55-64, wherein the fusion protein does not substantially mediate ADCC, does not substantially mediate CDC, and / or does not bind to one or more Fc receptor (e.g., an Fcγ receptor (e.g., FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and / or FcγRIIIb (e.g., FcγRI, FcγIIa, and / or FcγIIIa))).Attorney Docket No.62801.71WO01 66. The fusion protein of any one of claims 55-65, wherein the Ig of the first Ig Fc region and the second Ig Fc region is hIgG4 and the amino acid sequence of the first Fc region and the second Fc region each comprise an amino acid substitution at amino acid position S228, an amino acid substitution at amino acid position F234, and / or an amino acid substitution at amino acid position E235, numbering according to the EU index of Kabat.

67. The fusion protein of any one of claims 55-66, wherein the Ig of the first Ig Fc region and the second Ig Fc region is hIgG4 and the amino acid sequence of the first Fc region and the second Fc region each comprise a proline at amino acid position S228, an alanine at amino acid position F234, and / or an alanine at amino acid position E235, numbering according to EU index of Kabat.

68. The fusion protein of any one of claims 55-67, wherein the Ig of the first Ig Fc region and the second Ig Fc region is hIgG1 and the amino acid sequence of the first Fc region and the second Fc region each comprise an amino acid substitution at amino acid position L234, and / or an amino acid substitution at amino acid position L235, numbering according to the EU index of Kabat.

69. The fusion protein of any one of claims 55-68, wherein the Ig of the first Ig Fc region and the second Ig Fc region is hIgG1 and the amino acid sequence of the first Fc region and the second Fc region each comprise an alanine at amino acid position L234 and / or an alanine at amino acid position L235, numbering according to the EU index of Kabat.

70. The fusion protein of any one of claims 55-69, wherein the Ig of the first Ig Fc region and the second Ig Fc region is hIgG1 and the amino acid sequence of the first Fc region and the second Fc region each comprise a proline (or alanine) at amino acid position L234 and / or a proline (or alanine) at amino acid position L235, numbering according to the EU index of Kabat.

71. The fusion protein of any one of claims 55-70, wherein the Ig of the first Ig Fc region and the second Ig Fc region is hIgG1 and the amino acid sequence of the first Fc region and the second Fc region each comprise a proline (or alanine) at amino acid position L234 a proline (or alanine) at amino acid position L235, and / or a glycine at amino acid position P329, numbering according to the EU index of Kabat.

72. The fusion protein of any one of claims 55-71, wherein the first Ig (e.g., hIg) Fc region comprises an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of the second Ig (e.g., hIg) Fc region.Attorney Docket No.62801.71WO01 73. The fusion protein of any one of claims 55-72, wherein the first protein comprises from N- to C-terminus: the first Ig (e.g., hIg) Fc region and the first protein of any one of claims 1-15; and the second protein comprises from N- to C-terminus: the second Ig (e.g., hIg) Fc region and the second protein of any one of claims 1-15.

74. The fusion protein of any one of claims 55-73, wherein the first protein comprises from N- to C-terminus: the first Ig (e.g., hIg) Fc region, a first peptide linker, and the first protein of any one of claims 1-15; and the second protein comprises from N- to C-terminus: the second Ig (e.g., hIg) Fc region, a second peptide linker, and the second protein of any one of claims 1-15.

75. The fusion protein of any one of claims 55-74, wherein the first protein comprises from N- to C-terminus: the first protein of any one of claims 1-15 and the first Ig (e.g., hIg) Fc region; and the second protein comprises from N- to C-terminus: the second protein of any one of claims 1-15 and the second Ig (e.g., hIg) Fc region.

76. The fusion protein of any one of claims 55-75, wherein the first protein comprises from N- to C-terminus: the first protein of any one of claims 1-15, a first peptide linker, and the first Ig (e.g., hIg) Fc region; and the second protein comprises from N- to C-terminus: the second protein of any one of claims 1-15, a second peptide linker, and the second Ig (e.g., hIg) Fc region.

77. The fusion protein of claim 74 or 76, wherein the amino acid sequence of the first peptide linker and the second peptide linker each comprises or consists of glycine or glycine and serine amino acid residues.

78. The fusion protein of any one of claims 74, 76, or 77, wherein the amino acid of the first peptide linker and the second peptide linker each comprises or consists of (a) the amino acid sequence set forth in any one of SEQ ID NOS: 96-105; or (b) the amino acid sequence set forth in any one of SEQ ID NOS: 96-105 comprising or consisting of 1, 2, or 3 amino acid substitutions.

79. The fusion protein of any one of claims 55-78, wherein the amino acid sequence of the first protein is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 106-120 or set forth in Table 9; and the amino acid sequence of the first protein is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 106-120 or set forth in Table 9.Attorney Docket No.62801.71WO01 80. The fusion protein of any one of claims 55-79, wherein the amino acid sequence of the first protein is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 106-120; and the amino acid sequence of the first protein is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 106-120.

81. An immunogenic peptide or protein comprising at least an immunogenic fragment of the protein of any one of claims 1-15.

82. The immunogenic peptide or protein of claim 81, wherein the immunogenic peptide or protein does not specifically bind hTNFα or binds one or more of hTNFα with lower affinity relative to a reference protein of any one of claims 1-15.

83. The immunogenic peptide or protein of claim 81 or 82, wherein the immunogenic peptide or protein comprises a full-length protein of any one of claims 1-15.

84. The immunogenic peptide or protein of any one of claims 81-83, wherein the immunogenic peptide or protein comprises an immunogenic fragment of a protein of any one of claims 1-15.

85. The immunogenic peptide or protein of any one of claims 81-84, wherein the immunogenic peptide or protein comprises at least about 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, or 130 amino acids.

86. The immunogenic peptide or protein of any one of claims 81-85, wherein the immunogenic peptide or protein comprises from about 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, or 130 amino acids.

87. The immunogenic peptide or protein of any one of claims 81-86, wherein the amino acid sequence of the immunogenic peptide or protein comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acid variations (e.g., substitutions, additions, deletions) relative to a reference protein of any one of claims 1-15.

88. The immunogenic peptide or protein of any one of claims 81-87, wherein the immunogenic peptide or protein comprises an amino acid sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a contiguousAttorney Docket No.62801.71WO01 stretch of at least about 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, or 130 amino acids set forth in any one of SEQ ID NOS: 7-16.

89. The immunogenic peptide or protein of any one of claims 81-88, wherein the immunogenic peptide or protein comprises an amino acid sequence that, other than the one or more amino acid variation (e.g., substitution, addition, deletion), is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 7-16.

90. The immunogenic peptide or protein of any one of claims 81-89, wherein the immunogenic peptide or protein is formulated with an adjuvant.

91. An isolated antibody that specifically binds to a protein of any one of claims 1-15.

92. A nucleic acid molecule encoding the protein of any one of claims 1-15, the conjugate of claim 16, the radioligand of claim 17, the fusion protein of any one of claims 18-80, the immunogenic peptide or protein of any one of claims 81-90, or the antibody of claim 91.

93. The nucleic acid molecule of claim 92, wherein the nucleic acid molecule is an RNA (e.g., mRNA, circular RNA) molecule or a DNA molecule.

94. An mRNA molecule encoding the protein of any one of claims 1-15, the conjugate of claim 16, the radioligand of claim 17, the fusion protein of any one of claims 18-80, the immunogenic peptide or protein of any one of claims 81-90, or the antibody of claim 91.

95. The nucleic acid molecule of any one of claims 92-93 or the mRNA molecule of claim 94, comprising a heterologous 5'-untranslated region (UTR), 3'-UTR, or both a 5'-UTR and 3'- UTR.

96. The nucleic acid molecule of any one of claims 92-93 or 95 or the mRNA molecule of any one of claims 94-95, comprising a poly(A) sequence.

97. The nucleic acid molecule of any one of claims 92-93 or 95-96 or the mRNA molecule of any one of claims 94-96, comprising a 5' cap structure.

98. The nucleic acid molecule of any one of claims 92-93 or 95-97 or the mRNA molecule of any one of claims 94-97, comprising at least one variant nucleotide.

99. The nucleic acid molecule of any one of claims 92-93 or 95-98 or the mRNA molecule of any one of claims 94-98, wherein the sequence of the nucleic acid molecule or mRNA molecule, respectively, is codon optimized.Attorney Docket No.62801.71WO01 100. A vector (e.g., expression vector) comprising the nucleic acid molecule of any one of claims 92-93 or 95-99 or the mRNA molecule of any one of claims 94-99.

101. The vector of claim 100, wherein the vector is a viral vector or a non-viral vector (e.g., a plasmid).

102. A viral particle conjugated to the protein of any one of claims 1-15, the conjugate of claim 16, the radioligand of claim 17, or the fusion protein of any one of claims 18-80.

103. A cell (e.g., host cell) comprising the protein of any one of claims 1-15, the conjugate of claim 16, the radioligand of claim 17, the fusion protein of any one of claims 18-80, the immunogenic peptide or protein of any one of claims 81-90, the antibody of claim 91, the nucleic acid molecule of any one of claims 92-93 or 95-99, the mRNA molecule of any one of claims 94-99, the vector of any one of claims 100-101, the carrier of any one of claims 105-107, the lipid nanoparticle of any one of claims 108-109, the vaccine composition of claim 110, or the pharmaceutical composition of claim 111.

104. A cell (e.g., a therapeutic cell) (e.g., a CAR cell) expressing and / or genetically encoding the protein of any one of claims 1-15, the conjugate of claim 16, the radioligand of claim 17, or the fusion protein of any one of claims 18-80 (e.g., on the surface of the cell).

105. A carrier comprising the protein of any one of claims 1-15, the conjugate of claim 16, the radioligand of claim 17, the fusion protein of any one of claims 18-80, the immunogenic peptide or protein of any one of claims 81-90, the antibody of claim 91, the nucleic acid molecule of any one of claims 92-93 or 95-99, the mRNA molecule of any one of claims 94-99, the vector of any one of claims 100-101, the cell of any one of claims 103-104, the vaccine composition of claim 110, or the pharmaceutical composition of claim 111.

106. A carrier conjugated to the protein of any one of claims 1-15, the conjugate of claim 16, the radioligand of claim 17, or the fusion protein of any one of claims 18-80.

107. The carrier of claim 106, wherein the carrier is a lipid nanoparticle, liposome, lipoplex, or nanoliposome.

108. A lipid nanoparticle comprising the protein of any one of claims 1-15, the conjugate of claim 16, the radioligand of claim 17, the fusion protein of any one of claims 18-80, the immunogenic peptide or protein of any one of claims 81-90, the antibody of claim 91, the nucleic acid molecule of any one of claims 92-93 or 95-99, the mRNA molecule of any one of claims 94-99, the vector of any one of claims 100-101, the viral particle of claim 102, the cell of anyAttorney Docket No.62801.71WO01 one of claims 103-104, the vaccine composition of claim 110, or the pharmaceutical composition of claim 111.

109. A lipid nanoparticle conjugated to the protein of any one of claims 1-15, the conjugate of claim 16, the radioligand of claim 17, or the fusion protein of any one of claims 18-80.

110. A vaccine composition comprising the immunogenic peptide or protein of any one of claims 81-91 (or a nucleic acid molecule encoding the same (or a vector encoding the nucleic acid molecule) or a carrier comprising any of the foregoing).

111. A pharmaceutical composition comprising the protein of any one of claims 1-15, the conjugate of claim 16, the radioligand of claim 17, the fusion protein of any one of claims 18-80, the immunogenic peptide or protein of any one of claims 81-90, the antibody of claim 91, the nucleic acid molecule of any one of claims 92-93 or 95-99, the mRNA molecule of any one of claims 94-99, the vector of any one of claims 100-101, the viral particle of claim 102, the carrier of any one of claims 105-107, the lipid nanoparticle of any one of claims 108-109, the cell of any one of claims 103-104, or the vaccine composition of claim 110; and a pharmaceutically acceptable excipient.

112. A kit comprising the protein of any one of claims 1-15, the conjugate of claim 16, the radioligand of claim 17, the fusion protein of any one of claims 18-80, the immunogenic peptide or protein of any one of claims 81-90, the antibody of claim 91, the nucleic acid molecule of any one of claims 92-93 or 95-99, the mRNA molecule of any one of claims 94-99, the vector of any one of claims 100-101, the viral particle of claim 102, the carrier of any one of claims 105-107, the lipid nanoparticle of any one of claims 108-109, the cell of any one of claims 103-104, the vaccine composition of claim 110, or the pharmaceutical composition of claim 111; and optionally instructions for using any one or more of the foregoing.

113. A method of delivering a protein, fusion protein, conjugate, radioligand, nucleic acid molecule, mRNA molecule, expression vector, cell, viral particle, carrier, lipid nanoparticle, immunogenic peptide or protein, an antibody, a vaccine composition, or pharmaceutical composition to a subject, the method comprising administering to the subject the protein of any one of claims 1-15, the conjugate of claim 16, the radioligand of claim 17, the fusion protein of any one of claims 18-80, the immunogenic peptide or protein of any one of claims 81-90, the antibody of claim 91, the nucleic acid molecule of any one of claims 92-93 or 95-99, the mRNA molecule of any one of claims 94-99, the vector of any one of claims 100-101, the viral particleAttorney Docket No.62801.71WO01 of claim 102, the carrier of any one of claims 105-107, the lipid nanoparticle of any one of claims 108-109, the cell of any one of claims 103-104, the vaccine composition of claim 110, or the pharmaceutical composition of claim 111, to thereby deliver the protein, fusion protein, conjugate, radioligand, immunogenic peptide or protein, antibody, mRNA molecule, vector, cell, carrier, lipid nanoparticle, viral particle, vaccine composition, or pharmaceutical composition to a subject.

114. A method of inhibiting or reducing (e.g., preventing) binding of TNFα to TNFR1 and / or TNFR2 in a subject in need thereof, the method comprising administering to the subject the protein of any one of claims 1-15, the conjugate of claim 16, the radioligand of claim 17, the fusion protein of any one of claims 18-80, the immunogenic peptide or protein of any one of claims 81-90, the antibody of claim 91, the nucleic acid molecule of any one of claims 92-93 or 95-99, the mRNA molecule of any one of claims 94-99, the vector of any one of claims 100-101, the viral particle of claim 102, the carrier of any one of claims 105-107, the lipid nanoparticle of any one of claims 108-109, the cell of any one of claims 103-104, the vaccine composition of claim 110, or the pharmaceutical composition of claim 111, to thereby inhibit or reduce (e.g., prevent) binding of TNFα to TNFR1 and / or TNFR2 in the subject.

115. A method of inhibiting reducing (e.g., preventing) signaling mediated by the binding of TNFα to TNFR1 and / or TNFR2 in a subject in need thereof, the method comprising administering to the subject the protein of any one of claims 1-15, the conjugate of claim 16, the radioligand of claim 17, the fusion protein of any one of claims 18-80, the immunogenic peptide or protein of any one of claims 81-90, the antibody of claim 91, the nucleic acid molecule of any one of claims 92-93 or 95-99, the mRNA molecule of any one of claims 94-99, the vector of any one of claims 100-101, the viral particle of claim 102, the carrier of any one of claims 105-107, the lipid nanoparticle of any one of claims 108-109, the cell of any one of claims 103-104, the vaccine composition of claim 110, or the pharmaceutical composition of claim 111, to thereby inhibit or reduce (e.g., prevent) signaling mediated by the binding of TNFα to TNFR1 and / or TNFR2 in the subject.

116. A method of suppressing or preventing a pro-inflammatory immune response in a subject in need thereof, the method comprising administering to the subject the protein of any one of claims 1-15, the conjugate of claim 16, the radioligand of claim 17, the fusion protein of any one of claims 18-80, the immunogenic peptide or protein of any one of claims 81-90, the antibody ofAttorney Docket No.62801.71WO01 claim 91, the nucleic acid molecule of any one of claims 92-93 or 95-99, the mRNA molecule of any one of claims 94-99, the vector of any one of claims 100-101, the viral particle of claim 102, the carrier of any one of claims 105-107, the lipid nanoparticle of any one of claims 108-109, the cell of any one of claims 103-104, the vaccine composition of claim 110, or the pharmaceutical composition of claim 111, to thereby suppress or prevent a pro-inflammatory immune response in the subject.

117. A method of preventing, treating, or ameliorating a disease in a subject in need thereof, the method comprising administering to the subject the protein of any one of claims 1-15, the conjugate of claim 16, the radioligand of claim 17, the fusion protein of any one of claims 18-80, the immunogenic peptide or protein of any one of claims 81-90, the antibody of claim 91, the nucleic acid molecule of any one of claims 92-93 or 95-99, the mRNA molecule of any one of claims 94-99, the vector of any one of claims 100-101, the viral particle of claim 102, the carrier of any one of claims 105-107, the lipid nanoparticle of any one of claims 108-109, the cell of any one of claims 103-104, the vaccine composition of claim 110, or the pharmaceutical composition of claim 111, to thereby prevent, treat, or ameliorate the disease in the subject.

118. The method of claim 117, wherein the disease is a pro-inflammatory disease, an autoimmune disease, or a fibrotic disease.

119. The method of claim 117 or 118, wherein the disease is a proinflammatory (e.g., autoimmune disease).

120. The method of any one of claims 117-119, wherein the disease is inflammatory bowel disease, Crohn’s disease, ulcerative colitis, rheumatoid arthritis, psoriatic arthritis, plaque psoriasis, juvenile idiopathic arthritis, Hidradenitis suppurativa, uveitis, non-radiographic axial spondyloarthritis, ankylosing spondylitis, asthma, or systemic lupus erythematosus.

121. A method of inducing or enhancing an immune response in a subject in need thereof, the method comprising administering to the subject (i) the immunogenic peptide or protein of any one of claims 81-90 (or a conjugate or a fusion protein thereof); (ii) a nucleic acid molecule encoding (i); (iii) a vector comprising (ii); (iv) a carrier comprising (i), (ii), or (iii); a vaccine composition comprising (i), (ii), (iii), or (iv); or a pharmaceutical composition comprising (i), (ii), (iii), (iv), or (v), to thereby induce or enhance an immune response in the subject.

122. A method of vaccinating a subject in need thereof (e.g., against a viral infection), the method comprising administering to the subject (i) the immunogenic peptide or protein of anyAttorney Docket No.62801.71WO01 one of claims 81-90 (or a conjugate or a fusion protein thereof); (ii) a nucleic acid molecule encoding (i); (iii) a vector comprising (ii); (iv) a carrier comprising (i), (ii), or (iii); a vaccine composition comprising (i), (ii), (iii), or (iv); or a pharmaceutical composition comprising (i), (ii), (iii), (iv), or (v), to thereby vaccinate the subject in need thereof (e.g., against a virus).

123. A method of determining the presence of a virus in a subject, the method comprising (a) obtaining the sample from a subject or providing a sample that has been obtained from a subject, and (b) determining the presence or absence of the protein of any one of claims 1-15 (or a fragment or variant thereof) or a nucleic acid molecule encoding the protein of any one of claims 1-15 (or the fragment or variant thereof) in the sample.

124. A method of diagnosing a viral infection in a subject, the method comprising (a) obtaining a sample from a subject or providing a sample that has been obtained from a subject, (b) determining the presence or absence of the protein of any one of claims 1-15 (or a fragment or variant thereof) or a nucleic acid molecule encoding the protein of any one of claims 1-15 (or a fragment or variant thereof), and (c) diagnosing the subject as having the viral infection if the protein of any one of claims 1-15 (or a fragment or variant thereof) or a nucleic acid molecule encoding the protein of any one of claims 1-15 (or the fragment or variant thereof) is determined to be present in the sample in step (b).

125. The method of claim 123 or 124, wherein the method is an in vitro method.

126. A method of treating a viral infection in a subject, the method comprising (a) receiving testing results that determined the presence of the protein of any one of claims 1-15 (or a fragment or variant thereof) or a nucleic acid molecule encoding the protein of any one of claims 1-15 (or the fragment or variant thereof) in a sample from the subject, (b) diagnosing the subject as having the viral infection, and (c) administering a therapeutic agent to treat the viral infection.

127. The method or use of any one of claims 123-126, wherein the sample is a blood, cell, tissue, or saliva, or nasal swab.Attorney Docket No.62801.71WO01 128. The method or use of any one of claims 123-127, wherein the antibody of claim 91 is utilized to determine the presence or absence of the protein of any one of claims 1-15 (or the fragment or variant thereof).

129. The method or use of any one of claims 113-128, wherein the subject is a human.

Images