Immunomodulatory proteins and related methods

Abstract

Provided herein are immunomodulatory proteins and compositions (e.g., pharmaceutical compositions) comprising the same, as well as methods for making the immunomodulatory proteins and compositions. The immunomodulatory proteins provided herein are useful in pharmaceutical compositions and methods for treating IL-10-responsive disorders.

Description

[Technical Field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 394,155, filed August 1, 2022, U.S. Provisional Patent Application No. 63 / 483,419, filed February 6, 2023, and U.S. Provisional Patent Application No. 63 / 502,864, filed May 17, 2023, the entire contents of each of which are incorporated herein by reference.

[0002] 1.Technical Field The present disclosure relates to immunomodulatory polypeptides and proteins that bind to the human interleukin-10 receptor (hIL-10R), and polynucleotides encoding the immunomodulatory polypeptides and proteins. The disclosure further relates to methods of making and using the same. [Background technology]

[0003] 2.Background technology Human IL-10 (hIL-10) is a multifunctional immunoregulatory cytokine that primarily targets antigen-presenting cells, such as monocytes and macrophages, and inhibits the release of proinflammatory cytokines, such as TNFα, IL-1β, IL-6, and IL-8. hIL-10 also interferes with antigen presentation by downregulating the expression of major histocompatibility complex (MHC)-II and costimulatory molecules. hIL-10 also functions to suppress T cell responses by inhibiting the expression of IL-12 and IL-23, cytokines required for CD4+ T cell differentiation. In addition to these anti-inflammatory properties, hIL-10 is also known to have pro-inflammatory functions. For example, hIL-10 has been shown to stimulate CD8+ T cells and promote the survival, proliferation, and differentiation of B cells. Summary of the Invention

[0004] 3. Overview In particular, provided herein are immunomodulatory polypeptides and polynucleotides encoding same; fusions and conjugates comprising immunomodulatory polypeptides; methods of manufacture; pharmaceutical compositions; and methods of use, including, for example, methods of treating inflammatory diseases (e.g., autoimmune diseases), methods of promoting immune responses, methods of treating viral infections, and diagnostic methods.

[0005] Accordingly, in one aspect, provided herein is an isolated polypeptide or protein comprising an amino acid sequence that 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 of any one of SEQ ID NOs: 108-454, wherein the polypeptide or protein specifically binds to human IL-10 receptor (hIL-10R) (e.g., hIL-10Rα).

[0006] In some embodiments, the isolated polypeptide or protein consists of an amino acid sequence that 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 of any one of SEQ ID NOs: 108-454. In some embodiments, the isolated polypeptide or protein comprises or consists of an amino acid sequence that is at least about 85% identical to the amino acid sequence of any one of SEQ ID NOs: 108-454. In some embodiments, the isolated polypeptide or protein comprises or consists of an amino acid sequence that is at least about 90% identical to the amino acid sequence of any one of SEQ ID NOs: 108-454. In some embodiments, the isolated polypeptide or protein comprises or consists of an amino acid sequence that is at least about 95% identical to the amino acid sequence of any one of SEQ ID NOs: 108-454. In some embodiments, the isolated polypeptide or protein comprises or consists of an amino acid sequence that is 100% identical to the amino acid sequence of any one of SEQ ID NOs: 108-454.

[0007] In some embodiments, the isolated polypeptide or protein comprises or consists of an amino acid sequence that is at least about 85% identical to the amino acid sequence of any one of SEQ ID NOs: 108, 111-119, or 122-126. In some embodiments, the isolated polypeptide or protein comprises or consists of an amino acid sequence that is at least about 90% identical to the amino acid sequence of any one of SEQ ID NOs: 108, 111-119, or 122-126. In some embodiments, the isolated polypeptide or protein comprises or consists of an amino acid sequence that is at least about 95% identical to the amino acid sequence of any one of SEQ ID NOs: 108, 111-119, or 122-126. In some embodiments, the isolated polypeptide or protein comprises or consists of an amino acid sequence that is 100% identical to the amino acid sequence of any one of SEQ ID NOs: 108, 111-119, or 122-126.

[0008] In some embodiments, the isolated polypeptide or protein comprises or consists of an amino acid sequence that is at least about 85% identical to the amino acid sequence of any one of SEQ ID NOs: 112-115 or 123-126. In some embodiments, the isolated polypeptide or protein comprises or consists of an amino acid sequence that is at least about 90% identical to the amino acid sequence of any one of SEQ ID NOs: 112-115 or 123-126. In some embodiments, the isolated polypeptide or protein comprises or consists of an amino acid sequence that is at least about 95% identical to the amino acid sequence of any one of SEQ ID NOs: 112-115 or 123-126. In some embodiments, the isolated polypeptide or protein comprises or consists of an amino acid sequence that is 100% identical to the amino acid sequence of any one of SEQ ID NOs: 112-115 or 123-126.

[0009] In some embodiments, the isolated polypeptide or protein comprises or consists of an amino acid sequence that is at least about 85% identical to the amino acid sequence of any one of SEQ ID NOs: 114. In some embodiments, the isolated polypeptide or protein comprises or consists of an amino acid sequence that is at least about 90% identical to the amino acid sequence of any one of SEQ ID NOs: 114. In some embodiments, the isolated polypeptide or protein comprises or consists of an amino acid sequence that is at least about 95% identical to the amino acid sequence of any one of SEQ ID NOs: 114. In some embodiments, the isolated polypeptide or protein comprises or consists of an amino acid sequence that is 100% identical to the amino acid sequence of any one of SEQ ID NOs: 114.

[0010] In some embodiments, the isolated polypeptide or protein comprises or consists of an amino acid sequence that is at least about 85% identical to the amino acid sequence of any one of SEQ ID NOs: 125. In some embodiments, the isolated polypeptide or protein comprises or consists of an amino acid sequence that is at least about 90% identical to the amino acid sequence of any one of SEQ ID NOs: 125. In some embodiments, the isolated polypeptide or protein comprises or consists of an amino acid sequence that is at least about 95% identical to the amino acid sequence of any one of SEQ ID NOs: 125. In some embodiments, the isolated polypeptide or protein comprises or consists of an amino acid sequence that is 100% identical to the amino acid sequence of any one of SEQ ID NOs: 125.

[0011] In one aspect, provided herein is an isolated polypeptide or protein comprising an amino acid sequence that is at least about 85% identical to the amino acid sequence of any one of SEQ ID NOs: 119-126 or 291-454, wherein the polypeptide or protein specifically binds to hIL-10R (e.g., hIL-10Rα).

[0012] In some embodiments, the isolated polypeptide or protein consists of an amino acid sequence that 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 of any one of SEQ ID NOs: 119-126 or 291-454. In some embodiments, the isolated polypeptide or protein comprises or consists of an amino acid sequence that is at least about 85% identical to the amino acid sequence of any one of SEQ ID NOs: 119-126 or 291-454. In some embodiments, the isolated polypeptide or protein comprises or consists of an amino acid sequence that is at least about 90% identical to the amino acid sequence of any one of SEQ ID NOs: 119-126 or 291-454. In some embodiments, the isolated polypeptide or protein comprises or consists of an amino acid sequence that is at least about 95% identical to the amino acid sequence of any one of SEQ ID NOs: 119-126 or 291-454. In some embodiments, the isolated polypeptide or protein comprises or consists of an amino acid sequence that is 100% identical to the amino acid sequence of any one of SEQ ID NOs: 119-126 or 291-454.

[0013] In one aspect, provided herein is an isolated polypeptide or protein comprising an amino acid sequence that is at least about 85% identical to the amino acid sequence of any one of SEQ ID NOs: 108-118 or 127-290, wherein the polypeptide or protein specifically binds to hIL-10R (e.g., hIL-10Rα).

[0014] In some embodiments, the isolated polypeptide or protein consists of an amino acid sequence that 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 of any one of SEQ ID NOs: 108-118 or 127-290. In some embodiments, the isolated polypeptide or protein comprises or consists of an amino acid sequence that is at least about 85% identical to the amino acid sequence of any one of SEQ ID NOs: 108-118 or 127-290. In some embodiments, the isolated polypeptide or protein comprises or consists of an amino acid sequence that is at least about 90% identical to the amino acid sequence of any one of SEQ ID NOs: 108-118 or 127-290. In some embodiments, the isolated polypeptide or protein comprises or consists of an amino acid sequence that is at least about 95% identical to the amino acid sequence of any one of SEQ ID NOs: 108-118 or 127-290. In some embodiments, the isolated polypeptide or protein comprises or consists of an amino acid sequence that is 100% identical to the amino acid sequence of any one of SEQ ID NOs: 108-118 or 127-290.

[0015] It should be understood that any of the following embodiments are applicable to any of the aforementioned aspects relating to isolated polypeptides and proteins.

[0016] In some embodiments, the isolated polypeptide or protein (a) binds to cells expressing hIL-10R (e.g., hIL-10Rα) on the surface with an EC50 of less than about 500 pM, 400 pM, 300 pM, 200 pM, 100 pM, 50 pM, 40 pM, 30 pM, 20 pM, 10 pM, 9 pM, 8 pM, 7 pM, 6 pM, 5 pM, 4 pM, 3 pM, 2 pM, 1 pM, 0.9 pM, 0.8 pM, 0.7 pM, 0.6 pM, 0.5 pM, 0.4 pM, 0.3 pM, 0.2 pM, or 0.1 pM; or (b) binds to cells expressing hIL-10R (e.g., hIL-10Rα) on the surface with an EC50 of less than about 500 pM, 400 pM, 300 pM, 200 pM, 100 pM, 50 pM, 40 pM, 30 pM, 20 pM, 10 pM, 9 pM, 8 pM, 7 pM, 6 pM, 5 pM, 4 pM, 3 pM, 2 pM, 1 pM, 0.9 pM, 0.8 pM, 0.7 pM, 0.6 pM, 0.5 pM, 0.4 pM, 0.3 pM, 0.2 pM, or 0.1 pM. The antibody binds to cells expressing hIL-10R (e.g., hIL-10Rα) on its surface with an EC50 of 00 pM to 0.1 pM, 300 pM to 0.1 pM, 200 pM to 0.1 pM, 100 pM to 0.1 pM, 50 pM to 0.1 pM, 25 pM to 0.1 pM, 10 pM to 0.1 pM, 5 pM to 0.1 pM, or 1 pM to 0.1 pM, 500 pM to 0.5 pM, 400 pM to 0.5 pM, 300 pM to 0.5 pM, 200 pM to 0.5 pM, 100 pM to 0.5 pM, 50 pM to 0.5 pM, 25 pM to 0.5 pM, 10 pM to 0.5 pM, 5 pM to 0.5 pM, or 1 pM to 0.5 pM. In some embodiments, the EC50 is determined by the assay described in Example 3 herein.

[0017] In some embodiments, the isolated polypeptide or protein (a) binds to cells expressing hIL-10R (e.g., hIL-10Rα) on the surface with about an EC50 that is at least about 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 110-fold, 120-fold, 130-fold, 140-fold, or 150-fold higher than the EC50 of a reference hIL-10 polypeptide (e.g., SEQ ID NO: 2); or (b) binds to a cell expressing hIL-10R (e.g., hIL-10Rα) on the surface with about an EC50 that is at least about 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 110-fold, 120-fold, 130-fold, 140-fold, or 150-fold higher than the EC50 of a reference hIL-10 polypeptide (e.g., SEQ ID NO: 2). The EC50 of the hIL-10 protein or polypeptide (e.g., SEQ ID NO: 2) binds to cells expressing hIL-10R (e.g., hIL-10Rα) on their surface with an EC50 that is about 10-150 fold, 20-150 fold, 30-150 fold, 40-150 fold, 50-150 fold, 60-150 fold, 70-150 fold, 80-150 fold, 90-150 fold, 100-150 fold, 110-150 fold, 120-150 fold, 130-150 fold, or 140-150 fold higher than the EC50 of the hIL-10 protein or polypeptide (e.g., SEQ ID NO: 2). In some embodiments, the EC50 is determined by the assay described in Example 3 herein.

[0018] In some embodiments, the polypeptide or protein specifically binds to hIL-10Rα. In some embodiments, the polypeptide or protein specifically binds to hIL-10Rβ. In some embodiments, the polypeptide or protein specifically binds to hIL-10Rα and hIL-10Rβ.

[0019] In some embodiments, the polypeptide or protein is a hIL-10R (e.g., hIL-10Rα, hIL-10Rβ) agonist. In some embodiments, the polypeptide or protein is a hIL-10R (e.g., hIL-10Rα, hIL-10Rβ) antagonist.

[0020] In some embodiments, the isolated polypeptide or protein further comprises a homologous or heterologous signal peptide operably linked to the N-terminus of the polypeptide or protein.

[0021] In some embodiments, the isolated polypeptide or protein is an isolated protein.

[0022] In one aspect, provided herein is a polypeptide comprising a polypeptide or protein described herein operably linked to a heterologous moiety.

[0023] In one aspect, provided herein is a fusion polypeptide or protein comprising a polypeptide or protein described herein operably linked to a heterologous polypeptide or protein.

[0024] In some embodiments, the heterologous polypeptide or protein is a half-life extending polypeptide or protein.

[0025] In some embodiments, the heterologous polypeptide or protein comprises an immunoglobulin (Ig) (e.g., human Ig (hIg), murine Ig (mIg)) Fc region. In some embodiments, the Ig (e.g., hIg, mIg) 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, mIg) Fc region comprises a hinge region, a CH2 region, and a CH3 region.

[0026] In some embodiments, the Ig is hIg. In some embodiments, the hIg is human IgG (hIgG). In some embodiments, the hIgG is hIgG1 or hIgG4.

[0027] In some embodiments, the Ig is mIg. In some embodiments, the mIg is mIgG1. In some embodiments, the mIg is mIgG2a.

[0028] In some embodiments, the Ig (e.g., hIg, mIg) Fc region comprises one or more amino acid substitutions compared to a reference Ig (e.g., hIg, mIg) Fc region that reduce or eliminate one or more of the following effector functions: antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and / or affinity for one or more human Fc receptors (e.g., Fcγ receptors (e.g., FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and / or FcγRIIIb (e.g., FcγRI, FcγIIa, and / or FcγIIIa))) compared to the reference hIg Fc region.

[0029] In some embodiments, the Ig (e.g., hIg, mIg) Fc region does not substantially mediate ADCC, does not substantially mediate CDC, and / or does not bind to one or more human Fc receptors (e.g., Fcγ receptors (e.g., FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and / or FcγRIIIb (e.g., FcγRI, FcγIIa, and / or FcγIIIa))).

[0030] In some embodiments, the Ig is 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, numbered 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, numbered according to the EU index of Kabat.

[0031] 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, numbered 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, numbered 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, numbered according to the EU index of Kabat.

[0032] In some embodiments, the Ig is an mIgG2a 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, numbered according to the EU index of Kabat. In some embodiments, the Ig is an mIgG2a and the amino acid sequence of the Fc region comprises a proline (or alanine) at amino acid position L234 and / or a proline (or alanine) at amino acid position L235, numbered according to the EU index of Kabat. In some embodiments, the Ig is an mIgG2a and the amino acid sequence of the Fc region comprises a proline (or alanine) at amino acid position L234, a proline (or alanine) at amino acid position L235, and / or a glycine at position P329, numbered according to the EU index of Kabat.

[0033] In some embodiments, a polypeptide or protein described herein is directly operably linked to a heterologous polypeptide or protein via a peptide bond.

[0034] In some embodiments, a polypeptide or protein described herein is indirectly operably linked to a heterologous polypeptide or protein via 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 sequence of the peptide linker comprises or consists of (a) the amino acid sequence 57 of any one of SEQ ID NOs: 49-57; or (b) the amino acid sequence of any one of SEQ ID NOs: 49-57 comprising or consisting of one, two, or three amino acid substitutions.

[0035] In some embodiments, a fusion polypeptide or protein comprises, from N-terminus to C-terminus: a polypeptide or protein described herein, and a heterologous polypeptide or protein. In some embodiments, a fusion polypeptide or protein comprises, from N-terminus to C-terminus: a polypeptide or protein described herein, a peptide linker, and a heterologous polypeptide or protein. In some embodiments, a fusion polypeptide or protein comprises, from N-terminus to C-terminus: a signal peptide, a polypeptide or protein described herein, a peptide linker, and a heterologous polypeptide or protein. In some embodiments, a fusion polypeptide or protein comprises, from N-terminus to C-terminus: a heterologous polypeptide or protein and a polypeptide or protein described herein. In some embodiments, a fusion polypeptide or protein comprises, from N-terminus to C-terminus: a heterologous polypeptide or protein, a peptide linker, and a polypeptide or protein described herein. In some embodiments, a fusion polypeptide or protein comprises, from N-terminus to C-terminus: a signal peptide, a heterologous polypeptide or protein, a peptide linker, and an isolated polypeptide or protein described herein.

[0036] In one aspect, provided herein is a fusion protein comprising a first polypeptide or protein and a second polypeptide or protein, wherein the first polypeptide or protein comprises a first Ig (e.g., hIg, mIg) Fc region operably linked to a first polypeptide or protein described herein (e.g., a first immunomodulatory protein described herein), and the second polypeptide or protein comprises a second Ig (e.g., hIg, mIg) Fc region operably linked to a second polypeptide or protein described herein (e.g., a second immunomodulatory protein described herein).

[0037] In some embodiments, the first Fc region and the second Fc region combine to form a dimer.

[0038] In some embodiments, the first polypeptide or protein comprises an amino acid sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of the second polypeptide or protein.

[0039] In some embodiments, the first Ig (e.g., hIg, mIg) Fc region comprises at least a portion of the hinge region, CH2 region, and CH3 region; and the second Ig (e.g., hIg, mIg) Fc region comprises at least a portion of the hinge region, CH2 region, and CH3 region. In some embodiments, the first Ig (e.g., hIg, mIg) Fc region comprises at least a portion of the hinge region, CH2 region, and CH3 region; and the second Ig (e.g., hIg, mIg) Fc region comprises at least a portion of the hinge region, CH2 region, and CH3 region.

[0040] In some embodiments, the Ig of the first Ig Fc region is hIg, and the Ig of the second Ig Fc region is hIg. In some embodiments, the hIg of the first hIg Fc region is hIgG, and the hIg of the second hIg Fc region is 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.

[0041] In some embodiments, the Ig of the first Ig Fc region is hIg and the Ig of the second Ig Fc region is mIg. In some embodiments, the mIg of the first mIg Fc region is mIgG and the mIg of the second mIg Fc region is mIgG. In some embodiments, the mIg of the first mIg Fc region is mIgG1 and the mIg of the second mIg Fc region is mIgG1. In some embodiments, the mIg of the first mIg Fc region is mIgG2A and the mIg of the second mIg Fc region is mIgG2a.

[0042] In some embodiments, the first Ig (e.g., hIg, mIg) Fc region and the second Ig (e.g., hIg, mIg) Fc region each comprise one or more amino acid substitutions compared to a reference Ig (e.g., hIg, mIg) Fc region that reduce or eliminate one or more of the following effector functions: ADCC, CDC, and / or binding affinity to one or more Fc receptors (e.g., Fcγ receptors (e.g., FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and / or FcγRIIIb (e.g., FcγRI, FcγIIa, and / or FcγIIIa))) compared to the reference Ig (e.g., hIg, mIg) Fc region.

[0043] 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 receptors (e.g., an Fcγ receptor (e.g., FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and / or FcγRIIIb (e.g., FcγRI, FcγIIa, and / or FcγIIIa))).

[0044] In some embodiments, the Ig of the first Ig Fc region and the second Ig Fc region is hIgG4, and the amino acid sequences of the first Fc region and the second Fc region, respectively, 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, numbered 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 sequences of the first Fc region and the second Fc region, respectively, comprise a proline at amino acid position S228, an alanine at amino acid position F234, and / or an alanine at amino acid position E235, numbered according to the EU index of Kabat.

[0045] In some embodiments, the Ig of the first Ig Fc region and the second Ig Fc region is hIgG1, and the amino acid sequences of the first Fc region and the second Fc region, respectively, comprise an amino acid substitution at amino acid position L234 and / or an amino acid substitution at amino acid position L235, numbered 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 sequences of the first Fc region and the second Fc region, respectively, comprise an alanine at amino acid position L234 and / or an alanine at amino acid position L235, numbered 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 sequences of the first Fc region and the second Fc region, respectively, comprise a proline (or alanine) at amino acid position L234 and / or a proline (or alanine) at amino acid position L235, numbered 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 sequences of the first Fc region and the second Fc region, respectively, 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, numbered according to the EU index of Kabat.

[0046] In some embodiments, the Ig of the first Ig Fc region and the second Ig Fc region is mIgG2a, and the amino acid sequences of the first Fc region and the second Fc region, respectively, comprise an amino acid substitution at amino acid position L234 and / or an amino acid substitution at amino acid position L235, numbered according to the EU index of Kabat.

[0047] In some embodiments, the first Ig (e.g., hIg, mIg) Fc region comprises an amino acid sequence that is 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, mIg) Fc region.

[0048] In some embodiments, the first polypeptide or protein comprises, from N-terminus to C-terminus: a first Ig (e.g., hIg, mIg) Fc region and a first polypeptide or protein described herein (e.g., an immunomodulatory protein described herein); and the second polypeptide or protein comprises, from N-terminus to C-terminus: a second Ig (e.g., hIg, mIg) Fc region and a second polypeptide or protein described herein (e.g., an immunomodulatory protein described herein).

[0049] In some embodiments, the first polypeptide or protein comprises, from N-terminus to C-terminus: a first Ig (e.g., hIg, mIg) Fc region, a first peptide linker, and a first protein or polypeptide described herein (e.g., an immunomodulatory protein described herein); and the second polypeptide or protein comprises, from N-terminus to C-terminus: a second Ig (e.g., hIg, mIg) Fc region, a second peptide linker, and a second protein or polypeptide described herein (e.g., an immunomodulatory protein described herein).

[0050] In some embodiments, the first polypeptide or protein comprises, from N-terminus to C-terminus: a first polypeptide or protein described herein (e.g., an immunomodulatory protein described herein) and a first Ig (e.g., hIg, mIg) Fc region; and the second polypeptide or protein comprises, from N-terminus to C-terminus: a second polypeptide or protein described herein (e.g., an immunomodulatory protein described herein) and a second Ig (e.g., hIg, mIg) Fc region.

[0051] In some embodiments, the first polypeptide or protein comprises, from N-terminus to C-terminus: a first polypeptide or protein described herein (e.g., an immunomodulatory protein described herein), a first peptide linker, and a first Ig (e.g., hIg, mIg) Fc region; and the second polypeptide or protein comprises, from N-terminus to C-terminus: a second polypeptide or protein described herein (e.g., an immunomodulatory protein described herein), a second peptide linker, and a second Ig (e.g., hIg, mIg) Fc region.

[0052] In some embodiments, the amino acid sequences of the first and second peptide linkers each comprise or consist of glycine or glycine and serine amino acid residues. In some embodiments, the amino acid sequences of the first and second peptide linkers each comprise or consist of (a) the amino acid sequence 57 of any one of SEQ ID NOs: 49-57; or (b) the amino acid sequence of any one of SEQ ID NOs: 49-57, comprising or consisting of one, two, or three amino acid substitutions.

[0053] In some embodiments, a polypeptide or fusion protein comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of any one of SEQ ID NOs: 58, 61-69, or 72-79. In some embodiments, a polypeptide or fusion protein comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NOs: 58, 61-69, or 72-79. In some embodiments, a polypeptide or fusion protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of any one of SEQ ID NOs: 58, 61-69, or 72-79. In some embodiments, a polypeptide or fusion protein comprises an amino acid sequence that is 100% identical to the amino acid sequence of any one of SEQ ID NOs: 58, 61-69, or 72-79. In some embodiments, a polypeptide or fusion protein comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of any one of SEQ ID NOs: 62-65 or 73-76. In some embodiments, a polypeptide or fusion protein comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NOs: 62-65 or 73-76. In some embodiments, the polypeptide or fusion protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of any one of SEQ ID NOs: 62-65 or 73-76. In some embodiments, the polypeptide or fusion protein comprises an amino acid sequence that is 100% identical to the amino acid sequence of any one of SEQ ID NOs: 62-65 or 73-76.

[0054] In one aspect, provided herein is a conjugate comprising a polypeptide or protein described herein (e.g., an immunomodulatory polypeptide or protein described herein) operably linked to a heterologous moiety.

[0055] In one aspect, provided herein is an immunogenic peptide or protein comprising: (a) at least a portion of a polypeptide or protein described herein (e.g., an immunomodulatory polypeptide or protein described herein); and / or (b) a polypeptide or protein described herein (e.g., an immunomodulatory polypeptide or protein described herein) comprising at least one amino acid mutation (e.g., a substitution, addition, deletion), wherein the immunogenic peptide or protein does not specifically bind to hIL-10R (e.g., hIL-10Rα) or binds to hIL-10R (e.g., hIL-10Rα) with lower affinity compared to a reference polypeptide or protein.

[0056] In some embodiments, the immunogenic polypeptide or protein comprises at least about 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, or 250 amino acids. In some embodiments, the immunogenic peptide or protein comprises about 10 to 250, 10 to 200, 10 to 100, 10 to 90, 10 to 80, 10 to 70, 10 to 60, 10 to 50, 10 to 40, 10 to 30, 10 to 20, 10 to 250, 20 to 250, 30 to 250, 40 to 250, 50 to 250, 60 to 250, 70 to 250, 80 to 250, 90 to 250, 100 to 250, 10 to 200, 20 to 200, 30 to 200, 40 to 200, 50 to 200, 60 to 200, 70 to 200, 80 to 200, 90 to 200, or 100 to 200 amino acids.

[0057] In some embodiments, the amino acid sequence of an immunogenic peptide or protein contains at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid mutations (eg, substitutions, additions, deletions) compared to a reference polypeptide or protein.

[0058] 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, 150, or 200 amino acids of any one of SEQ ID NOs: 108-454.

[0059] 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 the amino acid sequence of any one of SEQ ID NOs: 108-454, except for at least one amino acid mutation (e.g., substitution, addition, deletion).

[0060] In some embodiments, the immunogenic polypeptide or protein is formulated with an adjuvant.

[0061] In one aspect, provided herein is an isolated antibody that specifically binds to a protein or polypeptide described herein (e.g., an immunomodulatory protein or polypeptide described herein).

[0062] In one aspect, provided herein is a polynucleotide encoding a polypeptide or protein described herein (e.g., an immunomodulatory protein or polypeptide described herein), a fusion polypeptide or protein described herein, a conjugate described herein, an immunogenic peptide or protein described herein, or an antibody described herein.

[0063] In some embodiments, the polynucleotide is RNA (eg, mRNA) or DNA.

[0064] In one aspect, provided herein is an mRNA polynucleotide encoding a polypeptide or protein described herein (e.g., an immunomodulatory protein or polypeptide described herein), a fusion polypeptide or protein described herein, a conjugate described herein, an immunogenic peptide or protein described herein, or an antibody described herein.

[0065] In some embodiments, the nucleic acid molecule comprises a heterologous 5'-untranslated region (UTR), a 3'-UTR, or both a 5'-UTR and a 3'-UTR. In some embodiments, the mRNA comprises a poly(A) sequence. In some embodiments, the mRNA comprises a 5'-cap structure.

[0066] In some embodiments, the polynucleotide or mRNA polynucleotide comprises at least one variant nucleotide. In some embodiments, the respective nucleic acid sequence of the polynucleotide or mRNA polynucleotide is codon optimized.

[0067] In one aspect, provided herein is an expression vector comprising a polynucleotide described herein or an mRNA polynucleotide described herein. In some embodiments, the expression vector is a viral vector or a plasmid.

[0068] In one aspect, provided herein is a cell (e.g., a host cell) comprising a polypeptide or protein described herein (e.g., an immunomodulatory protein or polypeptide described herein), a fusion polypeptide or protein described herein, a conjugate described herein, an immunogenic peptide or protein described herein, an antibody described herein, a polynucleotide described herein, an mRNA described herein, an expression vector described herein, or a carrier described herein.

[0069] In one aspect, provided herein is a carrier comprising a polypeptide or protein described herein (e.g., an immunomodulatory protein or polypeptide described herein), a fusion polypeptide or protein described herein, a conjugate described herein, an immunogenic peptide or protein described herein, an antibody described herein, a polynucleotide described herein, an mRNA described herein, an expression vector described herein, or a cell described herein. In some embodiments, the carrier is a lipid nanoparticle, a liposome, a lipoplex, or a nanoliposome.

[0070] In one aspect, provided herein is a lipid nanoparticle comprising a polypeptide or protein described herein (e.g., an immunomodulatory protein or polypeptide described herein), a fusion polypeptide or protein described herein, a conjugate described herein, an immunogenic peptide or protein described herein, an antibody described herein, a polynucleotide described herein, an mRNA described herein, an expression vector described herein, or a cell described herein.

[0071] In one aspect, provided herein is a vaccine composition comprising an immunogenic peptide or protein (or a nucleic acid molecule encoding same (or a vector encoding a nucleic acid molecule)) described herein.

[0072] In one aspect, provided herein is a pharmaceutical composition comprising a polypeptide or protein described herein (e.g., an immunomodulatory protein or polypeptide described herein), a fusion polypeptide or protein described herein, a conjugate described herein, an immunogenic peptide or protein described herein, an antibody described herein, a polynucleotide described herein, an mRNA described herein, an expression vector described herein, a cell described herein, a lipid nanoparticle described herein, a vaccine composition described herein, and a pharmaceutically acceptable excipient.

[0073] In one aspect, provided herein is a kit comprising a polypeptide or protein described herein (e.g., an immunomodulatory protein or polypeptide described herein), a fusion polypeptide or protein described herein, a conjugate described herein, an immunogenic peptide or protein described herein, an antibody described herein, a polynucleotide described herein, an mRNA described herein, an expression vector described herein, a cell described herein, a carrier described herein, a lipid nanoparticle described herein, a vaccine composition described herein, or a pharmaceutical composition described herein; and optionally, instructions for using any one or more of the foregoing.

[0074]

[0013] In one aspect, provided herein is a method of delivering a polypeptide, protein, fusion protein, conjugate, polynucleotide, mRNA polynucleotide, expression vector, cell, carrier, lipid nanoparticle, immunogenic peptide or protein, antibody, vaccine composition, or pharmaceutical composition to a subject, the method comprising administering a polypeptide or protein described herein (e.g., an immunomodulatory protein or polypeptide described herein), a fusion polypeptide or protein described herein, a conjugate described herein, an immunogenic peptide or protein described herein, an antibody described herein, an mRNA described herein, an expression vector described herein, a cell described herein, a carrier described herein, a lipid nanoparticle described herein, a vaccine composition described herein, or a pharmaceutical composition described herein to a subject, thereby delivering the polypeptide, protein, fusion polypeptide, fusion protein, conjugate, immunogenic peptide or protein, antibody, mRNA polynucleotide, expression vector, cell, carrier, lipid nanoparticle, vaccine composition, or pharmaceutical composition to the subject.

[0075] In one aspect, provided herein is the use of a polypeptide or protein described herein (e.g., an immunomodulatory protein or polypeptide described herein), a fusion polypeptide or protein described herein, a conjugate described herein, an immunogenic peptide or protein described herein, an antibody described herein, a polynucleotide described herein, an mRNA described herein, an expression vector described herein, a cell described herein, a carrier described herein, a lipid nanoparticle described herein, a vaccine composition described herein, or a pharmaceutical composition described herein in medicine.

[0076]

[0013] In one aspect, provided herein is a method of stimulating the IL-10 pathway in a subject in need thereof, the method comprising administering to the subject a polypeptide or protein described herein (e.g., an immunomodulatory protein or polypeptide described herein), a fusion polypeptide or protein described herein, a conjugate described herein, an immunogenic peptide or protein described herein, an antibody described herein, a polynucleotide described herein, an mRNA described herein, an expression vector described herein, a cell described herein, a carrier described herein, a lipid nanoparticle described herein, a vaccine composition described herein, or a pharmaceutical composition described herein, thereby stimulating the IL-10 pathway in the subject.

[0077]

[0014] In one aspect, provided herein is a polypeptide or protein described herein (e.g., an immunomodulatory protein or polypeptide described herein), a fusion polypeptide or protein described herein, a conjugate described herein, an immunogenic peptide or protein described herein, an antibody described herein, a polynucleotide described herein, an mRNA described herein, an expression vector described herein, a cell described herein, a carrier described herein, a lipid nanoparticle described herein, a vaccine composition described herein, or a pharmaceutical composition described herein for use in a method of stimulating the IL-10 pathway in a subject in need thereof, the method comprising administering to the subject the polypeptide or protein, the polypeptide or protein, the fusion polypeptide or protein, the fusion protein or protein, the conjugate, the immunogenic peptide or protein, the antibody, the polynucleotide, the mRNA polynucleotide, the expression vector, the cell, the carrier, the lipid nanoparticle, the vaccine composition, or the pharmaceutical composition, thereby stimulating the IL-10 pathway in the subject.

[0078]

[0014] In one aspect, provided herein is the use of a polypeptide or protein described herein (e.g., an immunomodulatory protein or polypeptide described herein), a fusion polypeptide or protein described herein, a conjugate described herein, an immunogenic peptide or protein described herein, an antibody described herein, a polynucleotide described herein, an mRNA described herein, an expression vector described herein, a cell described herein, a carrier described herein, a lipid nanoparticle described herein, a vaccine composition described herein, or a pharmaceutical composition described herein in the manufacture of a medicament for stimulating the IL-10 pathway in a subject in need thereof, comprising administering to the subject the polypeptide or protein, polypeptide or protein, fusion polypeptide or protein, fusion protein or protein, conjugate, immunogenic peptide or protein, antibody, polynucleotide, mRNA polynucleotide, expression vector, cell, carrier, lipid nanoparticle, vaccine composition, or pharmaceutical composition, thereby stimulating the IL-10 pathway in the subject.

[0079]

[0010] In one aspect, provided herein is a method of treating, preventing, or ameliorating an IL-10-responsive disorder in a subject in need thereof, the method comprising administering to the subject a polypeptide or protein described herein (e.g., an immunomodulatory protein or polypeptide described herein), a fusion polypeptide or protein described herein, a conjugate described herein, an immunogenic peptide or protein described herein, an antibody described herein, a polynucleotide described herein, an mRNA described herein, an expression vector described herein, a cell described herein, a carrier described herein, a lipid nanoparticle described herein, a vaccine composition described herein, or a pharmaceutical composition described herein, thereby treating, preventing, or ameliorating the IL-10-responsive disorder. In some embodiments, the IL-10-responsive disorder is an inflammatory disorder. In some embodiments, the IL-10-responsive disorder is an autoimmune disorder. In some embodiments, the IL-10-responsive disorder is a metabolic inflammatory disorder.

[0080]

[0014] In one aspect, provided herein is a polypeptide or protein described herein (e.g., an immunomodulatory protein or polypeptide described herein), a fusion polypeptide or protein described herein, a conjugate described herein, an immunogenic peptide or protein described herein, an antibody described herein, a polynucleotide described herein, an mRNA described herein, an expression vector described herein, a cell described herein, a carrier described herein, a lipid nanoparticle described herein, a vaccine composition described herein, or a pharmaceutical composition described herein for use in a method of treating, preventing, or ameliorating an IL-10-responsive disorder in a subject in need thereof, the method comprising administering the polypeptide or protein, polypeptide or protein, fusion polypeptide or protein, fusion protein, conjugate, immunogenic peptide or protein, antibody, polynucleotide, mRNA polynucleotide, expression vector, cell, carrier, lipid nanoparticle, vaccine composition, or pharmaceutical composition to the subject, thereby treating, preventing, or ameliorating the IL-10-responsive disorder. In some embodiments, the IL-10-responsive disorder is an inflammatory disorder. In some embodiments, the IL-10-responsive disorder is an autoimmune disorder. In some embodiments, the IL-10-responsive disorder is a metabolic inflammatory disorder.

[0081]

[0014] In one aspect, provided herein is the use of a polypeptide or protein described herein (e.g., an immunomodulatory protein or polypeptide described herein), a fusion polypeptide or protein described herein, a conjugate described herein, an immunogenic peptide or protein described herein, an antibody described herein, a polynucleotide described herein, an mRNA described herein, an expression vector described herein, a cell described herein, a carrier described herein, a lipid nanoparticle described herein, a vaccine composition described herein, or a pharmaceutical composition described herein in the manufacture of a medicament for treating, preventing, or ameliorating an IL-10-responsive disorder in a subject in need thereof, comprising administering the polypeptide or protein, polypeptide or protein, fusion polypeptide or protein, fusion protein, conjugate, immunogenic peptide or protein, antibody, polynucleotide, mRNA polynucleotide, expression vector, cell, carrier, lipid nanoparticle, vaccine composition, or pharmaceutical composition to the subject, thereby treating, preventing, or ameliorating the IL-10-responsive disorder. In some embodiments, the IL-10-responsive disorder is an inflammatory disorder. In some embodiments, the IL-10-responsive disorder is an autoimmune disorder. In some embodiments, the IL-10-responsive disorder is a metabolic inflammatory disorder.

[0082] In one aspect, provided herein is a method of suppressing or preventing an immune response in a subject in need thereof, the method comprising administering to the subject a polypeptide or protein described herein (e.g., an immunomodulatory protein or polypeptide described herein), a fusion polypeptide or protein described herein, a conjugate described herein, an immunogenic peptide or protein described herein, a polynucleotide described herein, an mRNA described herein, an expression vector described herein, a cell described herein, a carrier described herein, a lipid nanoparticle described herein, a vaccine composition described herein, or a pharmaceutical composition described herein, thereby suppressing or preventing an immune response in the subject.

[0083]

[0014] In one aspect, provided herein is a polypeptide or protein described herein (e.g., an immunomodulatory protein or polypeptide described herein), a fusion polypeptide or protein described herein, a conjugate described herein, an immunogenic peptide or protein described herein, an antibody described herein, a polynucleotide described herein, an mRNA described herein, an expression vector described herein, a cell described herein, a carrier described herein, a lipid nanoparticle described herein, a vaccine composition described herein, or a pharmaceutical composition described herein for use in a method of suppressing or preventing an immune response in a subject in need thereof, the method comprising administering to the subject the polypeptide or protein, polypeptide or protein, fusion polypeptide or protein, fusion, conjugate, immunogenic peptide or protein, said antibody, polynucleotide, mRNA polynucleotide, expression vector, cell, carrier, lipid nanoparticle, vaccine composition, or pharmaceutical composition, thereby suppressing or preventing an immune response in the subject.

[0084]

[0014] In one aspect, provided herein is the use of a polypeptide or protein described herein (e.g., an immunomodulatory protein or polypeptide described herein), a fusion polypeptide or protein described herein, a conjugate described herein, an immunogenic peptide or protein described herein, an antibody described herein, a polynucleotide described herein, an mRNA described herein, an expression vector described herein, a cell described herein, a carrier described herein, a lipid nanoparticle described herein, a vaccine composition described herein, or a pharmaceutical composition described herein in the manufacture of a medicament for suppressing or preventing an immune response in a subject in need thereof, comprising administering to the subject the polypeptide or protein, polypeptide or protein, fusion polypeptide or protein, fusion, conjugate, immunogenic peptide or protein, antibody, polynucleotide, mRNA polynucleotide, expression vector, cell, carrier, lipid nanoparticle, vaccine composition, or pharmaceutical composition, thereby suppressing or preventing an immune response in the subject.

[0085] In one aspect, provided herein is a method of inducing tolerance to an immunogen in a subject in need thereof, the method comprising administering to the subject a polypeptide or protein described herein (e.g., an immunomodulatory protein or polypeptide described herein), a fusion polypeptide or protein described herein, a conjugate described herein, an immunogenic peptide or protein described herein, an antibody described herein, a polynucleotide described herein, an mRNA described herein, an expression vector described herein, a cell described herein, a carrier described herein, a lipid nanoparticle described herein, a vaccine composition described herein, or a pharmaceutical composition described herein, thereby inducing tolerance to the immunogen in the subject.

[0086]

[0014] In one aspect, provided herein is a polypeptide or protein described herein (e.g., an immunomodulatory protein or polypeptide described herein), a fusion polypeptide or protein described herein, a conjugate described herein, an immunogenic peptide or protein described herein, an antibody described herein, a polynucleotide described herein, an mRNA described herein, an expression vector described herein, a cell described herein, a carrier described herein, a lipid nanoparticle described herein, a vaccine composition described herein, or a pharmaceutical composition described herein for use in a method of inducing tolerance to an immunogen in a subject in need thereof, the method comprising administering to the subject the polypeptide or protein, polypeptide or protein, fusion polypeptide or protein, fusion, conjugate, immunogenic peptide or protein, antibody, polynucleotide, mRNA polynucleotide, expression vector, cell, carrier, lipid nanoparticle, vaccine composition, or pharmaceutical composition, thereby inducing tolerance to the immunogen in the subject.

[0087]

[0014] In one aspect, provided herein is the use of a polypeptide or protein described herein (e.g., an immunomodulatory protein or polypeptide described herein), a fusion polypeptide or protein described herein, a conjugate described herein, an immunogenic peptide or protein described herein, an antibody described herein, a polynucleotide described herein, an mRNA described herein, an expression vector described herein, a cell described herein, a carrier described herein, a lipid nanoparticle described herein, a vaccine composition described herein, or a pharmaceutical composition described herein in the manufacture of a medicament for inducing tolerance to an immunogen in a subject in need thereof, comprising administering to the subject the polypeptide or protein, polypeptide or protein, fusion polypeptide or protein, fusion, conjugate, immunogenic peptide or protein, antibody, polynucleotide, mRNA polynucleotide, expression vector, cell, carrier, lipid nanoparticle, vaccine composition, or pharmaceutical composition, thereby inducing tolerance to an immunogen in the subject.

[0088] In one aspect, provided herein is a method of suppressing IgE expression in a subject in need thereof, the method comprising administering to the subject a polypeptide or protein described herein (e.g., an immunomodulatory protein or polypeptide described herein), a fusion polypeptide or protein described herein, a conjugate described herein, an immunogenic peptide or protein described herein, an antibody described herein, a polynucleotide described herein, an mRNA described herein, an expression vector described herein, a cell described herein, a carrier described herein, a lipid nanoparticle described herein, a vaccine composition described herein, or a pharmaceutical composition described herein, thereby suppressing IgE expression in the subject.

[0089]

[0023] In one aspect, provided herein is a polypeptide or protein described herein (e.g., an immunomodulatory protein or polypeptide described herein), a fusion polypeptide or protein described herein, a conjugate described herein, an immunogenic peptide or protein described herein, an antibody described herein, a polynucleotide described herein, an mRNA described herein, an expression vector described herein, a cell described herein, a carrier described herein, a lipid nanoparticle described herein, a vaccine composition described herein, or a pharmaceutical composition described herein for use in a method of suppressing IgE expression in a subject in need thereof, the method comprising administering to the subject the polypeptide or protein, polypeptide or protein, fusion polypeptide or protein, fusion, conjugate, immunogenic peptide or protein, antibody, polynucleotide, mRNA polynucleotide, expression vector, cell, carrier, lipid nanoparticle, vaccine composition, or pharmaceutical composition, thereby suppressing IgE expression in the subject.

[0090]

[0014] In one aspect, provided herein is the use of a polypeptide or protein described herein (e.g., an immunomodulatory protein or polypeptide described herein), a fusion polypeptide or protein described herein, a conjugate described herein, an immunogenic peptide or protein described herein, an antibody described herein, a polynucleotide described herein, an mRNA described herein, an expression vector described herein, a cell described herein, a carrier described herein, a lipid nanoparticle described herein, a vaccine composition described herein, or a pharmaceutical composition described herein in the manufacture of a medicament for suppressing IgE expression in a subject in need thereof, comprising administering to the subject the polypeptide or protein, polypeptide or protein, fusion polypeptide or protein, fusion, conjugate, immunogenic peptide or protein, antibody, polynucleotide, mRNA polynucleotide, expression vector, cell, carrier, lipid nanoparticle, vaccine composition, or pharmaceutical composition, thereby suppressing IgE expression in the subject.

[0091] In some embodiments, the levels of one or more inflammatory cytokines (eg, IFNγ, TNFα, IL-17A) are inhibited (eg, as measured by an assay described herein).

[0092] In some embodiments, levels of immunogen-specific immunoglobulin are suppressed (eg, as measured by an assay described herein).

[0093] In some embodiments, levels of IgE (eg, immunogen-specific IgE, eg, allergen-specific IgE) are suppressed (eg, as measured by an assay described herein).

[0094] In some embodiments, the subject has an IL-10 responsive disorder (e.g., an IL-10 responsive disorder described herein (e.g., an autoimmune disease (e.g., GvHD, an allergic disease, etc.)).

[0095] In some embodiments, prior to administering to the subject a polypeptide or protein described herein (e.g., an immunomodulatory protein or polypeptide described herein), a fusion polypeptide or protein described herein, a conjugate described herein, an immunogenic peptide or protein described herein, an antibody described herein, a polynucleotide described herein, an mRNA described herein, an expression vector described herein, a cell described herein, a lipid nanoparticle described herein, a vaccine composition described herein, or a pharmaceutical composition described herein, the subject has been administered one or more immunosuppressants (e.g., one or more steroids (e.g., corticosteroids)).

[0096] In some embodiments, the IL-10 responsive disorder is refractory to one or more immunosuppressive agents (eg, one or more steroids (eg, corticosteroids)).

[0097] In some embodiments, a polypeptide or protein described herein (e.g., an immunomodulatory protein or polypeptide described herein), a fusion polypeptide or protein described herein, a conjugate described herein, an immunogenic peptide or protein described herein, an antibody described herein, a polynucleotide described herein, an mRNA described herein, an expression vector described herein, a cell described herein, a carrier described herein, a lipid nanoparticle described herein, a vaccine composition described herein, or a pharmaceutical composition described herein is administered to a subject in combination with one or more immunosuppressants (e.g., one or more steroids (e.g., corticosteroids)).

[0098] In some embodiments, the immunogen is an allergen. In some embodiments, the subject has received an allergen-specific therapy (e.g., as described herein (e.g., the subject has received at least one (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more doses (e.g., escalating doses) of an allergen)). In some embodiments, the allergen-specific therapy has induced some degree of tolerance to the immunogen in the subject.

[0099] In some embodiments, a polypeptide or protein described herein (e.g., an immunomodulatory protein or polypeptide described herein), a fusion polypeptide or protein described herein, a conjugate described herein, an immunogenic peptide or protein described herein, an antibody described herein, a polynucleotide described herein, an mRNA described herein, an expression vector described herein, a cell described herein, a carrier described herein, a lipid nanoparticle described herein, a vaccine composition described herein, or a pharmaceutical composition described herein enhances the degree of tolerance induced by an immunogen-specific therapy.

[0100] In some embodiments, the subject has received an allogeneic or autologous cell, tissue, or organ transplant. In some embodiments, the immunogen is part of an allogeneic or autologous cell, tissue, or organ. In some embodiments, the subject has graft-versus-host disease. In some embodiments, the graft-versus-host disease is acute or chronic.

[0101] In one aspect, provided herein is a method of inducing an immune response in a subject in need thereof, the method comprising administering to the subject a polypeptide or protein described herein (e.g., an immunomodulatory protein or polypeptide described herein), a fusion polypeptide or protein described herein, a conjugate described herein, an immunogenic peptide or protein described herein, an antibody described herein, a polynucleotide described herein, an mRNA described herein, an expression vector described herein, a cell described herein, a carrier described herein, a lipid nanoparticle described herein, a vaccine composition described herein, or a pharmaceutical composition described herein, thereby inducing an immune response in the subject.

[0102]

[0014] In one aspect, provided herein is a polypeptide or protein described herein (e.g., an immunomodulatory protein or polypeptide described herein), a fusion polypeptide or protein described herein, a conjugate described herein, an immunogenic peptide or protein described herein, an antibody described herein, a polynucleotide described herein, an mRNA described herein, an expression vector described herein, a cell described herein, a carrier described herein, a lipid nanoparticle described herein, a vaccine composition described herein, or a pharmaceutical composition described herein for use in a method of inducing an immune response in a subject in need thereof, the method comprising administering the polypeptide or protein, polypeptide or protein, fusion polypeptide or protein, fusion, conjugate, immunogenic peptide or protein, antibody, polynucleotide, mRNA polynucleotide, expression vector, cell, carrier, lipid nanoparticle, vaccine composition, or pharmaceutical composition to the subject, thereby inducing an immune response in the subject.

[0103]

[0014] In one aspect, provided herein is the use of a polypeptide or protein described herein (e.g., an immunomodulatory protein or polypeptide described herein), a fusion polypeptide or protein described herein, a conjugate described herein, an immunogenic peptide or protein described herein, an antibody described herein, a polynucleotide described herein, an mRNA described herein, an expression vector described herein, a cell described herein, a carrier described herein, a lipid nanoparticle described herein, a vaccine composition described herein, or a pharmaceutical composition described herein in the manufacture of a medicament for inducing an immune response in a subject in need thereof, comprising administering to the subject the polypeptide or protein, polypeptide or protein, fusion polypeptide or protein, fusion, conjugate, immunogenic peptide or protein, antibody, polynucleotide, mRNA polynucleotide, expression vector, cell, carrier, lipid nanoparticle, vaccine composition, or pharmaceutical composition, thereby inducing an immune response in the subject.

[0104]

[0010] In one aspect, provided herein are methods of preventing, treating, or ameliorating a viral infection in a subject in need thereof, comprising administering to the subject a polypeptide or protein described herein (e.g., an immunomodulatory protein or polypeptide described herein), a fusion polypeptide or protein described herein, a conjugate described herein, an immunogenic peptide or protein described herein, an antibody described herein, a polynucleotide described herein, an mRNA described herein, an expression vector described herein, a cell described herein, a carrier described herein, a lipid nanoparticle described herein, a vaccine composition described herein, or a pharmaceutical composition described herein, thereby preventing, treating, or ameliorating the viral infection in the subject. In some embodiments, the viral infection is infection by parapoxvirus, cytomegalovirus, gammaherpesvirus, orf virus, pseudocowpoxvirus, betaherpesvirus, or Epstein-Barr virus.

[0105] In one aspect, provided herein is a polypeptide or protein described herein (e.g., an immunomodulatory protein or polypeptide described herein), a fusion polypeptide or protein described herein, a conjugate described herein, an immunogenic peptide or protein described herein, an antibody described herein, a polynucleotide described herein, an mRNA described herein, an expression vector described herein, a cell described herein, a carrier described herein, a lipid nanoparticle described herein, a vaccine composition described herein, or a pharmaceutical composition described herein for use in a method of preventing, treating, or ameliorating a viral infection in a subject in need thereof, the method comprising administering to the subject the polypeptide or protein, polypeptide or protein, fusion polypeptide or protein, fusion, conjugate, immunogenic peptide or protein, antibody, polynucleotide, mRNA polynucleotide, expression vector, cell, carrier, lipid nanoparticle, vaccine composition, or pharmaceutical composition, thereby preventing, treating, or ameliorating the viral infection in the subject. In some embodiments, the viral infection is infection by parapoxvirus, cytomegalovirus, gammaherpesvirus, orf virus, pseudocowpoxvirus, betaherpesvirus, or Epstein-Barr virus.

[0106] In one aspect, provided herein is the use of a polypeptide or protein described herein (e.g., an immunomodulatory protein or polypeptide described herein), a fusion polypeptide or protein described herein, a conjugate described herein, an immunogenic peptide or protein described herein, an antibody described herein, a polynucleotide described herein, an mRNA described herein, an expression vector described herein, a cell described herein, a carrier described herein, a lipid nanoparticle described herein, a vaccine composition described herein, or a pharmaceutical composition described herein in the manufacture of a medicament for preventing, treating, or ameliorating a viral infection in a subject in need thereof, comprising administering to the subject the polypeptide or protein, polypeptide or protein, fusion polypeptide or protein, fusion, conjugate, immunogenic peptide or protein, antibody, polynucleotide, mRNA polynucleotide, expression vector, cell, carrier, lipid nanoparticle, vaccine composition, or pharmaceutical composition, thereby preventing, treating, or ameliorating a viral infection in the subject. In some embodiments, the viral infection is infection by parapoxvirus, cytomegalovirus, gammaherpesvirus, orf virus, pseudocowpoxvirus, betaherpesvirus, or Epstein-Barr virus.

[0107] In one aspect, provided herein is a method of determining the presence of a virus in a subject, the method comprising: (a) obtaining a sample from the subject or providing a sample obtained from the subject; and (b) determining the presence or absence of a polypeptide or protein described herein (e.g., an immunomodulatory polypeptide or protein described herein) (or a fragment or variant thereof) or a polynucleotide encoding a polypeptide or protein described herein (e.g., an immunomodulatory polypeptide or protein described herein) (or a fragment or variant thereof) in the sample.

[0108] In some embodiments, the sample is blood, cells, tissue, or saliva, or a nasal swab. In some embodiments, the subject is a human. In some embodiments, the antibodies described herein are used to determine the presence or absence of a polypeptide or protein described herein (e.g., an immunomodulatory polypeptide or protein described herein) (or a fragment or variant thereof). In some embodiments, the viral infection is an infection by parapoxvirus, cytomegalovirus, gammaherpesvirus, orfvirus, pseudocowpoxvirus, betaherpesvirus, and Epstein-Barr virus.

[0109] In one aspect, provided herein is a method of diagnosing a viral infection in a subject, the method comprising: (a) obtaining a sample from the subject or providing a sample obtained from the subject; (b) determining the presence or absence of a polypeptide or protein described herein (e.g., an immunomodulatory polypeptide or protein described herein) (or a fragment or variant thereof) or a polynucleotide encoding a polypeptide or protein described herein (e.g., an immunomodulatory polypeptide or protein described herein) (or a fragment or variant thereof); and (c) diagnosing the subject as having a viral infection if a polypeptide or protein described herein (e.g., an immunomodulatory polypeptide or protein described herein) (or a fragment or variant thereof) or a polynucleotide encoding a polypeptide or protein described herein (e.g., an immunomodulatory polypeptide or protein described herein) (or a 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.

[0110] In some embodiments, the sample is blood, cells, tissue, or saliva, or a nasal swab. In some embodiments, the subject is a human. In some embodiments, the antibodies described herein are used to determine the presence or absence of a polypeptide or protein described herein (e.g., an immunomodulatory polypeptide or protein described herein) (or a fragment or variant thereof). In some embodiments, the viral infection is an infection by parapoxvirus, cytomegalovirus, gammaherpesvirus, orf virus, pseudocowpoxvirus, betaherpesvirus, and Epstein-Barr virus.

[0111] In one aspect, provided herein is a method of treating a viral infection in a subject, the method comprising: (a) receiving test results determining the presence of a polypeptide or protein described herein (e.g., an immunomodulatory polypeptide or protein described herein) (or a fragment or variant thereof) or a polynucleotide encoding a polypeptide or protein described herein (e.g., an immunomodulatory polypeptide or protein described herein) (or a fragment or variant thereof) in a sample from the subject; (b) diagnosing the subject as having a viral infection; and (c) administering a therapeutic agent to treat the viral infection.

[0112] In some embodiments, the sample is blood, cells, tissue, or saliva, or a nasal swab. In some embodiments, the subject is a human. In some embodiments, the antibodies described herein are used to determine the presence or absence of a polypeptide or protein described herein (e.g., an immunomodulatory polypeptide or protein described herein) (or a fragment or variant thereof). In some embodiments, the viral infection is an infection by parapoxvirus, cytomegalovirus, gammaherpesvirus, orf virus, pseudocowpoxvirus, betaherpesvirus, and Epstein-Barr virus.

[0113] In one aspect, provided herein is a therapeutic agent for treating a viral infection for use in a method of treating a viral infection in a subject, the method comprising: (a) receiving test results determining the presence of a polypeptide or protein described herein (e.g., an immunomodulatory polypeptide or protein) (or a fragment or variant thereof) or a polynucleotide encoding a polypeptide or protein described herein (e.g., an immunomodulatory polypeptide or protein described herein) (or a 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.

[0114] In some embodiments, the sample is blood, cells, tissue, or saliva, or a nasal swab. In some embodiments, the subject is a human. In some embodiments, the antibodies described herein are used to determine the presence or absence of a polypeptide or protein described herein (e.g., an immunomodulatory polypeptide or protein described herein) (or a fragment or variant thereof). In some embodiments, the viral infection is an infection by parapoxvirus, cytomegalovirus, gammaherpesvirus, orf virus, pseudocowpoxvirus, betaherpesvirus, and Epstein-Barr virus.

[0115] In one aspect, provided herein is the use of a therapeutic agent for treating a viral infection in the manufacture of a medicament for treating a viral infection in a subject, the use comprising: (a) receiving a test result determining the presence of a polypeptide or protein described herein (e.g., an immunomodulatory polypeptide or protein) (or a fragment or variant thereof), or a polynucleotide encoding a polypeptide or protein described herein (e.g., an immunomodulatory polypeptide or protein described herein) (or a fragment or variant thereof); (b) diagnosing the subject as having a viral infection; and (c) administering the therapeutic agent to treat the viral infection.

[0116] In some embodiments, the sample is blood, cells, tissue, or saliva, or a nasal swab. In some embodiments, the subject is a human. In some embodiments, the antibodies described herein are used to determine the presence or absence of a polypeptide or protein described herein (e.g., an immunomodulatory polypeptide or protein described herein) (or a fragment or variant thereof). In some embodiments, the viral infection is an infection by parapoxvirus, cytomegalovirus, gammaherpesvirus, orf virus, pseudocowpoxvirus, betaherpesvirus, and Epstein-Barr virus. 4. Brief description of the drawings [Brief explanation of the drawings]

[0117] [Figure 1] 1 is a bar graph showing the levels (ng / mL) of in vitro expressed immunomodulatory fusion proteins 1-11 (IFP-1-11) and a reference Fc-hIL-10 fusion protein, respectively. [Figure 2A] 1 is a line graph showing hIL-10R ligation by IFP-1 and a reference Fc-hIL-10 fusion protein in vitro. [Figure 2B] 1 is a line graph showing hIL-10R ligation by IFP-2 and a reference Fc-hIL-10 fusion protein in vitro. [Figure 2C] 1 is a line graph showing hIL-10R ligation by IFP-3 and a reference Fc-hIL-10 fusion protein in vitro. [Figure 2D] 1 is a line graph showing hIL-10R ligation by IFP-4 and a reference Fc-hIL-10 fusion protein in vitro. [Figure 2E] 1 is a line graph showing hIL-10R ligation by IFP-5 and a reference Fc-hIL-10 fusion protein in vitro. [Figure 2F] 1 is a line graph showing hIL-10R ligation by IFP-6 and a reference Fc-hIL-10 fusion protein in vitro. [Figure 2G] 1 is a line graph showing hIL-10R ligation by IFP-7 and a reference Fc-hIL-10 fusion protein in vitro. [Figure 2H] 1 is a line graph showing hIL-10R ligation by IFP-8 and a reference Fc-hIL-10 fusion protein in vitro. [Figure 2I] 1 is a line graph showing hIL-10R ligation by IFP-9 and a reference Fc-hIL-10 fusion protein in vitro. [Figure 2J] 1 is a line graph showing hIL-10R ligation by IFP-10 and a reference Fc-hIL-10 fusion protein in vitro. [Figure 2K] 1 is a line graph showing hIL-10R ligation by IFP-11 and a reference Fc-hIL-10 fusion protein in vitro. [Figure 3A] 1 is a line graph showing hIL-10R ligation in vitro by a reference Fc-hIL-10 fusion protein in the presence and absence of an anti-IL-10Rα antibody that blocks hIL-10 binding to hIL-10Rα. [Figure 3B] 1 is a line graph showing hIL-10R ligation by IFP-1 in vitro in the presence and absence of an anti-IL-10Rα antibody that blocks hIL-10 binding to hIL-10Rα. [Figure 3C] 1 is a line graph showing hIL-10R ligation by IFP-2 in vitro in the presence and absence of an anti-IL-10Rα antibody that blocks hIL-10 binding to hIL-10Rα. [Figure 3D] 1 is a line graph showing hIL-10R ligation by IFP-3 in vitro in the presence and absence of an anti-IL-10Rα antibody that blocks hIL-10 binding to hIL-10Rα. [Figure 3E] 1 is a line graph showing hIL-10R ligation by IFP-4 in vitro in the presence and absence of an anti-IL-10Rα antibody that blocks hIL-10 binding to hIL-10Rα. [Figure 3F] 1 is a line graph showing hIL-10R ligation by IFP-5 in vitro in the presence and absence of an anti-IL-10Rα antibody that blocks hIL-10 binding to hIL-10Rα. [Figure 3G] 1 is a line graph showing hIL-10R ligation by IFP-6 in vitro in the presence and absence of an anti-IL-10Rα antibody that blocks hIL-10 binding to hIL-10Rα. [Figure 3H] 1 is a line graph showing hIL-10R ligation by IFP-7 in vitro in the presence and absence of an anti-IL-10Rα antibody that blocks hIL-10 binding to hIL-10Rα. [Figure 3I] 1 is a line graph showing hIL-10R ligation by IFP-8 in vitro in the presence and absence of an anti-IL-10Rα antibody that blocks hIL-10 binding to hIL-10Rα. [Figure 3J] 1 is a line graph showing hIL-10R ligation by IFP-9 in vitro in the presence and absence of an anti-IL-10Rα antibody that blocks hIL-10 binding to hIL-10Rα. [Figure 3K] 1 is a line graph showing hIL-10R ligation by IFP-10 in vitro in the presence and absence of an anti-IL-10Rα antibody that blocks hIL-10 binding to hIL-10Rα. [Figure 3L] 1 is a line graph showing hIL-10R ligation by IFP-11 in vitro in the presence and absence of an anti-IL-10Rα antibody that blocks hIL-10 binding to hIL-10Rα. [Figure 4A] 4A is a radar chart showing IFN-γ, IL-1β, IL-8, IL-6, TNFα, and IL-4 from LPS-stimulated human peripheral blood mononuclear cells (hPBMCs) treated with a reference Fc-hIL-10 fusion protein or an Fc-GFP negative control (referred to as "Fc" in FIG. 4A). The scale indicates the percent of cytokines present relative to the levels in the Fc-GFP control group. [Figure 4B] 4B is a radar chart showing the expression of IL-1β, IL-8, IL-6, and TNFα from LPS-stimulated human peripheral blood mononuclear cells (hPBMCs) treated with IFP-2 or an Fc-GFP negative control (referred to as "Fc" in FIG. 4B). The scale indicates the percent of cytokines present relative to the levels in the Fc-GFP control group. [Figure 4C] 4C is a radar chart showing the expression of IFN-γ, IL-1β, IL-8, IL-6, TNFα, and IL-4 from LPS-stimulated human peripheral blood mononuclear cells (hPBMCs) treated with IFP-5 or an Fc-GFP negative control (referred to as "Fc" in FIG. 4C). The scale indicates the percent of cytokines present relative to the levels in the Fc-GFP control group. [Figure 4D] 4D is a radar chart showing the expression of IL-1β, IL-8, IL-6, and TNFα from LPS-stimulated human peripheral blood mononuclear cells (hPBMCs) treated with IFP-6 or an Fc-GFP negative control (referred to as "Fc" in FIG. 4D). The scale indicates the percent of cytokines present relative to the levels in the Fc-GFP control group. [Figure 4E] 4E is a radar chart showing the expression of IFN-γ, IL-1β, IL-8, IL-6, TNFα, and IL-4 from LPS-stimulated human peripheral blood mononuclear cells (hPBMCs) treated with IFP-7 or an Fc-GFP negative control (referred to as "Fc" in FIG. 4E). The scale indicates the percent of cytokines present relative to the levels in the Fc-GFP control group. [Figure 4F] 4F is a radar chart showing the expression of IL-1β, IL-8, IL-6, and TNFα from LPS-stimulated human peripheral blood mononuclear cells (hPBMCs) treated with IFP-8 or an Fc-GFP negative control (referred to as "Fc" in FIG. 4F). The scale indicates the percent of cytokines present relative to the levels in the Fc-GFP control group. [Figure 5]1 is a series of line graphs showing the expression of IFNγ, TNFα, and IL-17a from stimulated human peripheral blood mononuclear cells (hPBMCs) treated with IFP-7 (bottom row) or a reference hIL-10 Fc fusion protein (top row) alone or in combination with dexamethasone. [Figure 6] 1 is a PCA plot showing the clustering of IMPs 1-175 (SEQ ID NOS: 108-454) by sequence and structural similarity, visualized by principal component analysis (PCA). "Sampled" refers to the 97 IMPs (IMPs 1-12, 21, 26-27, 31-32, 36, 38, 41, 49-50, 53, 55, 58, 62, 67-70, 72-73, 75-82, 84, 86-87, 89, 91, 93, 95, 97-100, 102-103, 106-109, 111-112, 115, 117-118, 120-127, 129-130, 132, 134-136, and 138-158) generated and analyzed in, for example, Examples 1 and 3 herein. "No sampling" indicates the other 78 IMPs. The sequences are clustered into clusters 0 to 5, as shown in the plot. The IMPs in each cluster are described in Example 2. [Figure 7] This is a bar graph showing in vivo engraftment of human CD45+ immune cells (hCD45) in a xenograft mouse model. Mice were treated with dexamethasone (dexa) and IFP-7, dexamethasone (dexa) and a reference hIL-10 Fc fusion protein, dexamethasone (dexa) and a human Fc protein, or an untreated control. Data are presented as the ratio of hCD45+ immune cells to mouse CD45+ immune cells (mCD45). * indicates a p-value of approximately 0.02. * indicates a p-value of approximately 0.001-0.005. [Figure 8A]1 is a bar graph showing the percentage of human naive (CD27+ and CD45Ra+) CD4+ FOXP3- T cells in vivo in a xenograft mouse model. Mice were treated with dexamethasone (dexa) and IFP-7, dexamethasone (dexa) and a reference hIL-10 Fc fusion protein, dexamethasone (dexa) and a human Fc protein, or an untreated control. * indicates a P value of approximately 0.05. * indicates a P value of less than 0.0001. [Figure 8B] 1 is a bar graph showing the percentage of human effector (CD27- and CD45Ra-) CD4+FOXP3- T cells in vivo in a xenograft mouse model of GVHD. Mice were treated with dexamethasone (dexa) and IFP-7, dexamethasone and a reference hIL-10 Fc fusion protein, dexamethasone and human Fc, or an untreated control. "**" indicates a P value of approximately 0.001. "***" indicates a P value of approximately 0.0009. "****" indicates a P value less than 0.0001. [Figure 9] 1 is a bar graph showing in vivo IFNγ expression in a xenograft mouse model. Mice were treated with dexamethasone (dexa) and IFP-7, dexamethasone and a reference hIL-10 Fc fusion protein, dexamethasone and human Fc, or an untreated control. * indicates a P value of approximately 0.05. [Figure 10] 1 is a bar graph showing relative body weights of mice in a xenograft mouse model of graft-versus-host disease (GVHD). Mice were treated with dexamethasone (dexa) and IFP-7, dexamethasone and a reference hIL-10 Fc fusion protein, an untreated control, or control mice received no hPBMCs. [Figure 11] 1 is a bar graph showing mouse survival in a xenograft mouse model of GVHD. Mice were treated with dexamethasone (dexa) and IFP-7, dexamethasone and a reference hIL-10 Fc fusion protein, an untreated control, or control mice received no hPBMCs. * indicates a P value of approximately 0.02. DETAILED DESCRIPTION OF THE INVENTION

[0118] 5. Detailed Description hIL-10 has been evaluated as a therapeutic agent for the treatment of various autoimmune diseases, but has shown limited efficacy in all cases. The biologically active form of hIL-10 is an unstable homodimer, exhibiting a short half-life in vivo, facile degradation, and low potency. The present inventors have identified immunomodulatory proteins and polypeptides that specifically bind to hIL-10R (e.g., hIL-10Rα) and, in particular, exhibit one or more improved properties over hIL-10, such as increased binding affinity to hIL-10R (e.g., hIL-10Rα), increased potency, and enhanced suppression of one or more proinflammatory cytokines. Thus, the novel immunomodulatory proteins disclosed herein can be used to regulate immune responses and treat diseases (e.g., IL-10-responsive diseases). Thus, the present disclosure provides novel immunomodulatory proteins, particularly for use in pharmaceutical compositions for the treatment of diseases (e.g., IL-10-responsive diseases).

[0119] 5.1 Definition The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0120] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary 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 claimed subject matter.

[0121] The 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, the use of the terms "including" and other forms such as "include," "includes," and "included" is not limiting.

[0122] Whenever an embodiment is described herein with the term "comprising," it is understood that other similar embodiments described with the terms "consisting of" and "consisting essentially of" are also provided.

[0123] The term "and / or," as used herein, should be considered a specific disclosure of each of the two specified features or components, with or without the other. Thus, the term "and / or," when used in phrases such as "A and / or B," is intended herein to include "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, the term "and / or," when used in phrases such as "A, B, and / or C," is intended to encompass each of the following embodiments: 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).

[0124] As used herein, any concentration range, percentage range, ratio range, or integer range, unless otherwise indicated, should be understood to include any integer value within the recited range and, where appropriate, fractions thereof (tenths and hundredths of integers).

[0125] The term "about" refers to a value or composition that is within an acceptable error range for a 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 a particular value or composition is provided herein, unless otherwise specified, the meaning of "about" should be assumed to be within an acceptable error range for that particular value or composition.

[0126] Where proteins and / or polypeptides are described herein, it is understood that the polynucleotides (e.g., RNA (e.g., mRNA) or DNA nucleic acid molecules) that encode the proteins or polypeptides are also provided herein.

[0127] Where proteins, polypeptides, nucleic acid molecules, vectors, carriers, etc. are described herein, it is understood that isolated forms of the proteins, polypeptides, nucleic acid molecules, vectors, carriers, etc. are also provided herein.

[0128] Where a protein, polypeptide, nucleic acid molecule, or the like is described herein, it is understood that recombinant forms of the protein, polypeptide, nucleic acid molecule, or the like are also provided herein.

[0129] Where a polypeptide or set of polypeptides is described herein, it is understood that a protein comprising the polypeptide or set of polypeptides folded into their three-dimensional structure (i.e., tertiary or quaternary structure) is also provided herein, and vice versa.

[0130] As used herein, the term "adjuvant" refers to a substance that, when administered to a subject, causes stimulation of the subject's immune system.

[0131] As used herein, the term "administration" refers to the physical introduction of an agent, e.g., a therapeutic agent (or a precursor of a therapeutic agent that is metabolized or modified in the subject's body to produce the therapeutic agent in vivo) or a vaccine, into a subject using any of a variety of methods and delivery systems known to those of skill in the art. Administration can also be performed, for example, once, multiple times, and / or over one or more extended periods of time.

[0132] As used herein, the term "affinity" refers to the strength of binding of one protein (e.g., a ligand) to another protein (e.g., a receptor). Protein affinity 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 unbound ligand, and [receptor] is the molar concentration of unbound receptor. The affinity constant, Ka, is defined by 1 / Kd. Standard methods for measuring affinity are known to those of skill in the art. Exemplary methods for measuring affinity are described herein, see, e.g., §5.2.2.

[0133] As used herein, the term "antibody" or "antibodies" is used in the broadest sense and encompasses a variety of immunoglobulin (Ig) (e.g., human Ig (hIg), murine Ig (mIg)) structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific (e.g., bispecific, trispecific) antibodies, and antibody fragments (i.e., antigen-binding fragments or variants) so long as they exhibit the desired antigen-binding activity. Thus, the term antibody 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., fibronectin). Examples of antibodies include monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, camelized antibodies, intrabodies, affibodies, 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')2 fragments, 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, as well as conjugates or fusion proteins comprising any of the above. Antibodies may be of an Ig isotype (e.g., IgG, IgE, IgM, IgD, or IgA), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2), or any subclass of Ig (e.g., IgG 2a or IgG 2b)). In certain embodiments, the antibodies described herein are IgG antibodies, or a class (e.g., human IgG1 or IgG4) or subclass thereof. In certain embodiments, the 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 antibody refers to a monoclonal or polyclonal antibody population. The antibodies described herein can be produced by any standard method known in the art, for example, recombinant production in a host cell (see, e.g., §5.5); or synthetic production.

[0134] The terms "CH1" and "CH1 region" are used interchangeably herein to refer to the first constant region of an immunoglobulin heavy chain. An exemplary reference hIgG1 CH1 region amino acid sequence is set forth in SEQ ID NO:531; an exemplary reference hIgG4 CH1 region amino acid sequence is set forth in SEQ ID NO:534.

[0135] The terms "CH2" and "CH2 region" are used interchangeably herein to refer to the second constant region of an immunoglobulin heavy chain. An exemplary reference hIgG1 CH2 region amino acid sequence is set forth in SEQ ID NO: 13; an exemplary reference hIgG4 CH2 region amino acid sequence is set forth in SEQ ID NO: 24.

[0136] The terms "CH3" and "CH3 region" are used interchangeably herein to refer to the third constant region of an immunoglobulin heavy chain. An exemplary reference hIgG1 CH3 region amino acid sequence is set forth in SEQ ID NO: 14; an exemplary reference hIgG4 CH3 region amino acid sequence is set forth in SEQ ID NO: 25.

[0137] As used herein, the term "conjugation" refers to the chemical conjugation of a protein or polypeptide with a moiety (e.g., a small molecule, polypeptide, polynucleotide, carbohydrate, lipid, synthetic polymer (e.g., polymer of polyethylene glycol (PEG)), etc.). The moiety can be directly linked to the protein or polypeptide or can be indirectly linked via a linker, for example, as described herein. Chemical conjugation methods are well known in the art, as are commercially available conjugation reagents and kits, and detailed instructions for their use are readily available from commercial suppliers.

[0138] As used herein, the term "derived" with respect to a polynucleotide refers to a polynucleotide having at least 70% sequence identity to a reference polynucleotide (e.g., a naturally occurring polynucleotide) or a fragment thereof. The term "derived" with respect to a polypeptide or protein refers to a polypeptide or protein comprising an amino acid sequence that has at least 70% sequence identity to the amino acid sequence of a reference polypeptide or protein (e.g., a naturally occurring polypeptide or protein). The term "derived," as used herein, does not refer to any particular process or method for obtaining the polynucleotide, polypeptide, or protein. For example, a polynucleotide, polypeptide, or protein can be recombinantly produced or chemically synthesized.

[0139] As used herein, the term "diagnosing" or "diagnosis" refers to the 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.

[0140] As used herein, the term "disease" refers to any abnormal condition that disrupts physiological function. The term is used broadly to encompass any disorder, disease, abnormality, condition, disorder, state, or syndrome in which physiological function is disrupted, regardless of the nature of the etiology.

[0141] The terms "DNA" and "polydeoxyribonucleotide" are used interchangeably herein to refer to a polymer comprising multiple deoxyribonucleotides polymerized through phosphodiester bonds. A deoxyribonucleotide is a nucleotide in which the sugar is deoxyribose.

[0142] The term "EC50" or "half maximal effective concentration" is a measure of the potency of an agent (e.g., an immunomodulatory protein or polypeptide described herein) and refers to the concentration of an agent (e.g., an immunomodulatory protein or polypeptide described herein) required to induce a response halfway between the baseline and maximal response after a particular period of exposure. Assays for measuring the EC50 of a protein or polypeptide are standard in the art; see also, e.g., §5.2.2. In some embodiments, the EC50 is measured according to the method described in Example 3 herein.

[0143] The term "effector function" when used with respect to an antibody refers to a biological activity attributable to the Fc region of the antibody and therefore varies depending on 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.

[0144] As used herein, the term "Fc region" refers to the C-terminal region of an Ig heavy chain comprising at least one CH2 region operably linked from N-terminus to C-terminus 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 linked 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., §5.3.1.3. Additional examples of proteins with engineered Fc regions can be found in Saunders 2019 (KO Saunders, "Conceptual Approaches to Modulating Antibody Effector Functions and Circulation Half-Life," 2019, Frontiers in Immunology, Vol. 10, Art. 1296, pp. 1-20, the entire contents of which are incorporated herein by reference for all purposes).

[0145] The term "functional variant," as used herein with respect to a polypeptide or protein, refers to a polypeptide or protein that contains at least one, but not more than 15%, not more than 12%, not more than 10%, or not more than 8% amino acid mutations (e.g., substitutions, deletions, additions) compared to the amino acid sequence of a reference polypeptide or protein, wherein the polypeptide or protein retains at least one specific function of the reference polypeptide or protein. Not all functions of the reference polypeptide or protein (e.g., wild-type) need be retained by a functional variant of a protein. In some cases, one or more functions are selectively reduced or eliminated. In some embodiments, the reference polypeptide or protein is a wild-type protein. For example, a functional variant of a hIL-10 polypeptide or protein can refer to a hIL-10 protein that contains one or more amino acid substitutions compared to a reference hIL-10 protein (e.g., wild-type) that retains the ability to specifically bind to hIL-10R.

[0146] The term "functional fragment," as used herein with respect to a polypeptide or protein, refers to a fragment of a reference polypeptide or protein that retains at least one specific function. Not all functions of the reference polypeptide or protein need be retained by a functional fragment of a polypeptide or protein. In some cases, one or more functions are selectively reduced or eliminated. In some embodiments, the reference polypeptide or protein is a wild-type protein. For example, a functional fragment of hIL-10 can refer to a fragment of hIL-10 that retains the ability to specifically bind to IL-10R.

[0147] As used herein, the term "fused" and its grammatical equivalents refer to the operative linkage of at least one first polypeptide to a second polypeptide, where the first and second polypeptides are not operatively linked together in nature. For example, the first and second polypeptides are derived from different proteins. The term fused encompasses both the direct linkage of at least two polypeptides via a peptide bond and the indirect linkage via a linker (e.g., a peptide linker).

[0148] As used herein, the term "fusion protein" and its grammatical equivalents refer to a protein comprising at least one polypeptide operably linked to another polypeptide, wherein the first and second polypeptides are different and are not operably linked together in nature. For example, the first and second polypeptides of a fusion protein are each derived from different proteins. The at least two polypeptides of a fusion protein can be operably linked directly via a peptide bond; or indirectly via a linker (e.g., a peptide linker). Thus, for example, the term "fusion polypeptide" encompasses embodiments in which polypeptide A is operably linked directly to polypeptide B via a peptide bond (polypeptide A-polypeptide B) and embodiments in which polypeptide A is operably linked to polypeptide B via a peptide linker (polypeptide A-peptide linker-polypeptide B).

[0149] As used herein, the term "half-life extending moiety" refers to a moiety (e.g., a small molecule, polypeptide, polynucleotide, carbohydrate, lipid, synthetic polymer (e.g., polymer of PEG), etc.) that, when conjugated or otherwise operably linked (e.g., fused) to a polypeptide or protein (a subject polypeptide or protein), increases the half-life of the subject polypeptide or protein in vivo when administered to a subject (e.g., a human subject). The pharmacokinetic properties of a polypeptide or protein can be assessed utilizing in vivo models known in the art.

[0150] As used herein, the term "half-life extending polypeptide" or "half-life extending protein" refers to a polypeptide or protein that, when operably linked to another polypeptide or protein (a subject polypeptide or protein), increases the half-life of the subject polypeptide or protein in vivo when administered to a subject (e.g., a human subject). The pharmacokinetic properties of a polypeptide or protein can be assessed using in vivo models known in the art.

[0151] As used herein, the term "heterologous," when used to describe a first element in relation to a second element, means that the first element and second element are not naturally occurring in the described arrangement. For example, a polypeptide comprising a "heterologous moiety" refers to a polypeptide that is conjugated to a moiety that is not naturally conjugated to the polypeptide (e.g., a small molecule, polypeptide, polynucleotide, carbohydrate, lipid, synthetic polymer (e.g., a polymer of PEG), etc.). In one embodiment, the heterologous moiety is not derived from a polypeptide or protein comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 108-454. For example, a non-limiting example of a heterologous moiety is a heterologous polypeptide (as defined herein). In one embodiment, a heterologous polypeptide is a polypeptide derived from a polypeptide or protein other than a polypeptide or protein comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 108-454. For example, a non-limiting example of a heterologous polypeptide described herein is a human Ig Fc region.

[0152] As used herein, the term "heterologous signal peptide" refers to a signal peptide that is not operably linked to a native polypeptide or protein of interest. For example, in the context of a polypeptide comprising a signal peptide from human IL-2 operably linked 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.

[0153] The terms "hinge" or "hinge region" are used interchangeably herein and refer to the hinge region of an immunoglobulin heavy chain. An exemplary reference hIgG1 hinge region amino acid sequence is set forth in SEQ ID NO: 12; an exemplary reference hIgG4 hinge region amino acid sequence is set forth in SEQ ID NO: 22.

[0154] As used herein, the term "homologous signal peptide" refers to a signal peptide that is operably linked to a native polypeptide or protein of interest. For example, in the context of a polypeptide comprising a signal peptide from human IL-2 operably linked to human IL-2, the human IL-2 signal peptide would constitute a homologous signal peptide.

[0155] As used herein, the term "human interleukin-10" or "hIL-10" refers to a human immunoregulatory cytokine that mediates signaling through the human IL-10 receptor. An exemplary reference amino acid sequence of a mature hIL-10 protein is set forth in SEQ ID NO:2.

[0156] As used herein, the term "human IL-10 receptor" or "hIL-10R" refers to the human heterodimeric cell-surface complex comprising hIL-10Rα and hIL-10Rβ, through which hIL-10 mediates signal transduction.

[0157] As used herein, the term "human IL-10 receptor alpha" or "hIL-10Rα" refers to the alpha (α) subunit of the hIL-10 receptor. The amino acid sequence of an exemplary reference mature hIL-10Rα polypeptide is set forth in SEQ ID NO:4.

[0158] As used herein, the term "human IL-10 receptor beta" or "hIL-10Rβ" refers to the beta (β) subunit of the hIL-10 receptor. The amino acid sequence of an exemplary reference mature hIL-10Rβ polypeptide is set forth in SEQ ID NO:6.

[0159] As used herein, the term "variant Ig Fc fusion polypeptide or protein" refers to a fusion polypeptide or protein comprising an immunomodulatory polypeptide or protein described herein and an Ig Fc region, wherein the Ig Fc region comprises one or more mutations (e.g., one or more amino acid substitutions, deletions, or additions) that reduce or eliminate one or more Fc effector functions compared to a reference Ig Fc fusion protein that does not comprise the one or more mutations.

[0160] As used herein, the term "IL-10 responsive disease" refers to a disease in which one or more symptoms are reduced or ameliorated by stimulation of the IL-10 pathway.

[0161] As used herein, the term "immunogen" refers to a substance that can induce an immune response (e.g., an adaptive immune response) in humans. An immunogen may have one or more isoforms, sequence variants, or splice variants that have equivalent biological and immunological activity and, therefore, are also considered immunogenic equivalents of the immunogen for purposes of this disclosure.

[0162] As used herein, the term "immunogenic peptide or protein" refers to a peptide or protein that comprises an immunogen.

[0163] As used herein, the term "in combination with" means that two (or more) different agents or therapies are administered to a subject as part of a defined treatment regimen for a particular disease or condition. The treatment regimen defines the dosage and periodicity of administration of each agent so that the effects of the separate agents on the subject overlap. In some embodiments, delivery of two or more agents is simultaneous or concomitant, and the agents may be combined. In other embodiments, two or more agents are not combined but are administered in a sequential manner as part of a formulation. In some embodiments, administration of two or more agents or therapies in combination results in a reduction in symptoms or other parameters associated with the disease that is greater than that which would be observed if one agent or treatment were delivered alone or in the absence of the other. The effects of the two therapies may be partially additive, wholly additive, or greater than additive (e.g., synergistic). The sequential or substantially simultaneous administration of each therapeutic agent may be by any suitable route, including, but not limited to, oral, intravenous, and intramuscular. The therapeutic agents may be administered by the same route or by different routes.

[0164] As used herein, the term "isolated" with respect to a polypeptide, protein, or polynucleotide refers to a polypeptide, protein, or polynucleotide that is substantially free from other cellular components with which it is naturally associated.

[0165] As used herein, the term "moiety" is used generally to describe any macromolecule or small molecule that can be operably linked to a polypeptide or protein described herein. Exemplary moieties include, but are not limited to, small molecules, polypeptides, polynucleotides (e.g., DNA, RNA), carbohydrates, lipids, synthetic polymers (e.g., polymers of PEG).

[0166] As used herein, the use of the term "modified nucleotide," "nucleotide modification," or the term "modification," etc., in reference to a nucleotide or nucleic acid sequence refers to a nucleotide that includes 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., § 5.4.4.2. In certain embodiments of the present disclosure, the inclusion of deoxynucleotides (recognized as naturally occurring forms of nucleotides) is considered to constitute modified nucleotides when present in an RNA molecule.

[0167] As used herein, the term "obtaining a sample" refers to obtaining a sample. This term includes obtaining a sample directly from a subject and indirectly through one or more third parties, one of which obtains the sample directly from the subject.

[0168] As used herein, the term "operably linked" refers to the association of two moieties in a functional relationship. For example, a polypeptide is operably linked to another polypeptide when they are linked in-frame (directly or indirectly via a peptide linker) such that both polypeptides are functional (e.g., a fusion protein or polypeptide described herein). Or, for example, a transcriptional regulatory polynucleotide, e.g., a promoter, enhancer, or other expression control element, is operably linked to a polynucleotide encoding a protein if it affects the transcription of the polynucleotide encoding the protein. The term "operably linked" can also refer to the conjugation of moieties, e.g., a polynucleotide or a polypeptide (e.g., the conjugation of a PEG polymer to a protein or polypeptide).

[0169] The determination of "percent identity" between two sequences (e.g., peptides or proteins (amino acid sequences) or polynucleotides (nucleic acid sequences)) can be accomplished using a mathematical algorithm. A specific, non-limiting example of a mathematical algorithm utilized for comparing 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 incorporated herein 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 are incorporated herein by reference in their entirety. BLAST nucleotide searches can be performed with the NBLAST nucleotide program parameters set, e.g., score=100, wordlength=12, to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. BLAST protein searches can be performed with the XBLAST program parameters set, for example, to score 50 and word length = 3, to obtain amino acid sequences homologous to the protein molecules described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul SF et al., (1997) Nucleic Acids Res 25:3389-3402, which is incorporated herein by reference in its entirety. Alternatively, PSI BLAST can be used to perform an iterated search that detects distant relationships between molecules (Id.). When utilizing BLAST, Gapped BLAST, and PSI Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used (see, for example, the National Center for Biotechnology Information (NCBI) on the World Wide Web at ncbi.nlm.nih.gov).Another specific, non-limiting example of a mathematical algorithm utilized for comparing sequences is the algorithm of Myers and Miller, 1988, CABIOS 4:11-17, which is incorporated herein by reference in its entirety. Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When utilizing the ALIGN program to compare 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.

[0170] As used herein, the term "pharmaceutical composition" means a composition suitable for administration to an animal, e.g., a human subject, comprising a therapeutic agent and a pharmaceutically acceptable carrier or diluent. A "pharmaceutically acceptable carrier or diluent" means a material intended for use in contact with the tissues of humans and / or non-human animals without excessive toxicity, irritation, allergic response, or other problem or complication, consistent with a reasonable therapeutic benefit / risk ratio.

[0171] As used herein, the term "poly(A) sequence" refers to a sequence of adenosine nucleotides, typically up to about 1000 adenosine nucleotides, located at the 3' end of a coding linear RNA. In some embodiments, the poly(A) sequence is essentially homopolymeric, e.g., essentially 100 nucleotides in length, e.g., a poly(A) sequence of 100 adenosine nucleotides. In other embodiments, the poly(A) sequence may be interrupted by at least one nucleotide that is different from adenosine nucleotides, e.g., a poly(A) sequence of 100 adenosine nucleotides may have a length of more than 100 nucleotides (including 100 adenosine nucleotides in addition to the at least one nucleotide (or stretch of nucleotides) that is different from adenosine nucleotides). Although a "poly(A) sequence" as defined herein typically relates to mRNA, in the context of the present invention, it should be understood that the term also relates to the corresponding sequence in a DNA molecule (e.g., a "poly(T) sequence").

[0172] The terms "polynucleotide" and "nucleic acid molecule" are used interchangeably herein and refer to a polymer of DNA or RNA. Nucleic acid molecules may be single- or double-stranded; may contain natural, non-natural, or modified nucleotides; and may contain modified internucleotide linkages, such as phosphoramidate or phosphorothioate linkages, instead of the phosphodiesters found between nucleotides in natural, non-natural, or unmodified nucleic acid molecules. Nucleic acid molecules include, but are not limited to, all nucleic acid molecules obtained by any means available in the art, including, but not limited to, recombinant means, such as cloning nucleic acid molecules from recombinant libraries or cellular genomes using conventional cloning techniques and the polymerase chain reaction, and synthetic means. Those skilled in the art will understand that, unless otherwise noted, the nucleic acid sequences set forth herein will recite thymidine (T) in representative DNA sequences, but when the sequence represents RNA (e.g., mRNA), thymidine (T) will be substituted for uracil. Thus, any of the RNA polynucleotides encoded by a DNA identified by a particular sequence identification number may also include the corresponding RNA (e.g., mRNA) sequence encoded by the DNA, in which each thymidine (T) in the DNA sequence is replaced with uracil (U).

[0173] As used herein, the term "polypeptide" refers to a polymer of at least two (e.g., at least five) amino acids linked by peptide bonds. The term "polypeptide" does not indicate a specific length of the polymeric chain of amino acids. It is common in the art to refer to short polymers of amino acids (e.g., approximately 2 to 50 amino acids) as peptides and to refer to long polymers of amino acids (e.g., more than approximately 50 amino acids) as polypeptides. However, the terms "peptide" and "polypeptide" are used interchangeably herein.

[0174] As used herein, the term "protein" refers to one or more polypeptides folded into their three-dimensional structure. When a polypeptide is contemplated herein, it should be understood that proteins comprising the polypeptide (i.e., the polypeptide folded into its three-dimensional structure) are also provided herein. In some embodiments, the immunomodulatory polypeptide or protein herein is an immunomodulatory protein.

[0175] A "prophylactic" treatment is a treatment administered to a subject who does not show signs of the disease, or who shows only early signs of the disease, to reduce the risk of developing a pathological condition.

[0176] The terms "RNA" and "polyribonucleotide" are used interchangeably herein and refer to a polymer comprising multiple ribonucleotides polymerized via phosphodiester bonds. Ribonucleotides are nucleotides whose sugar is ribose. RNA may contain modified nucleotides; it may contain modified internucleotide bonds, such as phosphoramidate or phosphorothioate bonds, instead of the phosphodiester found between nucleotides in natural, non-natural, or unmodified nucleic acid molecules.

[0177] As used herein, the term "sample" encompasses a variety of biological specimens obtained from a subject. Exemplary sample types include, for example, 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 their progeny), tissue cultures (or cells derived therefrom and their progeny), and cell cultures (or cells derived therefrom and their progeny). The term also includes samples that have been manipulated in any way after their procurement from a subject, for example, by centrifugation, filtration, washing, precipitation, dialysis, chromatography, lysis, treatment with reagents, enrichment for particular cell populations, refrigeration, freezing, staining, etc.

[0178] 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. The translatable RNA can be an mRNA or a circular RNA that encodes a polypeptide.

[0179] As used herein, the term "(scFv)2" refers to an antibody comprising a first and a second scFv operably connected (e.g., via a peptide linker). The first and second scFv may specifically bind to the same or different antigens. In some embodiments, the first and second scFv are operably linked by a peptide linker.

[0180] As used herein, the term "scFv-Fc" refers to an antibody comprising an scFv operably linked (e.g., via a peptide linker) to an Fc domain or a subunit of an Fc domain. In some embodiments, the scFv is operably linked to only the first Fc domain of a first and second Fc domain pair. In some embodiments, the first scFv is operably linked to the first Fc domain and the second scFv is operably linked to the second Fc domain of the first and second Fc domain pair.

[0181] The term "(scFv)2-Fc," as used herein, refers to an (scFv)2 operably linked (e.g., via a peptide linker) to an Fc domain or a subunit of an Fc domain. In some embodiments, the (scFv)2 is operably linked to only the first Fc domain of a first and second Fc domain pair. In some embodiments, the first (scFv)2 is operably linked to the first Fc domain and the second (scFv)2 is operably linked to the second Fc domain of the first and second Fc domain pair.

[0182] As used herein, the term "single domain antibody" or "sdAb" refers to an antibody having a single monomeric variable antibody domain. An sdAb is capable of specifically binding to a specific antigen. A VHH (as defined herein) is an example of an sdAb.

[0183] 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 particular organelle, cellular compartment, or extracellular export. The term encompasses both signal sequence peptides and nucleic acid sequences that encode the signal peptide. Thus, reference to a signal peptide in the context of a nucleic acid refers to the nucleic acid sequence that encodes the signal peptide.

[0184] As used herein, the term "specifically binds" refers to a preferential interaction, i.e., a 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. As used herein, when a first protein or polypeptide is described as "specifically binding" to a second protein or polypeptide, it is understood that the first protein or polypeptide specifically binds to an epitope of the second protein or polypeptide. The term "epitope" refers to the portion of the second protein or polypeptide that the first protein or polypeptide specifically recognizes. The term "specifically binds" includes molecules that cross-react with the same epitope of different species. For example, an antibody that specifically binds to human IL-10 may cross-react with IL-10 from another species (e.g., cynomolgus monkey, mouse, etc.), but is still considered herein to specifically bind to human IL-10. A protein can specifically bind to two or more different proteins.

[0185] As used herein, the term "subject" includes any animal, such as a human or other animal. In some embodiments, the subject is a vertebrate (e.g., a mammal, a bird, a fish, a reptile, or an amphibian). In some embodiments, the subject is a human. In some embodiments, the subject of the method is a non-human mammal. In some embodiments, the subject is a non-human mammal such as a non-human primate (e.g., a monkey, an ape), an ungulate (e.g., a cow, a buffalo, a sheep, a goat, a pig, a camel, a llama, an alpaca, a deer, a horse, a donkey), a carnivore (e.g., a dog, a cat), a rodent (e.g., a rat, a mouse), or a lagomorph (e.g., a rabbit). In some embodiments, the subject is a bird, such as a member of the avian taxonomic order Galliformes (e.g., chickens, turkeys, pheasants, quail), Anseriformes (e.g., ducks, geese), Paleognatha (e.g., ostriches, emus), Columbiformes (e.g., house pigeons, pigeons), or Psittaciformes (e.g., parrots).

[0186] As used herein, the term "therapeutically effective amount" of a therapeutic agent, when used alone or in combination with another therapeutic agent, refers to any amount of a therapeutic agent that ameliorates a condition, e.g., protects a subject from developing a disease (or infection); ameliorates symptoms of a disease or infection, e.g., reduces the severity of disease or infectious symptoms, reduces the frequency or duration of disease or infectious symptoms, or increases the duration of disease or infectious symptom-free periods; prevents or reduces functional impairment or disability resulting from a disease or infection; or promotes the regression of a disease (or infection). The ability of a therapeutic agent to ameliorate a condition can be evaluated using a variety of methods known to those skilled in the art, for example, by assaying the activity of the agent in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or in in vitro assays.

[0187] As used herein, the terms "treat," "treating," "treatment," and the like refer to reducing or ameliorating a disease or infection and / or its associated symptoms or achieving a desired pharmacological and / or physiological effect. It will be understood, although not excluded, that treating a disease or infection does not require that the disease or infection or its associated symptoms be completely eliminated. In some embodiments, the effect is therapeutic, i.e., without limitation, the effect partially or completely reduces, reduces, inhibits, alleviates, relieves, lessens the intensity of, or cures the disease and / or adverse symptoms that may result from the disease or infection. In some embodiments, the effect is prophylactic, i.e., the effect protects against or prevents the occurrence or recurrence of the disease or infection. To this end, the methods of the present disclosure comprise administering a therapeutically effective amount of a composition described herein.

[0188] The terms "VL" and "VL domain" are used interchangeably to refer to the light chain variable region of an antibody.

[0189] The terms "VH" and "VH domain" are used interchangeably to refer to the heavy chain variable region of an antibody.

[0190] The term "VHH" as used herein refers to a type of single domain antibody (sdAb) having a single monomeric heavy chain variable antibody domain (VH). Such antibodies can be found in or produced from camelids (e.g., camels, llamas) and are naturally devoid of light chains or are produced synthetically.

[0191] As used herein, the term "(VHH)2" refers to an antibody comprising a first and a second VHH that are operably connected (e.g., via a peptide linker). The first and second VHHs may specifically bind to the same or different antigens. In some embodiments, the first and second VHHs are operably linked by a peptide linker.

[0192] As used herein, the term "VHH-Fc" refers to an antibody comprising a VHH operably linked (e.g., via a peptide linker) to an Fc domain or a subunit of an Fc domain. In some embodiments, the VHH is operably linked to only the first Fc domain of a first and second Fc domain pair. In some embodiments, the first VHH is operably linked to the first Fc domain and the second VHH is operably linked to the second Fc domain of the first Fc and second Fc pair.

[0193] As used herein, the term "(VHH)2-Fc" refers to a (VHH)2 operably linked (e.g., via a peptide linker) to an Fc domain or a subunit of an Fc domain. In some embodiments, a (VHH)2 is operably linked to only the first Fc domain of a first and second Fc domain pair. In some embodiments, a first (VHH)2 is operably linked to the first Fc domain and a second (VHH)2 is operably linked to the second Fc domain of the first Fc and second Fc pair.

[0194] As used herein, the term "5'-untranslated region" or "5'-UTR" refers to a portion of a nucleic acid molecule located 5' (i.e., "upstream") of a coding sequence that is not translated into protein. Typically, a 5'-UTR begins at the transcription initiation site and ends before the start codon of the coding sequence. A 5'-UTR may contain elements for controlling gene expression, also called regulatory elements. Such regulatory elements may be, for example, ribosome binding sites, miRNA binding sites, etc. A 5'-UTR may be post-transcriptionally modified, for example, by enzymatic or post-transcriptional addition of a 5'-cap structure.

[0195] As used herein, the term "3'-untranslated region" or "3'-UTR" refers to a portion of a nucleic acid molecule located 3' (i.e., "downstream") of a coding sequence that is not translated into protein. The 3'-UTR may be located between the coding sequence and an (optional) terminal poly(A) sequence of a nucleic acid sequence. The 3'-UTR may contain elements for controlling gene expression, also called regulatory elements. Such regulatory elements may be, for example, ribosome binding sites, miRNA binding sites, etc.

[0196] 5.2. Immunomodulatory Proteins IL-10 is the founding member of the IL-10 cytokine family, which includes IL-19, IL-20, IL-22, IL-24, and IL-26. hIL-10 is an important immunoregulatory cytokine that functions, in part, to suppress inflammatory immune responses. hIL-10 potently inhibits the production of proinflammatory cytokines such as IFN-γ, TNFα, IL-1β, and IL-6 in several cell types and prevents dendritic cell maturation, in part by inhibiting the expression of IL-12. hIL-10 also inhibits the expression of MHC and costimulatory molecules important for cell-mediated immunity. While functioning primarily as an anti-inflammatory cytokine, hIL-10 is multifunctional in nature and has been shown to inhibit CD8 + It is also known to mediate pro-inflammatory effects, including stimulation of IFN-γ and granzyme B production by T cells.

[0197] Cellular responses induced by hIL-10 require specific recognition and assembly of the hIL-10 receptor (hIL-10R). The hIL-10R contains two distinct subunits, the hIL-10Rα subunit and the hIL-10Rβ subunit. Although similar in overall structure, the hIL-10Rβ subunit exhibits lower affinity for hIL-10 compared to the α subunit. Due to this difference in the affinity of hIL-10Rα and hIL-10Rβ for hIL-10, each receptor subunit has a distinct function in activating hIL-10 cellular responses. More specifically, the hIL-10Rα generally functions as a hIL-10-binding subunit that controls the cell specificity and targeting of hIL-10 to immune cells that selectively express the hIL-10Rα subunit. On the other hand, the hIL-10Rβ subunit effectively detects hIL-10 bound to IL-10Rα and functions as a sensor that activates signal transduction based on the kinetics of hIL-10 / hIL-10Rα interaction.

[0198] The amino acid sequences of the reference immature hIL-10 polypeptide and mature hIL-10 polypeptide are set forth in SEQ ID NOs: 1 and 2, respectively. The amino acid sequences of the reference immature hIL-10Rα polypeptide and mature hIL-10Rα polypeptide are set forth in SEQ ID NOs: 3 and 4, respectively. The amino acid sequences of the reference immature hIL-10Rβ polypeptide and mature hIL-10Rβ polypeptide are set forth in SEQ ID NOs: 5 and 6, respectively. See Table 1 herein.

[0199] [Table 1]

[0200] [Table 2]

[0201] In one aspect, the present disclosure provides immunomodulatory proteins and polypeptides (and functional fragments and variants thereof) that specifically bind to hIL-10R (e.g., hIL-10Rα). The amino acid sequences of the immunomodulatory proteins and polypeptides provided herein are set forth in Table 2. The amino acid sequences of the immature forms of the immunomodulatory proteins and polypeptides (i.e., containing the native signal peptide) are set forth in SEQ ID NOs: 108-118 or 127-290. The amino acid sequences of the mature forms of the immunomodulatory proteins and polypeptides (i.e., lacking the native signal peptide) are set forth in SEQ ID NOs: 119-126 or 291-454.

[0202] Signal peptides have been computationally predicted using standard methods (see, e.g., Teufel, F., Almagro Armenteros, JJ, Johansen, AR et al. SignalP6.0 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 are incorporated herein by reference for all purposes). Those skilled in the art will know how to experimentally identify and / or validate computationally predicted signal peptides using standard methods known in the art, such as expressing an immunomodulatory protein from a host cell and sequencing the intracellular and extracellular forms 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 are incorporated herein by reference for all purposes).

[0203] [Table 3]

[0204]

Table 4

[0205]

Table 5

[0206]

Table 6

[0207]

Table 7

[0208]

Table 8

[0209]

Table 9

[0210]

Table 10

[0211]

Table 11

[0212]

Table 12

[0213]

Table 13

[0214]

Table 14

[0215]

Table 15

[0216] Table 16

[0217]

Table 17

[0218]

Table 18

[0219] Table 19

[0220] Table 20

[0221] Table 21

[0222] Table 22

[0223] Table 23

[0224] Table 24

[0225] Table 25

[0226] Table 26

[0227] Table 27

[0228] Table 28

[0229] Table 29

[0230]

Table 30

[0231] Table 31

[0232] In some embodiments, the immunomodulatory protein or polypeptide specifically binds to hIL-10Rα. In some embodiments, the immunomodulatory protein or polypeptide specifically binds to hIL-10Rβ. In some embodiments, the immunomodulatory protein or polypeptide specifically binds to both hIL-10Rα and hIL-10Rβ. In some embodiments, the immunomodulatory protein or polypeptide specifically binds to both hIL-10Rα and hIL-10Rβ, but binds to hIL-10Rα with higher affinity than hIL-10Rβ.

[0233] In some embodiments, the immunomodulatory protein or polypeptide is a hIL-10R agonist. In some embodiments, the immunomodulatory protein or polypeptide is a hIL-10Rα agonist. In some embodiments, the immunomodulatory protein or polypeptide is a hIL-10Rβ agonist. In some embodiments, the immunomodulatory protein or polypeptide is a hIL-10Rα agonist and a hIL-10Rβ agonist.

[0234] In some embodiments, the amino acid sequence of an immunomodulatory protein or polypeptide comprises an amino acid sequence that is 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 listed in Table 2. For example, the amino acid sequence of an immunomodulatory protein or polypeptide may comprise an amino acid sequence that is at least 85% identical to the amino acid sequence of a polypeptide listed in Table 2. The amino acid sequence of an immunomodulatory protein or polypeptide may comprise an amino acid sequence that is at least 90% identical to the amino acid sequence of a polypeptide listed in Table 2. The amino acid sequence of an immunomodulatory protein or polypeptide may comprise an amino acid sequence that is at least 95% identical to the amino acid sequence of a polypeptide listed in Table 2. In some embodiments, the amino acid sequence of an immunomodulatory protein or polypeptide may preferably comprise an amino acid sequence that is 100% identical to the amino acid sequence of a polypeptide listed in Table 2. In some embodiments, the amino acid sequence of an immunomodulatory protein or polypeptide consists of an amino acid sequence that is 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 listed in Table 2. For example, the amino acid sequence of an immunomodulatory protein or polypeptide may consist of an amino acid sequence that is at least 85% identical to the amino acid sequence of a polypeptide listed in Table 2. The amino acid sequence of an immunomodulatory protein or polypeptide may consist of an amino acid sequence that is at least 90% identical to the amino acid sequence of a polypeptide listed in Table 2. The amino acid sequence of an immunomodulatory protein or polypeptide may consist of an amino acid sequence that is at least 95% identical to the amino acid sequence of a polypeptide listed in Table 2. In some embodiments, the amino acid sequence of an immunomodulatory protein or polypeptide may preferably consist of an amino acid sequence that is 100% identical to the amino acid sequence of a polypeptide listed in Table 2.

[0235] In some embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of a polypeptide listed in Table 2, and further comprises one 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 immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of a polypeptide listed in Table 2, 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 immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of a polypeptide listed in Table 2, 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 immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of a polypeptide set forth in Table 2, and further comprises or consists of no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid mutations (e.g., substitutions, additions, deletions, etc.).

[0236] In some embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of a polypeptide listed in Table 2, and further comprises one 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 immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of a polypeptide listed in Table 2, 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 immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of a polypeptide listed in Table 2, 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 immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of a polypeptide listed in Table 2, and further comprises or consists of no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions.

[0237] In some embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide comprises an amino acid sequence that is 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 one of SEQ ID NOs: 108-454. For example, the amino acid sequence of the immunomodulatory protein or polypeptide may comprise an amino acid sequence that is at least 85% identical to the amino acid sequence of any one of SEQ ID NOs: 108-454. The amino acid sequence of the immunomodulatory protein or polypeptide may comprise an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NOs: 108-454. The amino acid sequence of the immunomodulatory protein or polypeptide may comprise an amino acid sequence that is at least 95% identical to the amino acid sequence of any one of SEQ ID NOs: 108-454. In some embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide may preferably comprise an amino acid sequence that is 100% identical to the amino acid sequence of any one of SEQ ID NOs: 108-454. In some embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide consists of an amino acid sequence that is 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 one of SEQ ID NOs: 108-454. For example, the amino acid sequence of the immunomodulatory protein or polypeptide may consist of an amino acid sequence that is at least 85% identical to the amino acid sequence of any one of SEQ ID NOs: 108-454. The amino acid sequence of the immunomodulatory protein or polypeptide may consist of an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NOs: 108-454. The amino acid sequence of the immunomodulating protein or polypeptide may consist of an amino acid sequence that is at least 95% identical to the amino acid sequence of any one of SEQ ID NOs: 108 to 454. In some embodiments, the amino acid sequence of the immunomodulating protein or polypeptide may preferably consist of an amino acid sequence that is 100% identical to the amino acid sequence of any one of SEQ ID NOs: 108 to 454.

[0238] In embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 108-454, and further comprises one or more, but less than 15% (less than 12%, less than 10%, less than 8%) amino acid mutations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 108-454, and further comprises or consists of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid mutations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 108-454, and further comprises or consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid mutations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 108-454, and further comprises or consists of no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid mutations (e.g., substitutions, additions, deletions, etc.).

[0239] In embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 108-454, and further comprises one 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 immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 108-454, 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 immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 108-454, 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 immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 108-454, and further comprises or consists of no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions.

[0240] In some embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide comprises an amino acid sequence that is 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 one of SEQ ID NOs: 108-114 or 116-125. For example, the amino acid sequence of the immunomodulatory protein or polypeptide may comprise an amino acid sequence that is at least 85% identical to the amino acid sequence of any one of SEQ ID NOs: 108-114 or 116-125. The amino acid sequence of the immunomodulatory protein or polypeptide may comprise an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NOs: 108-114 or 116-125. The amino acid sequence of the immunomodulatory protein or polypeptide may comprise an amino acid sequence that is at least 95% identical to the amino acid sequence of any one of SEQ ID NOs: 108-114 or 116-125. In some embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide may preferably comprise an amino acid sequence that is 100% identical to the amino acid sequence of any one of SEQ ID NOs: 108-114 or 116-125. In some embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide consists of an amino acid sequence that is 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 one of SEQ ID NOs: 108-114 or 116-125. For example, the amino acid sequence of the immunomodulatory protein or polypeptide may consist of an amino acid sequence that is at least 85% identical to the amino acid sequence of any one of SEQ ID NOs: 108-114 or 116-125. The amino acid sequence of the immunomodulatory protein or polypeptide may consist of an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NOs: 108-114 or 116-125. The amino acid sequence of the immunomodulating protein or polypeptide can consist of an amino acid sequence that is at least 95% identical to the amino acid sequence of any one of SEQ ID NOs: 108-114 or 116-125.In some embodiments, the amino acid sequence of the immunomodulating protein or polypeptide may preferably consist of an amino acid sequence that is 100% identical to the amino acid sequence of any one of SEQ ID NOs: 108-114 or 116-125.

[0241] In embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 108-114 or 116-125, and further comprises one 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 immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 108-114 or 116-125, 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 immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 108-114 or 116-125, and further comprises or consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid mutations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 108-114 or 116-125, and further comprises or consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or fewer amino acid mutations (e.g., substitutions, additions, deletions, etc.).

[0242] In embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 108-114 or 116-125, and further comprises one 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 immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 108-114 or 116-125, 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 immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 108-114 or 116-125, 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 immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 108-114 or 116-125, and further comprises or consists of no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions.

[0243] In some embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide comprises an amino acid sequence that is 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 one of SEQ ID NOs: 108-118 or 127-290. For example, the amino acid sequence of the immunomodulatory protein or polypeptide may comprise an amino acid sequence that is at least 85% identical to the amino acid sequence of any one of SEQ ID NOs: 108-118 or 127-290. The amino acid sequence of the immunomodulatory protein or polypeptide may comprise an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NOs: 108-118 or 127-290. The amino acid sequence of the immunomodulatory protein or polypeptide may comprise an amino acid sequence that is at least 95% identical to the amino acid sequence of any one of SEQ ID NOs: 108-118 or 127-290. In some embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide may preferably comprise an amino acid sequence that is 100% identical to the amino acid sequence of any one of SEQ ID NOs: 108-118 or 127-290. In some embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide consists of an amino acid sequence that is 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 one of SEQ ID NOs: 108-118 or 127-290. For example, the amino acid sequence of the immunomodulatory protein or polypeptide may consist of an amino acid sequence that is at least 85% identical to the amino acid sequence of any one of SEQ ID NOs: 108-118 or 127-290. The amino acid sequence of the immunomodulatory protein or polypeptide may consist of an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NOs: 108-118 or 127-290. The amino acid sequence of the immunomodulating protein or polypeptide can consist of an amino acid sequence that is at least 95% identical to the amino acid sequence of any one of SEQ ID NOs: 108-118 or 127-290.In some embodiments, the amino acid sequence of the immunomodulating protein or polypeptide may preferably consist of an amino acid sequence that is 100% identical to the amino acid sequence of any one of SEQ ID NOs: 108-118 or 127-290.

[0244] In embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 108-118 or 127-290, and further comprises one 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 immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 108-118 or 127-290, 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 immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 108-118 or 127-290, and further comprises or consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid mutations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 108-118 or 127-290, and further comprises or consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or fewer amino acid mutations (e.g., substitutions, additions, deletions, etc.).

[0245] In embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 108-118 or 127-290, and further comprises one 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 immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 108-118 or 127-290, 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 immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 108-118 or 127-290, 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 immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 108-118 or 127-290, and further comprises or consists of no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions.

[0246] In some embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide comprises an amino acid sequence that is 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 one of SEQ ID NOs: 108-114 or 116-118. For example, the amino acid sequence of the immunomodulatory protein or polypeptide may comprise an amino acid sequence that is at least 85% identical to the amino acid sequence of any one of SEQ ID NOs: 108-114 or 116-118. The amino acid sequence of the immunomodulatory protein or polypeptide may comprise an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NOs: 108-114 or 116-118. The amino acid sequence of the immunomodulatory protein or polypeptide may comprise an amino acid sequence that is at least 95% identical to the amino acid sequence of any one of SEQ ID NOs: 108-114 or 116-118. In some embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide may preferably comprise an amino acid sequence that is 100% identical to the amino acid sequence of any one of SEQ ID NOs: 108-114 or 116-118. In some embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide consists of an amino acid sequence that is 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 one of SEQ ID NOs: 108-114 or 116-118. For example, the amino acid sequence of the immunomodulatory protein or polypeptide may consist of an amino acid sequence that is at least 85% identical to the amino acid sequence of any one of SEQ ID NOs: 108-114 or 116-118. The amino acid sequence of the immunomodulatory protein or polypeptide may consist of an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NOs: 108-114 or 116-118. The amino acid sequence of the immunomodulating protein or polypeptide can consist of an amino acid sequence that is at least 95% identical to the amino acid sequence of any one of SEQ ID NOs: 108-114 or 116-118.In some embodiments, the amino acid sequence of the immunomodulating protein or polypeptide may preferably consist of an amino acid sequence that is 100% identical to the amino acid sequence of any one of SEQ ID NOs: 108-114 or 116-118.

[0247] In embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 108-114 or 116-118, and further comprises one 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 immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 108-114 or 116-118, 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 immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 108-114 or 116-118, and further comprises or consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid mutations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 108-114 or 116-118, and further comprises or consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or fewer amino acid mutations (e.g., substitutions, additions, deletions, etc.).

[0248] In embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 108-114 or 116-118, and further comprises one 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 immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 108-114 or 116-118, 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 immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 108-114 or 116-118, 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 immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 108-114 or 116-118, and further comprises or consists of no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions.

[0249] In some embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide comprises an amino acid sequence that is 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 one of SEQ ID NOs: 119-126 or 291-454. For example, the amino acid sequence of the immunomodulatory protein or polypeptide may comprise an amino acid sequence that is at least 85% identical to the amino acid sequence of any one of SEQ ID NOs: 119-126 or 291-454. The amino acid sequence of the immunomodulatory protein or polypeptide may comprise an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NOs: 119-126 or 291-454. The amino acid sequence of the immunomodulatory protein or polypeptide may comprise an amino acid sequence that is at least 95% identical to the amino acid sequence of any one of SEQ ID NOs: 119-126 or 291-454. In some embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide may preferably comprise an amino acid sequence that is 100% identical to the amino acid sequence of any one of SEQ ID NOs: 119-126 or 291-454. In some embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide consists of an amino acid sequence that is 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 one of SEQ ID NOs: 119-126 or 291-454. For example, the amino acid sequence of the immunomodulatory protein or polypeptide may consist of an amino acid sequence that is at least 85% identical to the amino acid sequence of any one of SEQ ID NOs: 119-126 or 291-454. The amino acid sequence of the immunomodulatory protein or polypeptide may consist of an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NOs: 119-126 or 291-454. The amino acid sequence of the immunomodulating protein or polypeptide can consist of an amino acid sequence that is at least 95% identical to the amino acid sequence of any one of SEQ ID NOs: 119-126 or 291-454.In some embodiments, the amino acid sequence of the immunomodulating protein or polypeptide may preferably consist of an amino acid sequence that is 100% identical to the amino acid sequence of any one of SEQ ID NOs: 119-126 or 291-454.

[0250] In embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 119-126 or 291-454, and further comprises one 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 immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 119-126 or 291-454, 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 immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 119-126 or 291-454, and further comprises or consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid mutations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 119-126 or 291-454, and further comprises or consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or fewer amino acid mutations (e.g., substitutions, additions, deletions, etc.).

[0251] In embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 119-126 or 291-454, and further comprises one 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 immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 119-126 or 291-454, 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 immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 119-126 or 291-454, 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 polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 119-126 or 291-454, and further comprises or consists of no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions.

[0252] In some embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide comprises an amino acid sequence that is 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 one of SEQ ID NOs: 119-125. For example, the amino acid sequence of the immunomodulatory protein or polypeptide may comprise an amino acid sequence that is at least 85% identical to the amino acid sequence of any one of SEQ ID NOs: 119-125. The amino acid sequence of the immunomodulatory protein or polypeptide may comprise an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NOs: 119-125. The amino acid sequence of the immunomodulatory protein or polypeptide may comprise an amino acid sequence that is at least 95% identical to the amino acid sequence of any one of SEQ ID NOs: 119-125. In some embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide may preferably comprise an amino acid sequence that is 100% identical to the amino acid sequence of any one of SEQ ID NOs: 119-125. In some embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide consists of an amino acid sequence that is 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 one of SEQ ID NOs: 119-125. For example, the amino acid sequence of the immunomodulatory protein or polypeptide may consist of an amino acid sequence that is at least 85% identical to the amino acid sequence of any one of SEQ ID NOs: 119-125. The amino acid sequence of the immunomodulatory protein or polypeptide may consist of an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NOs: 119-125. The amino acid sequence of the immunomodulating protein or polypeptide may consist of an amino acid sequence that is at least 95% identical to the amino acid sequence of any one of SEQ ID NOs: 119-125. In some embodiments, the amino acid sequence of the immunomodulating protein or polypeptide may preferably consist of an amino acid sequence that is 100% identical to the amino acid sequence of any one of SEQ ID NOs: 119-125.

[0253] In embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 119-125, and further comprises one 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 immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 119-125, 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 immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 119-125, 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 immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 119-125, and further comprises or consists of no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid mutations (e.g., substitutions, additions, deletions, etc.).

[0254] In embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 119-125, and further comprises one 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 immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 119-125, 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 immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 119-125, 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 polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 119-125, and further comprises or consists of no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions.

[0255] In some embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide comprises an amino acid sequence that is 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 one of SEQ ID NOs: 109-111, 113, or 115. For example, the amino acid sequence of the immunomodulatory protein or polypeptide may comprise an amino acid sequence that is at least 85% identical to the amino acid sequence of any one of SEQ ID NOs: 109-111, 113, or 115. The amino acid sequence of the immunomodulatory protein or polypeptide may comprise an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NOs: 109-111, 113, or 115. The amino acid sequence of the immunomodulatory protein or polypeptide may comprise an amino acid sequence that is at least 95% identical to the amino acid sequence of any one of SEQ ID NOs: 109-111, 113, or 115. In some embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide may preferably comprise an amino acid sequence that is 100% identical to the amino acid sequence of any one of SEQ ID NOs: 109-111, 113, or 115. In some embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide consists of an amino acid sequence that is 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 one of SEQ ID NOs: 109-111, 113, or 115. For example, the amino acid sequence of the immunomodulatory protein or polypeptide may consist of an amino acid sequence that is at least 85% identical to the amino acid sequence of any one of SEQ ID NOs: 109-111, 113, or 115. The amino acid sequence of the immunomodulatory protein or polypeptide may consist of an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NOs: 109-111, 113, or 115. The amino acid sequence of the immunomodulatory protein or polypeptide may consist of an amino acid sequence that is at least 95% identical to the amino acid sequence of any one of SEQ ID NOs: 109-111, 113, or 115.In some embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide may preferably consist of an amino acid sequence that is 100% identical to the amino acid sequence of any one of SEQ ID NOs: 109-111, 113, or 115.

[0256] In embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 109-111, 113, or 115, and further comprises one 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 immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 109-111, 113, or 115, 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 immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 109-111, 113, or 115, and further comprises or consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid mutations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 109-111, 113, or 115, and further comprises or consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or fewer amino acid mutations (e.g., substitutions, additions, deletions, etc.).

[0257] In embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 109-111, 113, or 115, and further comprises one 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 immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 109-111, 113, or 115, 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 immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 109-111, 113, or 115, 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 immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 109-111, 113, or 115, and further comprises or consists of no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions.

[0258] In some embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide comprises an amino acid sequence that is 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 one of SEQ ID NOs: 109-111 or 113. For example, the amino acid sequence of the immunomodulatory protein or polypeptide may comprise an amino acid sequence that is at least 85% identical to the amino acid sequence of any one of SEQ ID NOs: 109-111 or 113. The amino acid sequence of the immunomodulatory protein or polypeptide may comprise an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NOs: 109-111 or 113. The amino acid sequence of the immunomodulatory protein or polypeptide may comprise an amino acid sequence that is at least 95% identical to the amino acid sequence of any one of SEQ ID NOs: 109-111 or 113. In some embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide may preferably comprise an amino acid sequence that is 100% identical to the amino acid sequence of any one of SEQ ID NOs: 109-111, or 113. In some embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide consists of an amino acid sequence that is 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 one of SEQ ID NOs: 109-111, or 113. For example, the amino acid sequence of the immunomodulatory protein or polypeptide may consist of an amino acid sequence that is at least 85% identical to the amino acid sequence of any one of SEQ ID NOs: 109-111, or 113. The amino acid sequence of the immunomodulatory protein or polypeptide may consist of an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NOs: 109-111, or 113. The amino acid sequence of the immunomodulating protein or polypeptide can consist of an amino acid sequence that is at least 95% identical to the amino acid sequence of any one of SEQ ID NOs: 109-111, or 113.In some embodiments, the amino acid sequence of the immunomodulating protein or polypeptide may preferably consist of an amino acid sequence that is 100% identical to the amino acid sequence of any one of SEQ ID NOs: 109-111, or 113.

[0259] In embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 109-111, or 113, and further comprises one 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 immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 109-111, or 113, 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 immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 109-111, or 113, 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 immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 109-111, or 113, and further comprises or consists of no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid mutations (e.g., substitutions, additions, deletions, etc.).

[0260] In embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 109-111, or 113, and further comprises one 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 immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 109-111, or 113, 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 immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 109-111, or 113, 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 immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 109-111, or 113, and further comprises or consists of no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions.

[0261] In some embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide comprises an amino acid sequence that is 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 one of SEQ ID NOs: 108, 112, 114, or 116-118. For example, the amino acid sequence of the immunomodulatory protein or polypeptide may comprise an amino acid sequence that is at least 85% identical to the amino acid sequence of any one of SEQ ID NOs: 108, 112, 114, or 116-118. The amino acid sequence of the immunomodulatory protein or polypeptide may comprise an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NOs: 108, 112, 114, or 116-118. The amino acid sequence of the immunomodulatory protein or polypeptide may comprise an amino acid sequence that is at least 95% identical to the amino acid sequence of any one of SEQ ID NOs: 108, 112, 114, or 116-118. In some embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide may preferably comprise an amino acid sequence that is 100% identical to the amino acid sequence of any one of SEQ ID NOs: 108, 112, 114, or 116-118. In some embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide consists of an amino acid sequence that is 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 one of SEQ ID NOs: 108, 112, 114, or 116-118. For example, the amino acid sequence of the immunomodulatory protein or polypeptide may consist of an amino acid sequence that is at least 85% identical to the amino acid sequence of any one of SEQ ID NOs: 108, 112, 114, or 116-118. The amino acid sequence of the immunomodulatory protein or polypeptide may consist of an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NOs: 108, 112, 114, or 116-118. The amino acid sequence of the immunomodulatory protein or polypeptide may consist of an amino acid sequence that is at least 95% identical to the amino acid sequence of any one of SEQ ID NOs: 108, 112, 114, or 116-118.In some embodiments, the amino acid sequence of the immunomodulating protein or polypeptide may preferably consist of an amino acid sequence that is 100% identical to the amino acid sequence of any one of SEQ ID NOs: 108, 112, 114, or 116-118.

[0262] In embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 108, 112, 114, or 116-118, and further comprises one 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 immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 108, 112, 114, or 116-118, 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 immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 108, 112, 114, or 116-118, and further comprises or consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid mutations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 108, 112, 114, or 116-118, and further comprises or consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or fewer amino acid mutations (e.g., substitutions, additions, deletions, etc.).

[0263] In embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 108, 112, 114, or 116-118, and further comprises one 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 immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 108, 112, 114, or 116-118, 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 immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 108, 112, 114, or 116-118, 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 immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 108, 112, 114, or 116-118, and further comprises or consists of no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions.

[0264] In some embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide comprises an amino acid sequence that is 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 one of SEQ ID NOs: 120-122, 124, or 126. For example, the amino acid sequence of the immunomodulatory protein or polypeptide may comprise an amino acid sequence that is at least 85% identical to the amino acid sequence of any one of SEQ ID NOs: 120-122, 124, or 126. The amino acid sequence of the immunomodulatory protein or polypeptide may comprise an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NOs: 120-122, 124, or 126. The amino acid sequence of the immunomodulatory protein or polypeptide may comprise an amino acid sequence that is at least 95% identical to the amino acid sequence of any one of SEQ ID NOs: 120-122, 124, or 126. In some embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide may preferably comprise an amino acid sequence that is 100% identical to the amino acid sequence of any one of SEQ ID NOs: 120-122, 124, or 126. In some embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide consists of an amino acid sequence that is 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 one of SEQ ID NOs: 120-122, 124, or 126. For example, the amino acid sequence of the immunomodulatory protein or polypeptide may consist of an amino acid sequence that is at least 85% identical to the amino acid sequence of any one of SEQ ID NOs: 120-122, 124, or 126. The amino acid sequence of the immunomodulating protein or polypeptide may consist of an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NOs: 120-122, 124, or 126. The amino acid sequence of the immunomodulating protein or polypeptide may consist of an amino acid sequence that is at least 95% identical to the amino acid sequence of any one of SEQ ID NOs: 120-122, 124, or 126.In some embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide may preferably consist of an amino acid sequence that is 100% identical to the amino acid sequence of any one of SEQ ID NOs: 120-122, 124, or 126.

[0265] In embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 120-122, 124, or 126, and further comprises one 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 immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 120-122, 124, or 126, 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 immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 120-122, 124, or 126, and further comprises or consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid mutations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 120-122, 124, or 126, and further comprises or consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or fewer amino acid mutations (e.g., substitutions, additions, deletions, etc.).

[0266] In embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 120-122, 124, or 126, and further comprises one 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 immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 120-122, 124, or 126, 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 immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 120-122, 124, or 126, 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 immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 120-122, 124, or 126, and further comprises or consists of no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions.

[0267] In some embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide comprises an amino acid sequence that is 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 one of SEQ ID NOs: 120-122 or 124. For example, the amino acid sequence of the immunomodulatory protein or polypeptide may comprise an amino acid sequence that is at least 85% identical to the amino acid sequence of any one of SEQ ID NOs: 120-122 or 124. The amino acid sequence of the immunomodulatory protein or polypeptide may comprise an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NOs: 120-122 or 124. The amino acid sequence of the immunomodulatory protein or polypeptide may comprise an amino acid sequence that is at least 95% identical to the amino acid sequence of any one of SEQ ID NOs: 120-122 or 124. In some embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide may comprise an amino acid sequence that is 100% identical to the amino acid sequence of any one of SEQ ID NOs: 120-122, or 124. In some embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide consists of an amino acid sequence that is 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 one of SEQ ID NOs: 120-122, or 124. For example, the amino acid sequence of the immunomodulatory protein or polypeptide may consist of an amino acid sequence that is at least 85% identical to the amino acid sequence of any one of SEQ ID NOs: 120-122, or 124. The amino acid sequence of the immunomodulatory protein or polypeptide may consist of an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NOs: 120-122, or 124. The amino acid sequence of the immunomodulating protein or polypeptide can consist of an amino acid sequence that is at least 95% identical to the amino acid sequence of any one of SEQ ID NOs: 120-122, or 124.In some embodiments, the amino acid sequence of the immunomodulating protein or polypeptide may preferably consist of an amino acid sequence that is 100% identical to the amino acid sequence of any one of SEQ ID NOs: 120-122, or 124.

[0268] In embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 120-122 or 124, and further comprises one 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 immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 120-122 or 124, 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 immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 120-122 or 124, 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 immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 120-122, or 124, and further comprises or consists of no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid mutations (e.g., substitutions, additions, deletions, etc.).

[0269] In embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 120-122, or 124, and further comprises one 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 immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 120-122, or 124, 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 immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 120-122, or 124, 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 immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 120-122, or 124, and further comprises or consists of no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions.

[0270] In some embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide comprises an amino acid sequence that is 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 one of SEQ ID NOs: 119, 123, or 125. For example, the amino acid sequence of the immunomodulatory protein or polypeptide may comprise an amino acid sequence that is at least 85% identical to the amino acid sequence of any one of SEQ ID NOs: 119, 123, or 125. The amino acid sequence of the immunomodulatory protein or polypeptide may comprise an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NOs: 119, 123, or 125. The amino acid sequence of the immunomodulatory protein or polypeptide may comprise an amino acid sequence that is at least 95% identical to the amino acid sequence of any one of SEQ ID NOs: 119, 123, or 125. In some embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide may preferably comprise an amino acid sequence that is 100% identical to the amino acid sequence of any one of SEQ ID NOs: 119, 123, or 125. In some embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide consists of an amino acid sequence that is 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 one of SEQ ID NOs: 119, 123, or 125. For example, the amino acid sequence of the immunomodulatory protein or polypeptide may consist of an amino acid sequence that is at least 85% identical to the amino acid sequence of any one of SEQ ID NOs: 119, 123, or 125. The amino acid sequence of the immunomodulatory protein or polypeptide may consist of an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NOs: 119, 123, or 125. The amino acid sequence of the immunomodulatory protein or polypeptide can consist of an amino acid sequence that is at least 95% identical to the amino acid sequence of any one of SEQ ID NOs: 119, 123, or 125.In some embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide may preferably consist of an amino acid sequence that is 100% identical to the amino acid sequence of any one of SEQ ID NOs: 119, 123, or 125.

[0271] In embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 119, 123, or 125, and further comprises one 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 immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 119, 123, or 125, 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 immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 119, 123, or 125, 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 immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 119, 123, or 125, and further comprises or consists of no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid mutations (e.g., substitutions, additions, deletions, etc.).

[0272] In embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 119, 123, or 125, and further comprises one 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 immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 119, 123, or 125, 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 immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 119, 123, or 125, 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 immunomodulatory protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 119, 123, or 125, and further comprises or consists of no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions.

[0273] In some embodiments of the immunomodulatory proteins or polypeptides referred to herein, SEQ ID NOs: 108, 111-119, or 122-126 may be preferred. Advantageously, the immunomodulatory proteins or polypeptides may bind to hIL-10R with greater potency than hIL-10. Thus, in some embodiments, an immunomodulatory protein or polypeptide may comprise an amino acid sequence that is 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 one of SEQ ID NOs: 108, 111-119, or 122-126. For example, in some embodiments, the amino acid sequence of an immunomodulatory protein or polypeptide may comprise an amino acid sequence that is at least 85% identical to the amino acid sequence of any one of SEQ ID NOs: 108, 111-119, or 122-126. In some embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide may comprise an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NOs: 108, 111-119, or 122-126. In some embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide may comprise an amino acid sequence that is at least 95% identical to the amino acid sequence of any one of SEQ ID NOs: 108, 111-119, or 122-126. In some embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide may preferably comprise an amino acid sequence that is 100% identical to the amino acid sequence of any one of SEQ ID NOs: 108, 111-119, or 122-126. In some embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide consists of an amino acid sequence that is 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 one of SEQ ID NOs: 108, 111-119, or 122-126. For example, in some embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide can consist of an amino acid sequence that is at least 85% identical to the amino acid sequence of any one of SEQ ID NOs: 108, 111-119, or 122-126.In some embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide may consist of an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NOs: 108, 111-119, or 122-126. In some embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide may consist of an amino acid sequence that is at least 95% identical to the amino acid sequence of any one of SEQ ID NOs: 108, 111-119, or 122-126. In some embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide may preferably consist of an amino acid sequence that is 100% identical to the amino acid sequence of any one of SEQ ID NOs: 108, 111-119, or 122-126.

[0274] In some embodiments, the immunomodulatory proteins or polypeptides referred to herein, SEQ ID NOS: 112-115 or 123-126, may be more preferred. Advantageously, the immunomodulatory proteins or polypeptides may be particularly effective in suppressing the production of one or more of inflammatory cytokines, e.g., IFN-γ, IL-1β, IL-6, IL-8, TNF-α, and IL-4. For example, the immunomodulatory proteins or polypeptides may suppress the production of inflammatory cytokines more effectively than hIL-10. This is preferably in addition to binding hIL-10R with greater potency than hIL-10. Thus, in some embodiments, an immunomodulatory protein or polypeptide may comprise an amino acid sequence that is 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 one of SEQ ID NOs: 112-115 or 123-126. For example, in some embodiments, an immunomodulatory protein or polypeptide may comprise an amino acid sequence that is at least 85% identical to the amino acid sequence of any one of SEQ ID NOs: 112-115 or 123-126. In some embodiments, an immunomodulatory protein or polypeptide may comprise an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NOs: 112-115 or 123-126. In some embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide may comprise an amino acid sequence that is at least 95% identical to the amino acid sequence of any one of SEQ ID NOs: 112-115 or 123-126. In some embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide may comprise an amino acid sequence that is 100% identical to the amino acid sequence of any one of SEQ ID NOs: 112-115 or 123-126. In some embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide consists of an amino acid sequence that is 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 one of SEQ ID NOs: 112-115 or 123-126.For example, in some embodiments, the amino acid sequence of an immunomodulatory protein or polypeptide may consist of an amino acid sequence that is at least 85% identical to the amino acid sequence of any one of SEQ ID NOs: 112-115 or 123-126. In some embodiments, the amino acid sequence of an immunomodulatory protein or polypeptide may consist of an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NOs: 112-115 or 123-126. In some embodiments, the amino acid sequence of an immunomodulatory protein or polypeptide may consist of an amino acid sequence that is at least 95% identical to the amino acid sequence of any one of SEQ ID NOs: 112-115 or 123-126. In some embodiments, the amino acid sequence of an immunomodulatory protein or polypeptide may consist of an amino acid sequence that is 100% identical to the amino acid sequence of any one of SEQ ID NOs: 112-115 or 123-126.

[0275] In some embodiments of the immunomodulatory proteins or polypeptides referred to herein, SEQ ID NO: 114 or 125 may be preferred. Advantageously, the immunomodulatory protein or polypeptide may bind to hIL-10R with greater potency than hIL-10. Thus, in some embodiments, the immunomodulatory protein or polypeptide may comprise an amino acid sequence that is 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 one of SEQ ID NOs: 114 or 125. For example, in some embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide may comprise an amino acid sequence that is at least 85% identical to the amino acid sequence of any one of SEQ ID NOs: 114 or 125. In some embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide may comprise an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NOs: 114 or 125. In some embodiments, the amino acid sequence of an immunomodulatory protein or polypeptide may comprise an amino acid sequence that is at least 95% identical to the amino acid sequence of any one of SEQ ID NOs: 114 or 125. In some embodiments, the amino acid sequence of an immunomodulatory protein or polypeptide may preferably comprise an amino acid sequence that is 100% identical to the amino acid sequence of any one of SEQ ID NOs: 114 or 125. In some embodiments, the amino acid sequence of an immunomodulatory protein or polypeptide consists of an amino acid sequence that is 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 one of SEQ ID NOs: 114 or 125. For example, in some embodiments, the amino acid sequence of an immunomodulatory protein or polypeptide may consist of an amino acid sequence that is at least 85% identical to the amino acid sequence of any one of SEQ ID NOs: 114 or 125. In some embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide may consist of an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NOs:114 or 125.In some embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide may consist of an amino acid sequence that is at least 95% identical to the amino acid sequence of any one of SEQ ID NOs: 114 or 125. In some embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide may preferably consist of an amino acid sequence that is 100% identical to the amino acid sequence of any one of SEQ ID NOs: 114 or 125.

[0276] In some embodiments of the immunomodulatory proteins or polypeptides referred to herein, SEQ ID NO: 114 may be preferred. Advantageously, the immunomodulatory protein or polypeptide may bind to hIL-10R with greater potency than hIL-10. Thus, in some embodiments, the immunomodulatory protein or polypeptide may comprise an amino acid sequence that is 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 one of SEQ ID NO: 114. For example, in some embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide may comprise an amino acid sequence that is at least 85% identical to the amino acid sequence of any one of SEQ ID NO: 114. In some embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide may comprise an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NO: 114. In some embodiments, the amino acid sequence of an immunomodulatory protein or polypeptide may comprise an amino acid sequence that is at least 95% identical to the amino acid sequence of any one of SEQ ID NOs: 114. In some embodiments, the amino acid sequence of an immunomodulatory protein or polypeptide may preferably comprise an amino acid sequence that is 100% identical to the amino acid sequence of any one of SEQ ID NOs: 114. In some embodiments, the amino acid sequence of an immunomodulatory protein or polypeptide consists of an amino acid sequence that is 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 one of SEQ ID NOs: 114. For example, in some embodiments, the amino acid sequence of an immunomodulatory protein or polypeptide may consist of an amino acid sequence that is at least 85% identical to the amino acid sequence of any one of SEQ ID NOs: 114. In some embodiments, the amino acid sequence of an immunomodulatory protein or polypeptide may consist of an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NOs: 114.In some embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide may consist of an amino acid sequence that is at least 95% identical to the amino acid sequence of any one of SEQ ID NO: 114. In some embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide may preferably consist of an amino acid sequence that is 100% identical to the amino acid sequence of any one of SEQ ID NO: 114.

[0277] In some embodiments of the immunomodulatory proteins or polypeptides referred to herein, SEQ ID NO: 125 may be preferred. Advantageously, the immunomodulatory protein or polypeptide may bind to hIL-10R with greater potency than hIL-10. Thus, in some embodiments, the immunomodulatory protein or polypeptide may comprise an amino acid sequence that is 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 one of SEQ ID NO: 125. For example, in some embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide may comprise an amino acid sequence that is at least 85% identical to the amino acid sequence of any one of SEQ ID NO: 125. In some embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide may comprise an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NO: 125. In some embodiments, the amino acid sequence of an immunomodulatory protein or polypeptide may comprise an amino acid sequence that is at least 95% identical to the amino acid sequence of any one of SEQ ID NOs: 125. In some embodiments, the amino acid sequence of an immunomodulatory protein or polypeptide may preferably comprise an amino acid sequence that is 100% identical to the amino acid sequence of any one of SEQ ID NOs: 125. In some embodiments, the amino acid sequence of an immunomodulatory protein or polypeptide consists of an amino acid sequence that is 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 one of SEQ ID NOs: 125. For example, in some embodiments, the amino acid sequence of an immunomodulatory protein or polypeptide may consist of an amino acid sequence that is at least 85% identical to the amino acid sequence of any one of SEQ ID NOs: 125. In some embodiments, the amino acid sequence of an immunomodulatory protein or polypeptide may consist of an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NOs: 125.In some embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide may consist of an amino acid sequence that is at least 95% identical to the amino acid sequence of any one of SEQ ID NO: 125. In some embodiments, the amino acid sequence of the immunomodulatory protein or polypeptide may preferably consist of an amino acid sequence that is 100% identical to the amino acid sequence of any one of SEQ ID NO: 125.

[0278] 5.2.1 Signal peptide In some embodiments, an immunomodulatory protein or polypeptide comprises a homologous or heterologous signal peptide operably linked to the N-terminus of the immunomodulatory protein or polypeptide. In some embodiments, an immunomodulatory protein or polypeptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 108-454 and comprises a homologous signal peptide operably linked to the N-terminus of the polypeptide. In some embodiments, an immunomodulatory protein or polypeptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 108-454 and comprises a heterologous signal peptide operably linked to the N-terminus of the polypeptide. In some embodiments, an immunomodulatory protein or polypeptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 119-126 or 291-454 and comprises a homologous signal peptide operably linked to the N-terminus of the polypeptide. In some embodiments, an immunomodulatory protein or polypeptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 119-126 or 291-454 and comprises a heterologous signal peptide operably linked to the N-terminus of the polypeptide. In some embodiments, an immunomodulatory protein or polypeptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 108-118 or 127-290, and comprises a homologous signal peptide operably linked to the N-terminus of the polypeptide. In some embodiments, an immunomodulatory protein or polypeptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 108-118 or 127-290, and comprises a heterologous signal peptide operably linked to the N-terminus of the polypeptide.

[0279] In some embodiments, an immunomodulatory protein or polypeptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 114 or 125 and comprises a homologous signal peptide operably linked to the N-terminus of the polypeptide. In some embodiments, an immunomodulatory protein or polypeptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 114 or 125 and comprises a heterologous signal peptide operably linked to the N-terminus of the polypeptide.

[0280] Commonly used signal peptides are known in the art, such as the native signal peptides of human interleukin 2 (hIL-2), human oncostatin M (hOSM), human chymotrypsinogen (hCTRB1), human trypsinogen 2 (hTRY2), and human insulin (hINS). Those skilled in the art can determine an appropriate signal peptide using standard methods known in the art. The amino acid sequences of exemplary signal peptides are shown in Table 3.

[0281] [Table 32]

[0282] In some embodiments, the amino acid sequence of the signal peptide comprises or consists of the amino acid sequence of any one of the signal peptides listed in Table 3. In some embodiments, the amino acid sequence of the signal peptide comprises or consists of the amino acid sequence of any one of the signal peptides listed in Table 3, and further comprises one or more but less than 15% (less than 12%, less than 10%, less than 8%) amino acid mutations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the signal peptide comprises or consists of the amino acid sequence of any one of the signal peptides listed in Table 3, with one, two, or three amino acid mutations (e.g., substitutions, deletions, additions). In some embodiments, the amino acid sequence of the signal peptide comprises or consists of the amino acid sequence of any one of the signal peptides listed in Table 3, and further comprises one 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 or consists of the amino acid sequence of any one of the signal peptides listed in Table 3, with one, two, or three amino acid mutations (e.g., substitutions, deletions, additions).

[0283] In some embodiments, the amino acid sequence of the signal peptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 7-11. In some embodiments, the amino acid sequence of the signal peptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 7-11, and further comprises one or more but less than 15% (less than 12%, less than 10%, less than 8%) amino acid mutations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the signal peptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 7-11, containing one, two, or three amino acid mutations (e.g., substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the signal peptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 7-11, and further comprises one 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 or consists of the amino acid sequence of any one of SEQ ID NOs: 7-11, containing one, two, or three amino acid substitutions.

[0284] 5.2.2 Potency and Affinity of Immunomodulatory Proteins In some embodiments, an immunomodulatory protein or polypeptide (or fusion or conjugate described herein, e.g., an immunomodulatory fusion protein or polypeptide described herein) increases the level of STAT3 in cells expressing hIL-10R on their surface compared to the level of STAT3 in the absence of the immunomodulatory protein or polypeptide (or fusion or conjugate, e.g., an immunomodulatory fusion protein or polypeptide) or compared to the level of STAT3 in the presence of a suitable control (e.g., a reference hIL-10 protein or polypeptide (e.g., SEQ ID NO: 1 or 2) (or a reference fusion or conjugate, e.g., a reference immunomodulatory fusion protein or polypeptide (e.g., any one of SEQ ID NOs: 102-105))). In some embodiments, an immunomodulatory protein or polypeptide (or fusion or conjugate described herein, e.g., an immunomodulatory fusion protein or polypeptide described herein) increases the level of phosphorylated STAT3 in cells expressing hIL-10R on their surface compared to the level of phosphorylated STAT3 in the absence of the immunomodulatory protein or polypeptide (or fusion or conjugate, e.g., an immunomodulatory fusion protein or polypeptide) or in the presence of a suitable control (e.g., a reference hIL-10 protein or polypeptide, e.g., SEQ ID NO: 1 or 2) (or a reference fusion or conjugate, e.g., a reference immunomodulatory fusion protein or polypeptide, e.g., any one of SEQ ID NOs: 102-105)).

[0285] In some embodiments, an immunomodulatory protein or polypeptide (or a fusion or conjugate described herein (e.g., an immunomodulatory fusion protein or polypeptide described herein)) increases the level of phosphorylated STAT3 in cells expressing hIL-10R on their surface with an EC50 of less than about 500 pM, 400 pM, 300 pM, 200 pM, 100 pM, 50 pM, 40 pM, 30 pM, 20 pM, 10 pM, 9 pM, 8 pM, 7 pM, 6 pM, 5 pM, 4 pM, 3 pM, 2 pM, 1 pM, 0.9 pM, 0.8 pM, 0.7 pM, 0.6 pM, 0.5 pM, 0.4 pM, 0.3 pM, 0.2 pM, or 0.1 pM. In some embodiments, an immunomodulatory protein or polypeptide (or a fusion or conjugate described herein (e.g., an immunomodulatory fusion protein or polypeptide described herein)) increases the level of phosphorylated STAT3 in cells expressing hIL-10R on their surface, preferably with an EC50 of less than about 25 pM, e.g., less than about 10 pM. In some embodiments, an immunomodulatory protein or polypeptide (or a fusion or conjugate described herein (e.g., an immunomodulatory fusion protein or polypeptide described herein)) increases the level of phosphorylated STAT3 in cells expressing hIL-10R on their surface with an EC50 of less than about 500 pM to 0.1 pM, 400 pM to 0.1 pM, 300 pM to 0.1 pM, 200 pM to 0.1 pM, 1 ... Increase with an EC50 of 0 pM to 0.1 pM, 50 pM to 0.1 pM, 25 pM to 0.1 pM, 10 pM to 0.1 pM, 5 pM to 0.1 pM, or 1 pM to 0.1 pM, 500 pM to 0.5, 400 pM to 0.5, 300 pM to 0.5, 200 pM to 0.5, 100 pM to 0.5, 50 pM to 0.5, 25 pM to 0.5, 10 pM to 0.5, 5 pM to 0.5, or 1 pM to 0.5 pM.In some embodiments, the immunomodulatory protein or polypeptide (or fusion or conjugate described herein (e.g., an immunomodulatory fusion protein or polypeptide described herein)) increases the level of phosphorylated STAT3 in cells expressing hIL-10R on their surface with an EC50 of about 0.1 pM, 0.2 pM, 0.3 pM, 0.4 pM, 0.5 pM, 0.6 pM, 0.7 pM, 0.8 pM, 0.9 pM, 1.0 pM, 5 pM, 10 pM, 20 pM, 30 pM, 40 pM, 50 pM, 60 pM, 70 pM, 80 pM, 90 pM, 100 pM, 200 pM, 300 pM, 400 pM, or 500 pM or less.

[0286] In some embodiments, an immunomodulatory protein or polypeptide (or a fusion or conjugate described herein, e.g., an immunomodulatory fusion protein or polypeptide described herein) increases the level of phosphorylated STAT3 in a cell expressing hIL-10R on its surface with an EC50 that is at least about 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 110-fold, 120-fold, 130-fold, 140-fold, or 150-fold greater than the EC50 of a reference hIL-10 protein or polypeptide (e.g., a reference hIL-10 protein or polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or 2) (or a reference fusion or conjugate (e.g., a reference immunomodulatory fusion protein or polypeptide, e.g., a reference immunomodulatory fusion protein or polypeptide comprising the amino acid sequence set forth in any one of SEQ ID NOs: 102-105)). In embodiments, an immunomodulatory protein or polypeptide (or fusion or conjugate described herein (e.g., an immunomodulatory fusion protein or polypeptide described herein)) is used to increase the level of phosphorylated STAT3 in cells expressing hIL-10R on their surface relative to a reference hIL-10 protein or polypeptide (e.g., a reference hIL-10 protein or polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or 2) (or a reference fusion or conjugate described herein) The increase is by an EC50 that is about 4-150 fold, 10-150 fold, 20-150 fold, 30-150 fold, 40-150 fold, 50-150 fold, 60-150 fold, 70-150 fold, 80-150 fold, 90-150 fold, 100-150 fold, 110-150 fold, 120-150 fold, 130-150 fold, or 140-150 fold higher than the EC50 of the conjugate (e.g., a reference immunomodulatory fusion protein or polypeptide (e.g., any one of SEQ ID NOS: 102-105)).

[0287] In some embodiments, an immunomodulatory protein or polypeptide (or a fusion or conjugate described herein (e.g., an immunomodulatory fusion protein or polypeptide described herein)) binds to cells expressing hIL-10R on their surface with an EC50 of less than about 500 pM, 400 pM, 300 pM, 200 pM, 100 pM, 50 pM, 40 pM, 30 pM, 20 pM, 10 pM, 9 pM, 8 pM, 7 pM, 6 pM, 5 pM, 4 pM, 3 pM, 2 pM, 1 pM, 0.9 pM, 0.8 pM, 0.7 pM, 0.6 pM, 0.5 pM, 0.4 pM, 0.3 pM, 0.2 pM, or 0.1 pM. In some embodiments, an immunomodulatory protein or polypeptide (or a fusion or conjugate described herein (e.g., an immunomodulatory fusion protein or polypeptide described herein)) preferably binds to cells expressing hIL-10R on their surface with an EC50 of less than about 25 pM, e.g., less than about 10 pM. In some embodiments, an immunomodulatory protein or polypeptide (or a fusion or conjugate described herein (e.g., an immunomodulatory fusion protein or polypeptide described herein)) preferably binds to cells expressing hIL-10R on their surface with an EC50 of less than about 500 pM to 0.1 pM, 400 pM to 0.1 pM, 300 pM to 0.1 pM, 200 pM to 0.1 pM, 100 pM to 0.1 pM, 50 pM to 0.1 pM, 25 pM, and binds to cells expressing hIL-10R on their surface with an EC50 of 0.1 pM, 10 pM to 0.1 pM, 5 pM to 0.1 pM, or 1 pM to 0.1 pM, 500 pM to 0.5, 400 pM to 0.5, 300 pM to 0.5, 200 pM to 0.5, 100 pM to 0.5, 50 pM to 0.5, 25 pM to 0.5, 10 pM to 0.5, 5 pM to 0.5, or 1 pM to 0.5 pM.In some embodiments, an immunomodulatory protein or polypeptide (or a fusion or conjugate described herein (e.g., an immunomodulatory fusion protein or polypeptide described herein)) binds to cells expressing hIL-10R on their surface with an EC50 of about or less: 0.1 pM, 0.2 pM, 0.3 pM, 0.4 pM, 0.5 pM, 0.6 pM, 0.7 pM, 0.8 pM, 0.9 pM, 1.0 pM, 5 pM, 10 pM, 20 pM, 30 pM, 40 pM, 50 pM, 60 pM, 70 pM, 80 pM, 90 pM, 100 pM, 200 pM, 300 pM, 400 pM, or 500 pM.

[0288] In some embodiments, an immunomodulatory protein or polypeptide (or a fusion or conjugate described herein, e.g., an immunomodulatory fusion protein or polypeptide described herein) binds to a cell expressing hIL-10R on its surface with an EC50 that is at least about 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 110-fold, 120-fold, 130-fold, 140-fold, or 150-fold greater than the EC50 of a reference hIL-10 protein or polypeptide (e.g., a reference hIL-10 protein or polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or 2) (or a reference fusion or conjugate (e.g., a reference immunomodulatory fusion protein or polypeptide (e.g., any one of SEQ ID NOs: 102-105))). In some embodiments, an immunomodulatory protein A protein or polypeptide (or a fusion or conjugate described herein (e.g., an immunomodulatory fusion protein or polypeptide described herein)) may be a reference HIL-10 protein or polypeptide (e.g., a hIL-10 protein or polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or 2) (or a reference fusion or conjugate (e.g., a reference immunomodulatory fusion protein or polypeptide (e.g., a reference immunomodulatory fusion protein or polypeptide) and binds to cells expressing hIL-10R on the surface with an EC50 that is about 10-150 times, 20-150 times, 30-150 times, 40-150 times, 50-150 times, 60-150 times, 70-150 times, 80-150 times, 90-150 times, 100-150 times, 110-150 times, 120-150 times, 130-150 times, or 140-150 times higher than the EC50 of the recombinant protein or polypeptide (e.g., any one of SEQ ID NOs: 102-105).

[0289] In some embodiments, an immunomodulatory protein or polypeptide (or a fusion or conjugate described herein (e.g., an immunomodulatory fusion protein or polypeptide described herein)) binds to cells expressing hIL-10Rα on their surface with an EC50 of less than about 500 pM, 400 pM, 300 pM, 200 pM, 100 pM, 50 pM, 40 pM, 30 pM, 20 pM, 10 pM, 9 pM, 8 pM, 7 pM, 6 pM, 5 pM, 4 pM, 3 pM, 2 pM, 1 pM, 0.9 pM, 0.8 pM, 0.7 pM, 0.6 pM, 0.5 pM, 0.4 pM, 0.3 pM, 0.2 pM, or 0.1 pM. In some embodiments, an immunomodulatory protein or polypeptide (or a fusion or conjugate described herein (e.g., an immunomodulatory fusion protein or polypeptide described herein)) preferably binds to cells expressing hIL-10Rα on their surface with an EC50 of less than about 25 pM, e.g., less than about 10 pM. In some embodiments, an immunomodulatory protein or polypeptide (or a fusion or conjugate described herein (e.g., an immunomodulatory fusion protein or polypeptide described herein)) preferably binds to cells expressing hIL-10Rα on their surface with an EC50 of less than about 500 pM to 0.1 pM, 400 pM to 0.1 pM, 300 pM to 0.1 pM, 200 pM to 0.1 pM, 100 pM to 0.1 pM, 50 pM to 0.1 pM, 25 pM, and binds to cells expressing hIL-10Rα on their surface with an EC50 of 0.1 pM, 10 pM to 0.1 pM, 5 pM to 0.1 pM, or 1 pM to 0.1 pM, 500 pM to 0.5, 400 pM to 0.5, 300 pM to 0.5, 200 pM to 0.5, 100 pM to 0.5, 50 pM to 0.5, 25 pM to 0.5, 10 pM to 0.5, 5 pM to 0.5, or 1 pM to 0.5 pM.In some embodiments, an immunomodulatory protein or polypeptide (or a fusion or conjugate described herein (e.g., an immunomodulatory fusion protein or polypeptide described herein)) binds to cells expressing hIL-10Rα on their surface with an EC50 of about 0.1 pM, 0.2 pM, 0.3 pM, 0.4 pM, 0.5 pM, 0.6 pM, 0.7 pM, 0.8 pM, 0.9 pM, 1.0 pM, 5 pM, 10 pM, 20 pM, 30 pM, 40 pM, 50 pM, 60 pM, 70 pM, 80 pM, 90 pM, 100 pM, 200 pM, 300 pM, 400 pM, or 500 pM.

[0290] In some embodiments, an immunomodulatory protein or polypeptide (or a fusion or conjugate described herein, e.g., an immunomodulatory fusion protein or polypeptide described herein) binds to cells expressing hIL-10Rα on their surface with an EC50 that is at least about 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 110-fold, 120-fold, 130-fold, 140-fold, or 150-fold greater than the EC50 of a reference hIL-10 protein or polypeptide (e.g., a reference hIL-10 protein or polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or 2) (or a reference fusion or conjugate (e.g., a reference immunomodulatory fusion protein or polypeptide (e.g., a reference immunomodulatory fusion protein or polypeptide (e.g., any one of SEQ ID NOs: 102-105))). wherein the immunomodulatory protein or polypeptide (or fusion or conjugate described herein, e.g., an immunomodulatory fusion protein or polypeptide described herein) binds to cells expressing hIL-10Rα on their surface with an EC50 that is about 10-150 fold, 20-150 fold, 30-150 fold, 40-150 fold, 50-150 fold, 60-150 fold, 70-150 fold, 80-150 fold, 90-150 fold, 100-150 fold, 110-150 fold, 120-150 fold, 130-150 fold, or 140-150 fold higher than the EC50 of a reference hIL-10 protein or polypeptide (e.g., a reference hIL-10 protein or polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or 2) (or a reference fusion or conjugate described herein, e.g., a reference immunomodulatory fusion protein or polypeptide (e.g., any one of SEQ ID NOs: 102-105)).

[0291] Suitable assays for measuring the EC50 of the immunomodulatory proteins or polypeptides described herein are standard and known to those of skill in the art. For example, the EC50 can be determined by constructing a dose-response curve and testing the effect of different concentrations of the immunomodulatory protein or polypeptide on inducing activity in a particular functional assay (e.g., STAT3 signaling, STAT3 phosphorylation, STAT3-inducible SEAP expression). An exemplary method for determining the EC50 of the immunomodulatory proteins or polypeptides described herein (including the immunomodulatory fusion proteins described herein) is the use of the hIL-10 HEKBlue reporter cell line (InvivoGen #hkb-il10). The hIL-10 HEKBlue reporter cell line expresses the hIL-10Rα and hIL-10Rβ subunits, human STAT3, and a STAT3-inducible SEAP (secreted embryonic alkaline phosphatase) reporter. Protein binding to the hIL-10R thereby triggers JAK1 / STAT3 signaling and subsequent production of SEAP, which can be quantified using standard methods known in the art. Additionally, for example, the level of phosphorylated STAT3 can be assessed by contacting cells expressing hIL-10R with one or more concentrations of an immunomodulatory protein or polypeptide described herein, lysing the cells, and assessing the level of phosphorylated STAT3, for example, by Western blot, FRET-based assay, or chemiluminescence assay (e.g., AlphaLISA-based assay). Cells in cell-based assays can be cells such as HEK293 cells recombinantly expressing hIL-10R and / or human STAT3; or cells that naturally express hIL-10R and human STAT3.

[0292] In some embodiments, an immunomodulatory protein or polypeptide (or a fusion or conjugate described herein, e.g., an immunomodulatory fusion protein or polypeptide described herein) binds to hIL-10 (e.g., hIL-10Rα) with greater affinity than the affinity of a reference hIL-10 protein or polypeptide (e.g., a reference hIL-10 protein or polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or 2) (or a reference fusion or conjugate described herein, e.g., a reference immunomodulatory fusion protein or polypeptide (e.g., any one of SEQ ID NOs: 102-105)). 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., a BIAcore®-based assay), which is 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. Pat. Nos. 5,283,173 and 5,468,614, the entire contents of each of which are incorporated herein by reference for all purposes. SPR measures the change in molecular concentration at the sensor surface as molecules bind to or dissociate from the surface. The change in SPR signal is directly proportional to the change in mass concentration near the surface, thereby allowing for measurement of the binding reaction rate between two molecules (e.g., proteins). The dissociation constant of the complex can be determined by monitoring the change in refractive index over time as a buffer solution passes over the chip.

[0293] Other suitable assays for measuring binding of one protein to another (e.g., binding of a protein or polypeptide described herein to hIL-10R) include, for example, immunoassays such as enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA), or determining binding by monitoring changes in the spectroscopic or optical properties of the protein via fluorescence, UV absorbance, circular dichroism analysis, 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 detecting protein binding.

[0294] 5.3 Immunomodulatory Polypeptide / Protein Fusions and Conjugates In some embodiments, an immunomodulatory protein or polypeptide (e.g., as described herein) is operably linked to a heterologous moiety (e.g., a heterologous polypeptide) to form a fusion or conjugated protein or polypeptide, respectively. Thus, among other things, fusion proteins comprising an immunomodulatory protein or polypeptide (e.g., as described herein) and one or more heterologous proteins (or functional fragments, functional variants, or domains thereof) are further provided herein. Further provided herein are conjugates comprising an immunomodulatory protein or polypeptide (e.g., as described herein) (or a nucleic acid molecule encoding an immunomodulatory protein or polypeptide (e.g., as described herein)) and one or more heterologous moieties.

[0295] 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). In some embodiments, the heterologous moiety is a detectable moiety (e.g., a polypeptide or protein, e.g., a fluorescent polypeptide or protein).

[0296] In some embodiments, the heterologous moiety is a half-life extending moiety. Exemplary half-life extending moieties include, but are not limited to, immunoglobulins (e.g., human Ig (hIg), mouse Ig (mIg), fragments of Ig (e.g., hIg, mIg), Ig (e.g., hIg, mIg) constant regions, fragments of Ig (e.g., hIg, mIg) constant regions, Ig (e.g., hIg, mIg) Fc regions, human transferrin, human serum albumin (HSA), HSA binding proteins or peptides, and polyethylene glycol (PEG) (and polymers thereof). In some embodiments, the heterologous polypeptide is a half-life extending polypeptide. Exemplary half-life extending polypeptides include Ig, fragments of Ig, one or more Ig heavy chain constant regions, fragments of Ig constant regions, Ig Fc regions, hIg, fragments of hIg, one or more hIg heavy chain constant regions, fragments of hIg constant regions, hIg These include, but are not limited to, an Fc region, mIg, a fragment of mIg, one or more mIg heavy chain constant regions, a fragment of the mIg constant region, an mIg Fc region, human transferrin, human serum albumin (HSA), and an HSA-binding protein or peptide. The immunomodulating proteins or polypeptides or half-life extending moieties described herein fused or conjugated to a half-life extending moiety can be evaluated for their pharmacokinetic properties using standard in vivo methods known in the art.

[0297] 5.3.1 Ig Fusion Proteins and Polypeptides 5.3.1.1 Antibody Fusion Proteins and Polypeptides In some embodiments, the heterologous polypeptide comprises an antibody. Antibody fusions can serve to further target the immunomodulatory protein or polypeptide to a particular cell type, for example, expressing a specific cell surface protein. Exemplary antibodies include full-length antibodies, scFvs, Fabs, single domain antibodies (e.g., VHHs), scFv-Fc, Fab-Fc, and single domain antibody-Fc (e.g., VHH-Fc).

[0298] 5.3.1.2 Ig Fusion Proteins and Polypeptides In some embodiments, the heterologous polypeptide 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). In some embodiments, the Ig is an IgG. In some embodiments, the IgG is an IgG1, IgG2, IgG3, or IgG4 (e.g., in some embodiments, preferably IgG4).

[0299] In some embodiments, the heterologous polypeptide comprises or consists of an IgG CH2 region and an IgG CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of a partial IgG hinge region, an IgG CH2 region, and an IgG CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of an IgG hinge region, an IgG CH2 region, and an IgG CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of an IgG1 CH2 region and an IgG1 CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of a partial IgG1 hinge region, an IgG1 CH2 region, and an IgG1 CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of an IgG1 hinge region, an IgG1 CH2 region, and an IgG1 CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of an IgG4 CH2 region and an IgG4 CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of a partial IgG4 hinge region, an IgG4 CH2 region, and an IgG4 CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of an IgG4 hinge region, an IgG4 CH2 region, and an IgG4 CH3 region.

[0300] In some embodiments, the heterologous polypeptide comprises or consists of an Ig Fc region. In some embodiments, the Ig Fc region comprises or consists of at least a portion of a hinge region, a CH2 region, and a CH3 region. In some embodiments, the Ig Fc region comprises or consists of a hinge region, a CH2 region, and a CH3 region. In some embodiments, the Ig Fc region comprises or 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 comprises or consists of an IgG hinge region, an IgG CH2 region, and an IgG CH3 region. In some embodiments, the Ig Fc region comprises or 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 comprises or consists of an IgG1 hinge region, an IgG1 CH2 region, and an IgG1 CH3 region. In some embodiments, the Ig Fc region comprises or consists of at least a portion of an IgG4 hinge region, an IgG4 CH2 region, and an IgG4 CH3 region, hi some embodiments, the Ig Fc region comprises or consists of an IgG4 hinge region, an IgG4 CH2 region, and an IgG4 CH3 region.

[0301] In some embodiments, the heterologous polypeptide comprises one or more hIg heavy chain constant regions (e.g., CH2 region, CH3 region, hinge region, Fc region). In some embodiments, the hIg is 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.

[0302] In some embodiments, the heterologous polypeptide comprises or consists of an hIgG CH2 region and an hIgG CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of a partial hIgG hinge region, an hIgG CH2 region, and an hIgG CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of an hIgG hinge region, an hIgG CH2 region, and an hIgG CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of an hIgG1 CH2 region and an hIgG1 CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of a partial hIgG1 hinge region, an hIgG1 CH2 region, and an hIgG1 CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of an hIgG1 hinge region, an hIgG1 CH2 region, and an hIgG1 CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of an hIgG4 CH2 region and an hIgG4 CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of a partial hIgG4 hinge region, a hIgG4 CH2 region, and a hIgG4 CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of a hIgG4 hinge region, a hIgG4 CH2 region, and a hIgG4 CH3 region.

[0303] In some embodiments, the heterologous polypeptide comprises or consists of a hIg Fc region. In some embodiments, the hIg Fc region comprises or consists of at least a portion of a hinge region, a CH2 region, and a CH3 region. In some embodiments, the hIg Fc region comprises or consists of a hinge region, a CH2 region, and a CH3 region. In some embodiments, the hIg Fc region comprises or 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 comprises or consists of a hIgG hinge region, a hIgG CH2 region, and a hIgG CH3 region. In some embodiments, the hIg Fc region comprises or 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 comprises or consists of a hIgG1 hinge region, a hIgG1 CH2 region, and a hIgG1 CH3 region. In some embodiments, the hIg Fc region comprises or 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 comprises or consists of a hIgG4 hinge region, a hIgG4 CH2 region, and a hIgG4 CH3 region.

[0304] Exemplary reference hIgG1 and hIgG4 heavy chain constant region and hIg light chain constant region amino acid sequences that can be incorporated into one or more of the embodiments (e.g., fusion proteins and polypeptides) described herein are shown in Table 4.

[0305] [Table 33]

[0306] [Table 34]

[0307] [Table 35]

[0308] [Table 36]

[0309] [Table 37]

[0310] In some embodiments, the amino acid sequence of the heterologous polypeptide comprises an amino acid sequence set forth in Table 4. In some embodiments, the amino acid sequence of the heterologous polypeptide consists of an amino acid sequence set forth in Table 4. In some embodiments, the amino acid sequence of the heterologous polypeptide comprises or consists of an amino acid sequence set forth in Table 4, and further comprises one 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 polypeptide comprises or consists of an amino acid sequence set forth in Table 4, with 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 polypeptide comprises or consists of an amino acid sequence set forth in Table 4, with 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 polypeptide comprises or consists of an amino acid sequence set forth in Table 4, comprising or consisting of up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid variations (e.g., amino acid substitutions, deletions, or additions).

[0311] In some embodiments, the amino acid sequence of the heterologous polypeptide comprises or consists of an amino acid sequence set forth in Table 4, further comprising one 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 heterologous polypeptide comprises or consists of an amino acid sequence set forth in Table 4, comprising or consisting of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid substitutions. In some embodiments, the amino acid sequence of the heterologous polypeptide comprises or consists of an amino acid sequence set forth in Table 4, comprising or consisting of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid substitutions. In some embodiments, the amino acid sequence of the heterologous polypeptide comprises or consists of an amino acid sequence set forth in Table 4, comprising or consisting of no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid substitutions.

[0312] In some embodiments, the amino acid sequence of the heterologous polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 16-21 or 27-34. In some embodiments, the amino acid sequence of the heterologous polypeptide consists of the amino acid sequence of any one of SEQ ID NOs: 16-21 or 27-34. In some embodiments, the amino acid sequence of the heterologous polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 16-21 or 27-34, and further comprises one or more but less than 15% (less than 12%, less than 10%, less than 8%) amino acid mutations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the heterologous polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 16-21 or 27-34, comprising or consisting of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid mutations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the heterologous polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 16-21 or 27-34, which comprises or consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid mutations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the heterologous polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 16-21 or 27-34, which comprises or consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or fewer amino acid mutations (e.g., amino acid substitutions, deletions, or additions).

[0313] In some embodiments, the amino acid sequence of the heterologous polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 16-21 or 27-34, and further comprises one 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 heterologous polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 16-21 or 27-34. In some embodiments, the amino acid sequence of the heterologous polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 16-21 or 27-34, comprising or consisting of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid substitutions. In some embodiments, the amino acid sequence of the heterologous polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 16-21 or 27-34, comprising or consisting 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 heterologous polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 16-21 or 27-34, containing or consisting of no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions.

[0314] In some embodiments, the heterologous polypeptide comprises a CH3 region (e.g., an Fc region; including a hinge region, a CH2 region, and a CH3 region, etc.), wherein the CH3 region lacks a C-terminal lysine (e.g., residue 232 of SEQ ID NO:20, when numbered according to SEQ ID NO:20; or residue 229 of SEQ ID NO:31, when numbered according to SEQ ID NO:31, for example). In some embodiments, the CH3 region further lacks a C-terminal glycine (e.g., residue 231 of SEQ ID NO:20, when numbered according to SEQ ID NO:20; or residue 228 of SEQ ID NO:31, when numbered according to SEQ ID NO:31, for example).

[0315] In some embodiments, the heterologous polypeptide comprises one or more mIg heavy chain constant regions (e.g., CH2 region, CH3 region, hinge region, Fc region). In some embodiments, the mIg is mIgG (mIgG). In some aspects, 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.

[0316] In some embodiments, the heterologous polypeptide comprises or consists of an mIgG CH2 region and an mIgG CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of a partial mIgG hinge region, an mIgG CH2 region, and an mIgG CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of an mIgG hinge region, an mIgG CH2 region, and an mIgG CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of an mIgG1 CH2 region and an mIgG1 CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of a partial mIgG1 hinge region, an mIgG1 CH2 region, and an mIgG1 CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of an mIgG1 hinge region, an mIgG1 CH2 region, and an mIgG1 CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of an mIgG2a CH2 region and an mIgG2a CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of a partial mIgG2a hinge region, mIgG2a CH2 region, and mIgG2a CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of a mIgG2a hinge region, mIgG2a CH2 region, and mIgG2a CH3 region.

[0317] In some embodiments, the heterologous polypeptide comprises or consists of an mIg Fc region. In some embodiments, the mIg Fc region comprises or consists of at least a portion of a hinge region, a CH2 region, and a CH3 region. In some embodiments, the mIg Fc region comprises or consists of a hinge region, a CH2 region, and a CH3 region. In some embodiments, the mIG Fc region comprises or consists of at least a portion of an mIgG hinge region, an mIgG CH2 region, and an mIgG CH3 region. In some embodiments, the mIG Fc region comprises or consists of an mIgG hinge region, an mIgG CH2 region, and an mIgG CH3 region. In some embodiments, the mIG Fc region comprises or consists of at least a portion of an mIgG1 hinge region, an mIgG1 CH2 region, and an mIgG1 CH3 region. In some embodiments, the mIg Fc region comprises or consists of an mIgG1 hinge region, an mIgG1 CH2 region, and an mIgG1 CH3 region. In some embodiments, the mIG Fc region comprises or consists of at least a portion of the mIgG2a hinge region, mIgG2a CH2 region, and mIgG2a CH3 region, hi some embodiments, the mIg Fc region comprises or consists of the mIgG2a hinge region, mIgG2a CH2 region, and mIgG2a CH3 region.

[0318] Exemplary reference mIgG1 and mIgG2a heavy chain constant region amino acid sequences that can be incorporated into one or more of the embodiments (e.g., fusion proteins and polypeptides) described herein are provided in Table 10.

[0319] [Table 38]

[0320] [Table 39]

[0321] [Table 40]

[0322] In some embodiments, the amino acid sequence of the heterologous polypeptide comprises an amino acid sequence set forth in Table 10. In some embodiments, the amino acid sequence of the heterologous polypeptide consists of an amino acid sequence set forth in Table 10. In some embodiments, the amino acid sequence of the heterologous polypeptide comprises or consists of an amino acid sequence set forth in Table 10, and further comprises one 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 polypeptide comprises or consists of an amino acid sequence set forth in Table 10, comprising or consisting of 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 polypeptide comprises or consists of an amino acid sequence set forth in Table 10, comprising or consisting of 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 polypeptide comprises or consists of an amino acid sequence set forth in Table 10, comprising or consisting of up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid variations (e.g., amino acid substitutions, deletions, or additions).

[0323] In some embodiments, the amino acid sequence of the heterologous polypeptide comprises or consists of an amino acid sequence set forth in Table 10, further comprising one 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 heterologous polypeptide comprises or consists of an amino acid sequence set forth in Table 10, comprising or consisting of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid substitutions. In some embodiments, the amino acid sequence of the heterologous polypeptide comprises or consists of an amino acid sequence set forth in Table 10, comprising or consisting of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid substitutions. In some embodiments, the amino acid sequence of the heterologous polypeptide comprises or consists of an amino acid sequence set forth in Table 10, comprising or consisting of no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid substitutions.

[0324] In some embodiments, the amino acid sequence of the heterologous polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 459-462 or 467-470. In some embodiments, the amino acid sequence of the heterologous polypeptide consists of the amino acid sequence of any one of SEQ ID NOs: 459-462 or 467-470. In some embodiments, the amino acid sequence of the heterologous polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 459-462 or 467-470, and further comprises one or more but less than 15% (less than 12%, less than 10%, less than 8%) amino acid mutations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the heterologous polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 459-462 or 467-470, comprising or consisting of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid mutations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the heterologous polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 459-462 or 467-470, which comprises or consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid mutations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the heterologous polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 459-462 or 467-470, which comprises or consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or fewer amino acid mutations (e.g., amino acid substitutions, deletions, or additions).

[0325] In some embodiments, the amino acid sequence of the heterologous polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 459-462 or 467-470, and further comprises one 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 heterologous polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 459-462 or 467-470. In some embodiments, the amino acid sequence of the heterologous polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 459-462 or 467-470, comprising or consisting of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid substitutions. In some embodiments, the amino acid sequence of the heterologous polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 459-462 or 467-470, which 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 heterologous polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 459-462 or 467-470, which comprises or consists of no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions.

[0326] In some embodiments, the heterologous polypeptide comprises a CH3 region (e.g., including an Fc region; a hinge region, a CH2 region, and a CH3 region, etc.), wherein the CH3 region lacks a C-terminal lysine (e.g., residue 227 of SEQ ID NO: 461, when numbered according to SEQ ID NO: 461; or residue 223 of SEQ ID NO: 469, when numbered according to SEQ ID NO: 469). In some embodiments, the CH3 region further lacks a C-terminal glycine (e.g., residue 226 of SEQ ID NO: 461, when numbered according to SEQ ID NO: 461; or residue 222 of SEQ ID NO: 469, when numbered according to SEQ ID NO: 469).

[0327] 5.3.1.3 Ig effector functions In some embodiments, the Ig (e.g., hIg, mIg) Fc region of a fusion protein or polypeptide described herein exhibits a reduction in one or more Fc effector functions compared 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 receptors (e.g., Fcγ receptors (e.g., FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and / or FcγRIIIb (e.g., FcγRI, FcγIIa, and / or FcγIIIa))).

[0328] Standard in vitro and / or in vivo assays known in the art can be performed to assess Fc effector function, including any one or more of ADCC, CDC, ADCP, Fc receptor (e.g., Fcγ receptor) binding affinity, and C1q binding affinity.

[0329] For example, ADCC activity can be assessed using standard methods (both radioactive and non-radioactive) known in the art (see, e.g., WO 2006 / 082515, WO 2012 / 130831, the entire contents of each of which are incorporated herein by reference for all purposes). For example, ADCC activity can be assessed using chromium-5 ( 51 Cr) assay. 51Cr is preloaded onto CD20-expressing target cells, NK cells are added to the culture, and radioactivity in the cell culture supernatant is assessed (indicating lysis of the target cells by the NK cells). Similar non-radioactive assays using similar methods are also available, but the target cells are preloaded with a fluorescent dye such as calcein-AM, CFSE, BCECF, or a lanthanide fluorescent dye (europium). See, for example, 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 are incorporated herein by reference for all purposes. Exemplary commercially available non-radioactive assays include, for example, the ACTI™ Non-Radioactive Cytotoxicity Assay for Flow Cytometry (Cell Technology, Inc. Mountain View, Calif.); and the CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega, Madison, Wis.). Further non-limiting examples of in vitro assays that can be used to assess ADCC activity of the fusion proteins described herein are described in U.S. Pat. No. 5,500,362; U.S. Pat. No. 5,821,337; 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. al., J. Exp. Med. 166 (1987) 1351-1361, the entire contents of each of which are incorporated herein by reference.Alternatively, or in addition, the ADCC activity of the fusion proteins described herein can 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 each of which are incorporated herein by reference for all purposes.

[0330] C1q binding assays can be used to assess the ability of hIg fusion proteins or polypeptides described herein to bind to C1q (or bind with lower affinity than a reference fusion protein) and thus lack (or have reduced) CDC activity. Binding of hIg fusion proteins or polypeptides described herein to C1q can be determined by a variety of in vitro assays (e.g., biochemical or immunological assays) known in the art for determining Fc-C1q interactions, including, for example, equilibrium methods (e.g., enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA)), or kinetic methods (e.g., surface plasmon resonance (SPR) analysis), as well as 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 utilize labels in one or more of the components being examined and / or may use a variety of detection methods, including, but not limited to, chromogenic, fluorescent, luminescent, or isotopic labels. A detailed description of binding affinity and binding kinetics can be found, for example, in Paul, WE, ed., Fundamental Immunology, 4 thEd., Lippincott-Raven, Philadelphia (1999), the entire contents of which are incorporated herein by reference. See, for example, the C1q and C3c binding ELISAs described in WO 2006 / 029879 and WO 2005 / 100402, the entire contents of each of which are incorporated herein by reference for all purposes. Additional CDC activity assays include those described, for example, in Gazzano-Santoro, et al., J. Immunol. Methods 202 (1996) 163; Cragg, MS, et al., Blood 101 (2003) 1045-1052; and Cragg, MS, and Glennie, MJ, Blood 103 (2004) 2738-2743, the entire contents of each of which are incorporated herein by reference for all purposes.

[0331] ADCP activity can be measured by in vitro or in vivo methods known in the art and by commercially available assays (see, for example, 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 are incorporated herein by reference for all purposes). For example, a primary cell-based ADCP assay can be used in which fresh human peripheral blood mononuclear cells (PBMCs) are isolated, and monocytes are isolated and differentiated into macrophages in culture using standard procedures. Macrophages are then cultured containing fluorescently labeled target cells expressing CD20 and the fusion proteins described herein. Phagocytic events can be analyzed using FACS screening and / or microscopy. A modified reporter version of the above assay can also be used, using an engineered cell line (e.g., an engineered T cell line, e.g., THP-1) stably expressing FcγRIIa (CD32a) as the effector cell line, thereby eliminating the requirement for primary cells. Exemplary ADCP assays are described, for example, in Ackerman, ME 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. Get 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 are incorporated herein by reference.

[0332] Binding of the hIg fusion proteins or polypeptides described herein to Ig (e.g., hIg, mIg) Fc receptors can be determined by a variety of in vitro assays (e.g., biochemical or immunological 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), as well as 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 utilize labels in one or more of the components being examined and / or may use a variety of detection methods, including, but not limited to, chromogenic, fluorescent, luminescent, or isotopic labels. For a detailed description of binding affinities and kinetics, see, e.g., Paul, W.E., ed., Fundamental Immunology, 4th Ed., Lippincott-Raven, Philadelphia (1999), the entire contents of which are incorporated herein by reference for all purposes.

[0333] In some embodiments, the Ig (e.g., hIg, mIg) Fc region of a fusion protein or polypeptide described herein is mutated (e.g., includes one or more mutations (e.g., one or more amino acid substitutions, deletions, additions, etc.)) (referred to herein as a "variant Ig (e.g., hIg, mIg) Fc fusion polypeptide or protein"). In some embodiments, the one or more mutations (e.g., one or more amino acid substitutions, deletions, additions, etc.)) reduce or eliminate one or more Fc effector functions compared to a reference Ig (e.g., hIg, mIg) Fc that does not include the modification (e.g., one or more mutations (e.g., one or more amino acid substitutions, deletions, additions, etc.)).

[0334] In some embodiments, a variant Ig (e.g., hIg, mIg) Fc fusion protein or polypeptide exhibits no detectable or reduced ADCC compared to a reference fusion protein or polypeptide that does not include an Ig (e.g., hIg, mIg) Fc modification (e.g., one or more mutations (e.g., one or more amino acid substitutions, deletions, or additions)). In some embodiments, a variant Ig (e.g., hIg, mIg) Fc fusion protein or polypeptide exhibits no detectable or reduced CDC compared to a reference fusion protein or polypeptide that does not include an Ig (e.g., hIg, mIg) Fc modification (e.g., one or more mutations (e.g., one or more amino acid substitutions, deletions, or additions)). In some embodiments, the variant Ig (e.g., hIg, mIg) Fc fusion protein or polypeptide exhibits no detectable or reduced ADCP compared to a reference fusion protein or polypeptide that does not include an Ig (e.g., hIg, mIg) Fc modification (e.g., one or more mutations (e.g., one or more amino acid substitutions, deletions, or additions)). In some embodiments, the variant Ig (e.g., hIg, mIg) Fc fusion protein or polypeptide exhibits reduced or no binding affinity to one or more Fc receptors (e.g., human Fc receptors) (e.g., Fcγ receptors (e.g., FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and / or FcγRIIIb (e.g., FcγRI, FcγIIa, and / or FcγIIIa))) compared to a reference fusion protein or polypeptide that does not include an Ig (e.g., hIg, mIg) Fc modification (e.g., one or more mutations (e.g., one or more amino acid substitutions, deletions, or additions)). In some embodiments, the variant Ig (e.g., hIg, mIg) Fc fusion protein or polypeptide exhibits reduced or no binding affinity to FcγRI, FcγIIa, and / or FcγIIIa compared to a reference fusion protein or polypeptide that does not contain a hIg Fc modification (e.g., one or more mutations (e.g., one or more amino acid substitutions, deletions, or additions)).In some embodiments, the variant Ig (e.g., hIg, mIg) Fc fusion protein or polypeptide exhibits reduced or no binding affinity to FcγRI compared to a reference fusion protein or polypeptide that does not include an Ig (e.g., hIg, mIg) Fc modification (e.g., one or more mutations (e.g., one or more amino acid substitutions, deletions, or additions)). In some embodiments, the variant Ig (e.g., hIg, mIg) Fc fusion protein or polypeptide exhibits reduced or no binding affinity to FcγIIa compared to a reference fusion protein or polypeptide that does not include an Ig (e.g., hIg, mIg) Fc modification (e.g., one or more mutations (e.g., one or more amino acid substitutions, deletions, or additions)). In some embodiments, the variant Ig (e.g., hIg, mIg) Fc fusion protein or polypeptide exhibits reduced or no binding affinity for FcγIIIa compared to a reference fusion protein or polypeptide that does not include an Ig (e.g., hIg, mIg) Fc modification (e.g., one or more mutations (e.g., one or more amino acid substitutions, deletions, or additions)). In some embodiments, the variant Ig (e.g., hIg, mIg) Fc fusion protein or polypeptide exhibits reduced or no binding affinity for C1q compared to a reference fusion protein or polypeptide that does not include an Ig (e.g., hIg, mIg) Fc modification (e.g., one or more mutations (e.g., one or more amino acid substitutions, deletions, or additions)).

[0335] Amino acid substitutions that reduce or eliminate one or more Ig (e.g., hIg, mIg) Fc effector functions are known in the art (see, e.g., 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 entire contents of which are incorporated herein by reference for all purposes; see, e.g., Table 3 of Saunders, for more details).

[0336] In some embodiments, a variant Ig Fc fusion protein or polypeptide comprises an hIg Fc region comprising one or more amino acid mutations. In some embodiments, a variant hIg Fc fusion protein or polypeptide comprises an hIg4 Fc region comprising one or more amino acid mutations. In some embodiments, the hIgG4 Fc region comprises amino acid substitutions 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, EU 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 region comprises the following amino acid substitutions, S228P and / or L235E, EU numbering according to Kabat.

[0337] In some embodiments, a variant hIg Fc fusion protein or polypeptide comprises an hIgG1 Fc region comprising one or more amino acid mutations. In some embodiments, the hIgG1 Fc region comprises amino acid substitutions 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.

[0338] The amino acid sequences of exemplary variant hIg Fc regions known in the art to exhibit reduced one or more effector functions are shown in Table 5.

[0339] [Table 41]

[0340] [Table 42]

[0341] [Table 43]

[0342] In some embodiments, a variant hIg Fc fusion protein or polypeptide comprises a hIg 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 listed in Table 5. For example, a variant hIg Fc fusion protein or polypeptide may comprise a hIg Fc region comprising an amino acid sequence at least 85% identical to the amino acid sequence of a polypeptide listed in Table 5. A variant hIg Fc fusion protein or polypeptide may comprise a hIg Fc region comprising an amino acid sequence at least 90% identical to the amino acid sequence of a polypeptide listed in Table 5. A variant hIg Fc fusion protein or polypeptide may comprise a hIg Fc region comprising an amino acid sequence at least 95% identical to the amino acid sequence of a polypeptide listed in Table 5. In some embodiments, a variant hIg Fc fusion protein or polypeptide may preferably comprise an hIg Fc region comprising an amino acid sequence that is 100% identical to the amino acid sequence of a polypeptide set forth in Table 5.

[0343] In some embodiments, the amino acid sequence of a variant hIg Fc fusion protein or polypeptide comprises a hIg Fc region comprising or consisting of the amino acid sequence of a polypeptide listed in Table 5, and further comprising one or more, but less than 15% (less than 12%, less than 10%, less than 8%) amino acid mutations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of a variant hIg Fc fusion protein or polypeptide comprises a hIg Fc region comprising or consisting of the amino acid sequence of a polypeptide listed in Table 5, and further comprising or consisting of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid mutations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of a variant hIg Fc fusion protein or polypeptide comprises a hIg Fc region comprising or consisting of the amino acid sequence of a polypeptide listed in Table 5, and further comprising or consisting of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid mutations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the variant hIg Fc fusion protein or polypeptide comprises an hIg Fc region that comprises or consists of the amino acid sequence of a polypeptide listed in Table 5 and further comprises or consists of no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid mutations (e.g., substitutions, additions, deletions, etc.).

[0344] In some embodiments, the amino acid sequence of the variant hIg Fc fusion protein or polypeptide comprises an hIg Fc region that comprises or consists of the amino acid sequence of a polypeptide listed in Table 5 and further comprises one or more, 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 hIg Fc fusion protein or polypeptide comprises an hIg Fc region that comprises or consists of the amino acid sequence of a polypeptide listed in Table 5 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 hIg Fc fusion protein or polypeptide comprises an hIg Fc region that comprises or consists of the amino acid sequence of a polypeptide listed in Table 5 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 hIg Fc fusion protein or polypeptide comprises a hIg Fc region that comprises or consists of the amino acid sequence of a polypeptide set forth in Table 5 and further comprises or consists of no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions.

[0345] In some embodiments, the amino acid sequence of a variant hIg Fc fusion protein or polypeptide comprises a hIg Fc region comprising 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 of any one of SEQ ID NOs: 35-48. For example, the amino acid sequence of a variant hIg Fc fusion protein or polypeptide may comprise a hIg Fc region comprising an amino acid sequence at least 85% identical to the amino acid sequence of any one of SEQ ID NOs: 35-48. The amino acid sequence of a variant hIg Fc fusion protein or polypeptide may comprise a hIg Fc region comprising an amino acid sequence at least 90% identical to the amino acid sequence of any one of SEQ ID NOs: 35-48. The amino acid sequence of a variant hIg Fc fusion protein or polypeptide may comprise a hIg Fc region comprising an amino acid sequence at least 95% identical to the amino acid sequence of any one of SEQ ID NOs: 35-48. In some embodiments, the amino acid sequence of a variant hIg Fc fusion protein or polypeptide may preferably comprise a hIg Fc region comprising an amino acid sequence that is 100% identical to the amino acid sequence of any one of SEQ ID NOs: 35 to 48. In some embodiments, the amino acid sequence of a variant hIg Fc fusion protein or polypeptide may comprise a hIg Fc region consisting of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 35 to 48. For example, the amino acid sequence of a variant hIg Fc fusion protein or polypeptide may comprise a hIg Fc region consisting of an amino acid sequence that is at least 85% identical to the amino acid sequence of any one of SEQ ID NOs: 35 to 48. The amino acid sequence of the mutant hIg Fc fusion protein or polypeptide may comprise a hIg Fc region consisting of an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NOs: 35 to 48. The amino acid sequence of the mutant hIg Fc fusion protein or polypeptide may comprise a hIg Fc region consisting of an amino acid sequence that is at least 95% identical to the amino acid sequence of any one of SEQ ID NOs: 35 to 48.In some embodiments, the amino acid sequence of the mutant hIg Fc fusion protein or polypeptide may preferably comprise a hIg Fc region consisting of an amino acid sequence that is 100% identical to the amino acid sequence of any one of SEQ ID NOs: 35 to 48.

[0346] In some embodiments, the amino acid sequence of the variant hIg Fc fusion protein or polypeptide comprises an hIg Fc region that comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 35-48 and further comprises one or more, but less than 15% (less than 12%, less than 10%, less than 8%) amino acid mutations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the variant hIg Fc fusion protein or polypeptide comprises an hIg Fc region that comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 35-48 and further comprises or consists of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid mutations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the variant hIg Fc fusion protein or polypeptide comprises an hIg Fc region that comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 35-48 and further comprises or consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid mutations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the variant hIg Fc fusion protein or polypeptide comprises an hIg Fc region that comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 35-48 and further comprises or consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or fewer amino acid mutations (e.g., substitutions, additions, deletions, etc.).

[0347] In some embodiments, the amino acid sequence of the variant hIg Fc fusion protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 35-48 and comprises a hIg Fc region further comprising one 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 hIg Fc fusion protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 35-48 and comprises a hIg Fc region further comprising or consisting 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 hIg Fc fusion protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 35-48 and comprises a hIg Fc region further comprising or consisting 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 hIg Fc fusion protein or polypeptide comprises an hIg Fc region comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 35 to 48, and further comprising or consisting of no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions.

[0348] In some embodiments, a variant mIgG Fc fusion protein or polypeptide comprises an mIgG2a Fc region comprising one or more amino acid mutations. In some embodiments, the mIgG2a Fc region comprises amino acid substitutions 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.

[0349] In some embodiments, a variant mIgG Fc fusion protein or polypeptide comprises an mIgG2a Fc region comprising one or more amino acid mutations. In some embodiments, the mIgG2a Fc region comprises amino acid substitutions 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.

[0350] The amino acid sequences of exemplary variant hIg Fc regions known in the art to exhibit reduced one or more effector functions are shown in Table 11.

[0351] [Table 44]

[0352] [Table 45]

[0353] In some embodiments, a variant mIg Fc fusion protein or polypeptide comprises an 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 listed in Table 11. For example, a variant mIg Fc fusion protein or polypeptide may comprise an mIg Fc region comprising an amino acid sequence at least 85% identical to the amino acid sequence of a polypeptide listed in Table 11. A variant mIg Fc fusion protein or polypeptide may comprise an mIg Fc region comprising an amino acid sequence at least 90% identical to the amino acid sequence of a polypeptide listed in Table 11. A variant mIg Fc fusion protein or polypeptide may comprise an mIg Fc region comprising an amino acid sequence at least 95% identical to the amino acid sequence of a polypeptide listed in Table 11. In some embodiments, a variant mIg Fc fusion protein or polypeptide may preferably comprise an mIg Fc region comprising an amino acid sequence that is 100% identical to the amino acid sequence of a polypeptide set forth in Table 11.

[0354] In some embodiments, the amino acid sequence of the variant mIg Fc fusion protein or polypeptide comprises an mIg Fc region that comprises or consists of the amino acid sequence of a polypeptide set forth in Table 11 and further comprises one or more, but less than 15% (less than 12%, less than 10%, less than 8%) amino acid mutations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the variant mIg Fc fusion protein or polypeptide comprises an mIg Fc region that comprises or consists of the amino acid sequence of a polypeptide set forth in Table 11 and further comprises or consists of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid mutations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the variant mIg Fc fusion protein or polypeptide comprises an mIg Fc region that comprises or consists of the amino acid sequence of a polypeptide set forth in Table 11 and further comprises or consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid mutations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the variant mIg Fc fusion protein or polypeptide comprises an mIg Fc region that comprises or consists of the amino acid sequence of a polypeptide set forth in Table 11 and further comprises or consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or fewer amino acid mutations (e.g., substitutions, additions, deletions, etc.).

[0355] In some embodiments, the amino acid sequence of a variant mIg Fc fusion protein or polypeptide comprises an mIg Fc region that comprises or consists of the amino acid sequence of a polypeptide listed in Table 11 and further comprises one or more, less than 15% (less than 12%, less than 10%, less than 8%) amino acid substitutions. In some embodiments, the amino acid sequence of a variant mIg Fc fusion protein or polypeptide comprises an mIg Fc region that comprises or consists of the amino acid sequence of a polypeptide listed in Table 11 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 a variant mIg Fc fusion protein or polypeptide comprises an mIg Fc region that comprises or consists of the amino acid sequence of a polypeptide listed in Table 11 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 or polypeptide comprises an mIg Fc region that comprises or consists of the amino acid sequence of a polypeptide set forth in Table 11 and further comprises or consists of no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions.

[0356] In some embodiments, the amino acid sequence of a variant mIg Fc fusion protein or polypeptide comprises an mIg Fc region comprising 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 of any one of SEQ ID NOs: 471-478. For example, the amino acid sequence of a variant mIg Fc fusion protein or polypeptide may comprise an mIg Fc region comprising an amino acid sequence at least 85% identical to the amino acid sequence of any one of SEQ ID NOs: 471-478. The amino acid sequence of a variant mIg Fc fusion protein or polypeptide may comprise an mIg Fc region comprising an amino acid sequence at least 90% identical to the amino acid sequence of any one of SEQ ID NOs: 471-478. The amino acid sequence of the variant mIg Fc fusion protein or polypeptide may comprise an mIg Fc region comprising an amino acid sequence at least 95% identical to the amino acid sequence of any one of SEQ ID NOs: 471 to 478. In some embodiments, the amino acid sequence of the variant mIg Fc fusion protein or polypeptide may preferably comprise an mIg Fc region comprising an amino acid sequence 100% identical to the amino acid sequence of any one of SEQ ID NOs: 471 to 478. In some embodiments, the amino acid sequence of the variant mIg Fc fusion protein or polypeptide comprises an mIg Fc region consisting 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 of any one of SEQ ID NOs: 471 to 478. For example, the amino acid sequence of the mutant mIg Fc fusion protein or polypeptide may comprise an mIg Fc region consisting of an amino acid sequence that is at least 85% identical to the amino acid sequence of any one of SEQ ID NOs: 471 to 478. The amino acid sequence of the mutant mIg Fc fusion protein or polypeptide may comprise an hIg Fc region consisting of an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NOs: 471 to 478.The amino acid sequence of the mutant mIg Fc fusion protein or polypeptide may comprise an mIg Fc region consisting of an amino acid sequence that is at least 95% identical to the amino acid sequence of any one of SEQ ID NOs: 471 to 478. In some embodiments, the amino acid sequence of the mutant mIg Fc fusion protein or polypeptide may preferably comprise an mIg Fc region consisting of an amino acid sequence that is 100% identical to the amino acid sequence of any one of SEQ ID NOs: 471 to 478.

[0357] In some embodiments, the amino acid sequence of the variant mIg Fc fusion protein or polypeptide comprises an mIg Fc region that comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 471-478 and further comprises one or more, but less than 15% (less than 12%, less than 10%, less than 8%) amino acid mutations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the variant mIg Fc fusion protein or polypeptide comprises an mIg Fc region that comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 471-478 and further comprises or consists of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid mutations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of a variant mIg Fc fusion protein or polypeptide comprises an mIg Fc region that comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 471-478 and further comprises or consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid mutations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of a variant mIg Fc fusion protein or polypeptide comprises an mIg Fc region that comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 471-478 and further comprises or consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or fewer amino acid mutations (e.g., substitutions, additions, deletions, etc.).

[0358] In some embodiments, the amino acid sequence of the variant mIg Fc fusion protein or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 471-478 and comprises an mIg Fc region further comprising one 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 or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 471-478 and comprises an mIg Fc region further comprising or consisting 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 or polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 471-478 and comprises an mIg Fc region further comprising or consisting 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 or polypeptide comprises an mIg Fc region comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 471-478 and further comprising or consisting of no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions.

[0359] 5.3.2 Linker As described herein, a heterologous moiety (e.g., a heterologous polypeptide) can be directly or indirectly operably linked to an immunomodulatory protein or polypeptide (e.g., as described herein). In some embodiments, a heterologous polypeptide is directly operably linked to an immunomodulatory protein or polypeptide (e.g., as described herein) via a peptide bond. In some embodiments, a heterologous polypeptide is indirectly operably linked to an immunomodulatory protein or polypeptide (e.g., as described herein) via a peptide linker.

[0360] In some embodiments, the peptide linker is a cleavable linker, a non-cleavable linker, a flexible linker, a rigid linker, a helical linker, and / or a non-helical linker.

[0361] In some embodiments, the peptide linker comprises 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, or 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 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 linker comprises or 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 linker comprises or 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.

[0362] In some embodiments, the amino acid sequence of the peptide linker comprises or consists of glycine, serine, or both glycine and serine amino acid residues, hi some embodiments, the amino acid sequence of the peptide linker comprises or consists of glycine, serine, and proline amino acid residues.

[0363] Table 6 provides amino acid sequences of exemplary peptide linkers that can be incorporated into one or more of the embodiments (eg, fusion proteins and polypeptides) described herein.

[0364] [Table 46]

[0365] In some embodiments, the amino acid sequence of the peptide linker comprises the amino acid sequence of any one of the linkers listed in Table 6. In some embodiments, the amino acid sequence of the peptide linker consists of the amino acid sequence of any one of the linkers listed in Table 6. In some embodiments, the amino acid sequence of the peptide linker comprises or consists of the amino acid sequence of any one of the linkers listed in Table 6, and further comprises one or more but less than 15% (less than 12%, less than 10%, less than 8%) amino acid mutations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the peptide linker comprises or consists of the amino acid sequence of any one of the linkers listed in Table 6, with one, two, or three amino acid mutations (e.g., substitutions, deletions, additions). In some embodiments, the amino acid sequence of the peptide linker comprises or consists of the amino acid sequence of any one of the linkers listed in Table 6, and further comprises one 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 or consists of the amino acid sequence of any one of the linkers listed in Table 6, including one, two, or three amino acid substitutions.

[0366] In some embodiments, the amino acid sequence of the peptide linker comprises the amino acid sequence of any one of SEQ ID NOs: 49-57. In some embodiments, the amino acid sequence of the peptide linker consists of the amino acid sequence of any one of SEQ ID NOs: 49-57. In some embodiments, the amino acid sequence of the peptide linker comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 49-57, and further comprises one or more but less than 15% (less than 12%, less than 10%, less than 8%) amino acid mutations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the peptide linker comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 49-57, and further comprises one, two, or three amino acid mutations (e.g., substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the peptide linker comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 49-57, and further comprises one 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 or consists of the amino acid sequence of any one of SEQ ID NOs: 49-57, including one, two, or three amino acid substitutions.

[0367] In some embodiments, the amino acid sequence of the peptide linker comprises the amino acid sequence of SEQ ID NO: 51, and further comprises one 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 SEQ ID NO: 51. In some embodiments, the amino acid sequence of the peptide linker comprises the amino acid sequence of any one of SEQ ID NO: 51, comprising one, two, or three amino acid mutations (e.g., substitutions, additions, deletions). In some embodiments, the amino acid sequence of the peptide linker comprises the amino acid sequence of any one of SEQ ID NO: 51, comprising one, two, or three amino acid substitutions. In some embodiments, the amino acid sequence of the peptide linker consists of the amino acid sequence of SEQ ID NO: 51, and further comprises one 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 SEQ ID NO: 51. In some embodiments, the amino acid sequence of the peptide linker consists of the amino acid sequence of any one of SEQ ID NO: 51 with one, two, or three amino acid mutations (e.g., substitutions, additions, deletions). In some embodiments, the amino acid sequence of the peptide linker consists of the amino acid sequence of any one of SEQ ID NO: 51 with one, two, or three amino acid substitutions.

[0368] 5.3.3 Orientation The heterologous moiety (e.g., a heterologous polypeptide) and the immunomodulatory protein or polypeptide (e.g., as described herein) can be arranged in any configuration or order, so long as the immunomodulatory protein or polypeptide (e.g., as described herein) maintains the ability to mediate its function (e.g., bind to hIL-10R) and, in embodiments in which the heterologous moiety (e.g., a heterologous polypeptide) has a particular function, the heterologous moiety (e.g., a heterologous polypeptide) can mediate that function.

[0369] In some embodiments, the heterologous moiety is a heterologous polypeptide (e.g., an Ig (e.g., hIg, mIg) Fc region (e.g., an Ig (e.g., hIg, mIg) Fc region described herein)) that forms a fusion polypeptide or protein. In some embodiments, the fusion polypeptide or protein comprises, from N-terminus to C-terminus: an immunomodulatory polypeptide (e.g., as described herein) and a heterologous polypeptide (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 polypeptide or protein comprises, from N-terminus to C-terminus: an immunomodulatory polypeptide (e.g., as described herein), a peptide linker (e.g., as described herein), and a heterologous polypeptide (e.g., an Ig (e.g., hIg, mIg) Fc region (e.g., an Ig (e.g., hIg, mIg) Fc region described herein)). In this particular orientation, the N-terminus of the immunomodulatory polypeptide (e.g., as described herein) is operably linked directly or indirectly via a peptide linker (e.g., as described herein) to the C-terminus of a heterologous polypeptide (e.g., an Ig (e.g., hIg, mIg) Fc region (e.g., an Ig (e.g., hIg, mIg) Fc region described herein)).

[0370] In some embodiments, the fusion polypeptide comprises, from N-terminus to C-terminus: a heterologous polypeptide (e.g., an Ig (e.g., hIg, mIg) Fc region (e.g., an Ig (e.g., hIg, mIg) Fc region described herein)) and an immunomodulatory polypeptide (e.g., as described herein). In some embodiments, the fusion polypeptide comprises, from N-terminus to C-terminus: a heterologous polypeptide (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., as described herein), and an immunomodulatory polypeptide (e.g., as described herein). In this particular orientation, the C-terminus of the immunomodulatory polypeptide (e.g., as described herein) is operably linked directly or indirectly via a peptide linker (e.g., as described herein) to the N-terminus of a heterologous polypeptide (e.g., an Ig (e.g., hIg, mIg) Fc region (e.g., an Ig (e.g., hIg, mIg) Fc region described herein)).

[0371] 5.3.4 Multimeric Fusion Proteins In one aspect, provided herein is a multimeric (e.g., dimeric) protein comprising at least two polypeptide or protein fusions or conjugates described herein (e.g., an Ig (e.g., hIg, mIg) Fc fusion protein or polypeptide described herein). In some embodiments, the protein is a dimer. In some embodiments, the protein is a homodimer. In some embodiments, the protein is a heterodimer. In some embodiments, at least two polypeptide or protein fusions (e.g., an Ig (e.g., hIg, mIg) Fc fusion protein or polypeptide described herein) or conjugates herein are associated via covalent or non-covalent interactions. In some embodiments, at least two polypeptide or protein fusions (e.g., an Ig (e.g., hIg, mIg) Fc fusion protein or polypeptide described herein) or conjugates herein are associated via at least one covalent interaction. In some embodiments, at least two polypeptide or protein fusions (e.g., Ig (e.g., hIg, mIg) Fc fusion proteins or polypeptides) or conjugates are associated through one or more disulfide bonds. In some embodiments, at least two polypeptide or protein fusions (e.g., Ig (e.g., hIg, mIg) Fc fusion proteins or polypeptides) or conjugates are associated through one, two, three, four, or more disulfide bonds.

[0372] In some embodiments, the protein is a dimer comprising a first polypeptide or protein fusion (e.g., an hIg Fc fusion protein or polypeptide) or conjugate described herein and a second polypeptide or protein fusion (e.g., an Ig (e.g., hIg, mIg) Fc fusion protein or polypeptide) or conjugate described herein, wherein the first polypeptide comprises an amino acid sequence that 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 polypeptide. For example, the first polypeptide may comprise an amino acid sequence that is at least about 85% identical to the amino acid sequence of the second polypeptide. For example, the first polypeptide may comprise an amino acid sequence that is at least about 90% identical to the amino acid sequence of the second polypeptide. For example, the first polypeptide may comprise an amino acid sequence that is at least about 95% identical to the amino acid sequence of the second polypeptide. In some embodiments, the first polypeptide may preferably comprise an amino acid sequence that is 100% identical to the amino acid sequence of the second polypeptide.

[0373] In some embodiments, the protein is a dimer comprising a first Ig (e.g., hIg, mIg) Fc fusion protein or polypeptide and a second Ig (e.g., hIg, mIg) Fc fusion protein or polypeptide. In some embodiments, the dimeric protein is a homodimer. In some embodiments, the dimeric protein is a heterodimer. In some embodiments, the first Ig (e.g., hIg, mIg) Fc fusion protein or polypeptide comprises an amino acid sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86...

Claims

1. A fusion polypeptide or protein comprising (i) a protein or polypeptide having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of the amino acid sequences of SEQ ID NOs: 125, 114, 108-113, 115-124, or 136-454, wherein the polypeptide or protein specifically binds to a human IL-10 receptor (hIL-10R) operably linked to a heterologous polypeptide or protein.

2. An isolated polypeptide or protein comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of the amino acid sequences of SEQ ID NOs: 125, 114, 108-113, 115-124, or 136-454, wherein the polypeptide or protein specifically binds to the human IL-10 receptor (hIL-10R).

3. The fusion polypeptide or protein according to claim 1, wherein the protein or polypeptide of (i) comprises an amino acid sequence that is 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 either SEQ ID NO: 125 or 114.

4. The fusion polypeptide or protein according to claim 1, wherein the heterogeneous polypeptide or protein comprises a half-life extension polypeptide or protein.

5. The fusion polypeptide or protein according to claim 1, wherein the heterologous polypeptide or protein includes an immunoglobulin (Ig) Fc region.

6. The fusion polypeptide or protein according to claim 5, wherein the IgFc region includes at least a portion of the hinge region, the CH2 region, and the CH3 region.

7. The fusion polypeptide or protein according to claim 5, wherein the IgFc region comprises a hinge region, a CH2 region, and a CH3 region.

8. The fusion polypeptide or protein according to claim 5, wherein the Ig is hIg.

9. The fusion polypeptide or protein according to claim 8, wherein the hIg is human IgG (hIgG).

10. The fusion polypeptide or protein according to claim 9, wherein the IgG is IgG1 or IgG4.

11. The fusion polypeptide or protein according to claim 5, wherein the IgFc region comprises one or more amino acid substitutions compared to the reference IgFc region, which reduces or eliminates one or more of the following effector functions: antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and / or affinity for one or more human Fc receptors.

12. The fusion polypeptide or protein according to claim 5, wherein the IgFc region is substantially non-mediating of ADCC, substantially non-mediating of CDC, and / or does not bind to one or more human Fc receptors.

13. The fusion polypeptide or protein according to claim 1, wherein a polypeptide or protein is directly and operably linked to the heterologous polypeptide or protein via a peptide bond.

14. The fusion polypeptide or protein according to claim 1, wherein a polypeptide or protein is indirectly and operably linked to the heterologous polypeptide or protein via a peptide linker.

15. A fusion protein comprising a first polypeptide or protein and a second polypeptide or protein, The first polypeptide or protein comprises a first IgFc region operably linked to the first polypeptide or protein described in claim 2; A fusion protein comprising a second polypeptide or protein operably linked to the second polypeptide or protein described in claim 2.

16. A conjugate comprising a polypeptide or protein according to claim 2, operably linked to a different portion.

17. (a) at least a portion of the polypeptide described in claim 2; and / or (b) The polypeptide or protein according to claim 2, comprising at least one amino acid mutation (e.g., substitution, addition, deletion). An immunogenic peptide or protein containing, An immunogenic peptide or protein which does not specifically bind to the hIL-10R (e.g., hIL-10Rα), or which binds to the hIL-10R (e.g., hIL-10Rα) with lower affinity than the reference polypeptide or protein described in claim 2.

18. An isolated antibody that specifically binds to the polypeptide described in claim 2.

19. A polynucleotide encoding a fusion polypeptide or protein as described in claim 1.

20. An expression vector comprising the polynucleotide described in claim 19.

21. A cell comprising the fusion polypeptide or protein described in claim 1.

22. A carrier comprising the fusion polypeptide or protein described in claim 1.

23. A vaccine composition comprising the immunogenic peptide or protein (or a nucleic acid molecule encoding the same (or a vector encoding the nucleic acid molecule)) according to claim 17.

24. A pharmaceutical composition comprising a fusion polypeptide or protein as described in claim 1, and a pharmaceutically acceptable excipient.

25. A kit comprising the fusion polypeptide or protein described in claim 1, and optionally, instructions for using one or more of the foregoing.

26. A pharmaceutical agent for stimulating the IL-10 pathway in a target subject, comprising the fusion polypeptide or protein described in Claim 1.

27. ​​A pharmaceutical product comprising the fusion polypeptide or protein described in Claim 1, for treating, preventing, or improving IL-10 responsive diseases in a target as needed.

28. The pharmaceutical product according to claim 27, wherein the IL-10 responsive disease is an inflammatory disease.

29. The pharmaceutical product according to claim 27, wherein the IL-10 responsive disease is an autoimmune disease.

30. The pharmaceutical product according to claim 27, wherein the IL-10 responsive disease is a metabolic inflammatory disease.

31. A pharmaceutical agent for suppressing or preventing an immune response in a target, comprising the fusion polypeptide or protein described in Claim 1.

32. A pharmaceutical agent for inducing resistance to an immunogen in a target subject, comprising the fusion polypeptide or protein described in Claim 1.

33. A pharmaceutical agent for suppressing IgE expression in a target, comprising the fusion polypeptide or protein described in Claim 1.

34. A pharmaceutical agent for inducing an immune response in a target, comprising the fusion polypeptide or protein described in Claim 1.

35. A pharmaceutical agent comprising the fusion polypeptide or protein described in Claim 1, for preventing, treating, or improving viral infection in a target area as needed.

36. A kit for determining the presence of a virus in a subject, (a) to obtain a sample from the subject, or to provide a sample obtained from the subject, and (b) Determining the presence or absence of the polypeptide or protein (or fragment or variant thereof) described in claim 2, or the polynucleotide encoding the polypeptide or protein (or fragment or variant thereof) described in claim 2, in the sample. A kit that includes the means to do so.

Images