Immunoreceptor targeting proteins and related methods

Abstract

Provided herein are immunoreceptor targeting proteins and compositions (e.g., pharmaceutical compositions) comprising the same; as well as methods of making the immunoreceptor targeting proteins and compositions. The immunoreceptor targeting proteins provided herein are useful in pharmaceutical compositions and methods of use (including, e.g., binding to (e.g., thereby targeting) cells expressing CD30L, inhibiting binding or reducing (e.g., preventing) of one or more TNSRSF member to one or more TNFLSF member (e.g., binding of CD30L to CD30).

Description

IMMUNORECEPTOR TARGETING PROTEINS AND RELATED METHODSRELATED APPLICATIONS

[0001] This application claims priority to U.S. Serial No.: 63 / 650,602, filed May 22, 2024; the entire contents of which is incorporated herein by reference.1. FIELD

[0002] This disclosure relates to immunoreceptor targeting proteins that bind to CD30L (e.g., human CD30L (hCD30L)) and nucleic acid molecules encoding the same. The disclosure further relates to methods of making and utilizing the same.2. BACKGROUND

[0003] The TNF superfamily (TNFSF) comprises 19 ligands and 29 receptors that regulate multiple cellular functions, including e.g., immune responses, cell proliferation, cell survival, cell differentiation, and programmed cell death. Exemplary TNFSF ligands and exemplary cognate receptors, include, e.g., CD30 and CD30L. CD30 is a type 1 transmembrane receptor expressed by, e.g., activated immune cells (e.g., activated T-cells, activated B-cells). CD30 engages in various biological processes through interaction with CD30L (also known as CD153). CD30L is a type II transmembrane protein belonging to the TNF superfamily 8 (TNFSF8). CD30L is expressed by, e.g., activated immune cells e.g., activated T-cells, activated B-cells).3. SUMMARY

[0004] Provided herein are, inter alia, immunoreceptor targeting proteins and nucleic acid molecules encoding the same; fusions and conjugates comprising the immunoreceptor targeting proteins; methods of manufacturing; pharmaceutical compositions; and methods of use including e.g., methods of inhibiting or reducing (e.g., preventing) binding of CD30L to CD30, inhibiting or reducing (e.g., preventing) signaling of CD30 (including, e.g., signaling mediated through the binding of CD30 to CD30L), and modulating (e.g., suppressing or reducing (e.g., preventing)) an immune response, as well as diagnostics.

[0005] Accordingly, in one aspect provided herein are proteins (e.g., recombinant and / or isolated) comprising an amino acid sequence at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequenceof any protein set forth in Table 2 or set forth in any one of SEQ TD NOS: 129-240.

[0006] In some embodiments, the protein comprises an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any protein set forth in Table 2 or set forth in any one of SEQ ID NOS: 129-240. In some embodiments, the protein comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any protein set forth in Table 2 or set forth in any one of SEQ ID NOS: 129-240. In some embodiments, the protein comprises an amino acid sequence about 100% identical to the amino acid sequence of any protein set forth in Table 2 or set forth in any one of SEQ ID NOS: 129-240.

[0007] In some embodiments, the protein comprises an amino acid sequence at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any protein set forth in Table 2 or set forth in any one of SEQ ID NOS: 129-186. In some embodiments, the protein comprises an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any protein set forth in Table 2 or set forth in any one of SEQ ID NOS: 129-186. In some embodiments, the protein comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any protein set forth in Table 2 or set forth in any one of SEQ ID NOS: 129-186. In some embodiments, the protein comprises an amino acid sequence about 100% identical to the amino acid sequence of any protein set forth in Table 2 or set forth in any one of SEQ ID NOS: 129-186.

[0008] In some embodiments, the protein specifically binds CD30L (e.g., human CD30L (hCD30L)). In some embodiments, the protein exhibits anti-inflammatory properties (e.g., upon administration to a subject). In some embodiments, the protein inhibits or reduces (e.g., prevents) binding of hCD30L to human CD30 (hCD30). In some embodiments, the protein inhibits or reduces (e.g., prevents) activation of hCD30 signaling.

[0009] In some embodiments, the protein comprises a homologous or heterologous signal peptide (e.g., operably connected to the N-terminus of the protein). In some embodiments, the protein is operably connected to a heterologous moiety (e.g., described herein). In some embodiments, the heterologous moiety is a protein, peptide, small molecule, nucleic acid molecule (e.g., DNA, RNA, DNA / RNA hybrid molecule), lipid, or synthetic polymer. In some embodiments, the heterologous moiety is a protein.

[0010] In one aspect, provided herein are conjugates comprising a protein described herein (e.g., an ITP described herein) operably connected to a heterologous moiety (e.g., described herein).

[0011] In one aspect, provided herein are radioligand comprising a protein described herein (e.g., an ITP described herein) operably connected to a radionuclide.

[0012] In one aspect, provided herein are fusion protein comprising a protein described herein (e.g., an ITP described herein) operably connected to a heterologous protein. In some embodiments, the heterologous protein comprises a half-life extension protein.

[0013] In some embodiments, the heterologous protein comprises an immunoglobulin (Ig) (e.g., a human Ig (hlg)) Fc region. In some embodiments, the Ig (e.g., hlg) 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., hlg) Fc region comprises a hinge region, a CH2 region, and a CH3 region. In some embodiments, the Ig is a hlg. In some embodiments, the hlg is a human IgG (hlgG). In some embodiments, the hlgG is hlgGl or hIgG4.

[0014] In some embodiments, the Ig (e.g., hlg) Fc region does not substantially mediate ADCC, does not substantially mediate CDC, and / or does not bind to one or more human Fc receptor (e.g., an Fey receptor (e.g., FcyRI, FcyRIIa, FcyRIIc, FcyRIIIa, and / or FcyRIIIb (e.g., FcyRI, FcyRIIa, and / or FcyRIIIa))). In some embodiments, the Ig (e.g., hlg) Fc region comprises one or more amino acid variation relative to a reference Ig (e.g., hlg) Fc region that reduces or abolishes one or more of the following effector functions relative to the reference Ig (e.g., hlg) Fc region: antibody dependent cell mediated cytotoxicity (ADCC), complement dependent cytotoxicity (CDC), and / or affinity to one or more human Fc receptor (e.g., an Fey receptor (e.g., FcyRI, FcyRIIa, FcyRIIc, FcyRIIIa, and / or FcyRIIIb (e.g., FcyRI, FcyRIIa, and / or FcyRIIIa))).

[0015] In some embodiments, the Ig (e.g., hlg) Fc region is capable of mediating ADCC, capable of mediating CDC, and / or binds to one or more human Fc receptor (e.g., an Fey receptor (e.g., FcyRI, FcyRIIa, FcyRIIc, FcyRIIIa, and / or FcyRIIIb (e.g., FcyRI, FcyRIIa, and / or FcyRIIIa))). In some embodiments, the Ig (e.g., hlg) Fc region comprises one or more amino acid variation relative to a reference Ig (e.g., hlg) Fc region that enhances one or more of the following effector functions relative to the reference Ig (e.g., hlg) Fc region: ADCC, CDC, and / or affinity to one or more human Fc receptor (e.g., an Fey receptor (e.g., FcyRI, FcyRIIa, FcyRIIc, FcyRIIIa, and / or FcyRIIIb (e.g., FcyRI, FcyRIIa, and / or FcyRIIIa))).

[0016] In some embodiments, the protein described herein (e.g., an ITP described herein) is directly operably connected to the heterologous protein through a peptide bond.

[0017] In some embodiments, the protein described herein (e.g., an ITP described herein) is indirectly operably connected to the heterologous protein through a peptide linker. In some embodiments, the amino acid sequence of the peptide linker comprises or consists of glycine or glycine and serine amino acid residues.

[0018] In some embodiments, the fusion protein comprises from N- to C-terminus: the protein described herein (e.g., an ITP described herein) and the heterologous protein. In some embodiments, the fusion protein comprises from N- to C-terminus: the protein described herein (e.g., an ITP described herein), a peptide linker, and the heterologous protein. In some embodiments, the fusion protein comprises from N- to C-terminus: a signal peptide, the protein described herein (e.g., an ITP described herein), a peptide linker, and the heterologous protein. In some embodiments, the fusion protein comprises from N- to C-terminus: the heterologous protein and the protein described herein (e.g., an ITP described herein). In some embodiments, the fusion protein comprises from N- to C-terminus: the heterologous protein, a peptide linker, and the protein described herein (e.g., an ITP described herein). In some embodiments, the fusion protein comprises from N- to C-terminus: a signal peptide, the heterologous protein, a peptide linker, and the protein described herein (e.g., an ITP described herein).

[0019] In some embodiments, the amino acid sequence of the fusion protein is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 93-128 or set forth in Table 10. In some embodiments, the amino acid sequence of the fusion protein is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 94, 97, 100, 103, 106, 109, 112, 115, 118, 121, 124, or 127.

[0020] In one aspect, provided herein are fusion proteins comprising a first protein and a second protein, wherein the first protein comprises a first Ig (e.g., hlg) Fc region operably connected to a first protein described herein (e.g., an ITP described herein); and wherein the second protein comprises a second Ig (e.g., hlg) Fc region operably connected to a second protein described herein (e.g., an ITP described herein).

[0021] In some embodiments, the first Fc region and the second Fc region associate to form adimer. In some embodiments, the first protein comprises an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of the second protein.

[0022] In some embodiments, the first Ig (e.g., hlg, mlg) Fc region comprises at least a portion of a hinge region, a CH2 region, and a CH3 region; and the second Ig (e.g., hlg) Fc region comprises at least a portion of a hinge region, a CH2 region, and a CH3 region.

[0023] In some embodiments, the first Ig (e.g., hlg) Fc region comprises a hinge region, a CH2 region, and a CH3 region; and the second Ig (e.g., hlg) Fc region comprises a hinge region, a CH2 region, and a CH3 region. In some embodiments, the Ig of the first Ig Fc region is a hlg and the Ig of the second Ig Fc region is a hlg. In some embodiments, the hlg of the first hlg Fc region is a hlgG and the hlg of the second hlg Fc region is a hlgG. In some embodiments, the hlgG of the first hlg Fc region is hIgG4 and the hlgG of the first hlg Fc region is hIgG4. In some embodiments, the hlgG of the first hlg Fc region is hlgGl and the hlgG of the first hlg Fc region is hlgGl.

[0024] In some embodiments, the fusion protein does not substantially mediate ADCC, does not substantially mediate CDC, and / or does not bind to one or more Fc receptor (e.g., an Fey receptor (e.g., FcyRI, FcyRIIa, FcyRIIc, FcyRIIIa, and / or FcyRIIIb (e.g., FcyRI, FcyRIIa, and / or FcyRIIIa))). In some embodiments, the first Ig (e.g., hlg) Fc region and the second Ig (e.g., hlg) Fc region each comprises one or more amino acid variation relative to a reference Ig (e.g., hlg) Fc region that reduces or abolishes one or more of the following effector functions relative to the reference Ig (e.g., hlg) Fc region: ADCC, CDC, and / or binding affinity to one or more Fc receptor (e.g., an Fey receptor (e.g., FcyRI, FcyRIIa, FcyRIIc, FcyRIIIa, and / or FcyRIIIb (e.g., FcyRI, FcyRIIa, and / or FcyRIIIa))).

[0025] In some embodiments, the fusion protein is capable of mediating ADCC, capable of mediating CDC, and / or binds to one or more Fc receptor (e.g., an Fey receptor (e.g., FcyRI, FcyRIIa, FcyRIIc, FcyRIIIa, and / or FcyRIIIb (e.g., FcyRI, FcyRIIa, and / or FcyRIIIa))). In some embodiments, the first Ig (e.g., hlg) Fc region and the second Ig (e.g., hlg) Fc region each comprises one or more amino acid variation relative to a reference Ig (e.g., hlg) Fc region that reduces or abolishes one or more of the following effector functions relative to the reference Ig (e.g., hlg) Fc region: ADCC, CDC, and / or binding affinity to one or more Fc receptor (e.g., an Fey receptor (e.g., FcyRI, FcyRIIa, FcyRIIc, FcyRIIIa, and / or FcyRIIIb (e.g., FcyRI, FcyRIIa, and / or FcyRIIIa))).

[0026] In some embodiments, the first Ig (e.g., hlg) Fc region comprises an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of the second Ig (e.g., hlg) Fc region.

[0027] In some embodiments, the first protein comprises from N- to C-terminus: the first Ig (e.g., hlg) Fc region and the first protein described herein (e.g., an ITP described herein); and the second protein comprises from N- to C-terminus: the second Ig (e.g., hlg) Fc region and the second protein described herein (e.g., an ITP described herein). In some embodiments, the first protein comprises from N- to C-terminus: the first Ig (e.g., hlg) Fc region, a first peptide linker, and the first protein described herein (e.g., an ITP described herein); and the second protein comprises from N- to C-terminus: the second Ig (e.g., hlg) Fc region, a second peptide linker, and the second protein described herein (e.g., an ITP described herein). In some embodiments, the first protein comprises from N- to C-terminus: the first protein described herein (e.g., an ITP described herein) and the first Ig (e.g., hlg) Fc region; and the second protein comprises from N- to C-terminus: the second protein described herein (e.g., an ITP described herein) and the second Ig (e.g., hlg) Fc region. In some embodiments, the first protein comprises from N- to C-terminus: the first protein described herein (e.g., an ITP described herein), a first peptide linker, and the first Ig (e.g., hlg) Fc region; and the second protein comprises from N- to C-terminus: the second protein described herein (e.g., an ITP described herein), a second peptide linker, and the second Ig (e.g., hlg) Fc region.

[0028] In some embodiments, the amino acid sequence of the first peptide linker and the second peptide linker each comprises or consists of glycine or glycine and serine amino acid residues.

[0029] In some embodiments, the amino acid sequence of the first protein is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 93-128 or set forth in Table 10; and the amino acid sequence of the first protein is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 93-128 or set forth in Table 10.

[0030] In some embodiments, the amino acid sequence of the first protein is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 94, 97, 100, 103, 106, 109, 112, 115, 118, 121, 124, or 127; and the amino acid sequence of the first protein is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 94, 97, 100, 103, 106, 109, 112, 115, 118, 121, 124, or 127.

[0031] In one aspect, provided herein are immunogenic peptides or proteins comprising at least an immunogenic fragment of the protein described herein (e.g., an ITP described herein). In some embodiments, the immunogenic peptide or protein is formulated with an adjuvant.

[0032] In some embodiments, the immunogenic peptide or protein does not specifically bind hCD30L or binds one or more of hCD30L with lower affinity relative to a reference protein described herein (e.g., an ITP described herein).

[0033] In some embodiments, the immunogenic peptide or protein comprises a full-length protein described herein (e.g., an ITP described herein). In some embodiments, the immunogenic peptide or protein comprises an immunogenic fragment of a protein described herein (e.g., an ITP described herein). In some embodiments, the immunogenic peptide or protein comprises at least about 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, or 130 amino acids. In some embodiments, the immunogenic peptide or protein comprises from about 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, or 130 amino acids.

[0034] In some embodiments, the amino acid sequence of the immunogenic peptide or protein comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acid variations (e.g., substitutions, additions, deletions) relative to a reference protein described herein (e.g., an ITP described herein).

[0035] In some embodiments, the immunogenic peptide or protein comprises an amino acid sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a contiguous stretch of at least about 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, or 130 amino acids set forth in any one of SEQ ID NOS: 129-240. In some embodiments, the immunogenic peptide or protein comprises an amino acid sequence that, other than the one or more amino acid variation (e.g., substitution, addition, deletion), is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 129-240.

[0036] In one aspect, provided herein are isolated antibodies that specifically binds to a proteindescribed herein (e.g., an ITP described herein).

[0037] In one aspect, provided herein arc nucleic acid molecules encoding a protein described herein (e.g., an ITP described herein), a conjugate described herein, a radioligand described herein, a fusion protein described herein, an immunogenic peptide or protein described herein, or an antibody described herein.

[0038] In some embodiments, the nucleic acid molecule is an RNA (e.g., mRNA, circular RNA) molecule or a DNA molecule. In some embodiments, the nucleic acid molecule comprises a heterologous 5'-untranslated region (UTR), 3'-UTR, or both a 5'-UTR and 3'-UTR. In some embodiments, the nucleic acid molecule comprises a poly(A) sequence. In some embodiments, the nucleic acid molecule comprises a 5' cap structure. In some embodiments, the nucleic acid molecule comprises at least one variant nucleotide. In some embodiments, the sequence of the nucleic acid molecule is codon optimized.

[0039] In one aspect, provided herein are mRNA molecules encoding a protein described herein (e.g., an ITP described herein), a conjugate described herein, a radioligand described herein, a fusion protein described herein, an immunogenic peptide or protein described herein, or an antibody described herein.

[0040] In some embodiments, the mRNA molecule comprises a heterologous 5 '-untranslated region (UTR), 3'-UTR, or both a 5'-UTR and 3'-UTR.In some embodiments, the mRNA molecule comprises a poly(A) sequence. In some embodiments, the mRNA molecule comprises a 5' cap structure. In some embodiments, the mRNA molecule comprises at least one variant nucleotide. In some embodiments, the sequence of the mRNA molecule is codon optimized.

[0041] In one aspect, provided herein are vectors (e.g., expression vectors) comprising an nucleic acid molecule described herein or an mRNA molecule described herein.

[0042] In some embodiments, the vector is a viral vector or a non- viral vector (e.g., a plasmid).

[0043] In one aspect, provided herein are viral particles conjugated to a protein described herein (e.g., an ITP described herein), a conjugate described herein, a radioligand described herein, a fusion protein described herein, an immunogenic peptide or protein described herein, or an antibody described herein.

[0044] In one aspect, provided herein are cells (e.g., host cells) comprising a protein described herein (e.g., an ITP described herein), a conjugate described herein, a radioligand described herein, a fusion protein described herein, an immunogenic peptide or protein described herein, or anantibody described herein, a nucleic acid molecule described herein, an mRNA molecule described herein, a vector described herein, a viral particle described herein, a carrier described herein, a lipid nanoparticle described herein, a vaccine composition described herein, or a pharmaceutical composition described herein.

[0045] In one aspect, provided herein are cells (e.g., a therapeutic cells) expressing a protein described herein (e.g., an ITP described herein), a conjugate described herein, a radioligand described herein, a fusion protein described herein, an immunogenic peptide or protein described herein, or an antibody described herein, on the surface of the cell.

[0046] In one aspect, provided herein are carriers comprising a protein described herein (e.g., an ITP described herein), a conjugate described herein, a radioligand described herein, a fusion protein described herein, an immunogenic peptide or protein described herein, or an antibody described herein, a nucleic acid molecule described herein, an mRNA molecule described herein, a vector described herein, a viral particle described herein, a cell described herein, a vaccine composition described herein, or a pharmaceutical composition described herein.

[0047] In one aspect, provided herein are carriers conjugated to expressing a protein described herein (e.g., an ITP described herein), a conjugate described herein, a radioligand described herein, a fusion protein described herein, an immunogenic peptide or protein described herein, or an antibody described herein. In some embodiments, the carrier is a lipid nanoparticle, liposome, lipoplex, or nanoliposome.

[0048] In one aspect, provided herein are lipid nanoparticles comprising a protein described herein (e.g., an ITP described herein), a conjugate described herein, a radioligand described herein, a fusion protein described herein, an immunogenic peptide or protein described herein, or an antibody described herein, a nucleic acid molecule described herein, an mRNA molecule described herein, a vector described herein, a viral particle described herein, a cell described herein, a vaccine composition described herein, or a pharmaceutical composition described herein.

[0049] In one aspect, provided herein are lipid nanoparticles conjugated to a protein described herein (e.g., an ITP described herein), a conjugate described herein, a radioligand described herein, a fusion protein described herein, an immunogenic peptide or protein described herein, or an antibody described herein.

[0050] In one aspect, provided herein are vaccine compositions comprising an immunogenic peptide or protein described herein (or a nucleic acid molecule encoding the same (or a vectorencoding the nucleic acid molecule) or a carrier comprising any of the foregoing).

[0051] In one aspect, provided herein arc pharmaceutical compositions comprising a protein described herein (e.g., an ITP described herein), a conjugate described herein, a radioligand described herein, a fusion protein described herein, an immunogenic peptide or protein described herein, or an antibody described herein, a nucleic acid molecule described herein, an mRNA molecule described herein, a vector described herein, a viral particle described herein, a carrier described herein, a lipid nanoparticle described herein, a vaccine composition described herein, or a cell described herein; and a pharmaceutically acceptable excipient.

[0052] In one aspect, provided herein are kits comprising a protein described herein (e.g., an ITP described herein), a conjugate described herein, a radioligand described herein, a fusion protein described herein, an immunogenic peptide or protein described herein, or an antibody described herein, a nucleic acid molecule described herein, an mRNA molecule described herein, a vector described herein, a viral particle described herein, a carrier described herein, a lipid nanoparticle described herein, a vaccine composition described herein, a cell described herein, or a pharmaceutical composition described herein; and optionally instructions for using any one or more of the foregoing.

[0053] In one aspect, provided herein are methods of delivering a protein, fusion protein, conjugate, radioligand, nucleic acid molecule, mRNA molecule, expression vector, viral particle, cell, carrier, lipid nanoparticle, immunogenic peptide or protein, an antibody, a vaccine composition, or pharmaceutical composition to a subject, the method comprising administering to a protein described herein (e.g., an ITP described herein), a conjugate described herein, a radioligand described herein, a fusion protein described herein, an immunogenic peptide or protein described herein, or an antibody described herein, a nucleic acid molecule described herein, an mRNA molecule described herein, a vector described herein, a viral particle described herein, a carrier described herein, a lipid nanoparticle described herein, a vaccine composition described herein, a cell described herein, or a pharmaceutical composition described herein, to thereby deliver the protein, fusion protein, conjugate, radioligand, immunogenic peptide or protein, antibody, mRNA molecule, vector, viral particle, cell, carrier, lipid nanoparticle, vaccine composition, or pharmaceutical composition to a subject.

[0054] In one aspect, provided herein are methods of inhibiting or reducing (e.g., preventing) binding of CD30L to CD30 in a subject in need thereof, the method comprising administering tothe subject a protein described herein (e.g., an ITP described herein), a conjugate described herein, a radioligand described herein, a fusion protein described herein, an immunogenic peptide or protein described herein, or an antibody described herein, a nucleic acid molecule described herein, an mRNA molecule described herein, a vector described herein, a viral particle described herein, a carrier described herein, a lipid nanoparticle described herein, a vaccine composition described herein, a cell described herein, or a pharmaceutical composition described herein, to thereby inhibit or reduce (e.g., prevent) binding of CD30L to CD30 in the subject.

[0055] In one aspect, provided herein are methods of inhibiting or reducing (e.g., preventing) signaling mediated by the binding of CD30L to CD30 in a subject in need thereof, the method comprising administering to the subject a protein described herein (e.g., an ITP described herein), a conjugate described herein, a radioligand described herein, a fusion protein described herein, an immunogenic peptide or protein described herein, or an antibody described herein, a nucleic acid molecule described herein, an mRNA molecule described herein, a vector described herein, a viral particle described herein, a carrier described herein, a lipid nanoparticle described herein, a vaccine composition described herein, a cell described herein, or a pharmaceutical composition described herein, to thereby inhibit or reduce (e.g., prevent) signaling mediated by the binding of CD30L to CD30 in the subject.

[0056] In one aspect, provided herein are methods of suppressing or reducing (e.g., preventing) a pro-inflammatory immune response in a subject in need thereof, the method comprising administering to the subject a protein described herein (e.g., an ITP described herein), a conjugate described herein, a radioligand described herein, a fusion protein described herein, an immunogenic peptide or protein described herein, or an antibody described herein, a nucleic acid molecule described herein, an mRNA molecule described herein, a vector described herein, a viral particle described herein, a carrier described herein, a lipid nanoparticle described herein, a vaccine composition described herein, a cell described herein, or a pharmaceutical composition described herein, to thereby suppress or reduce (e.g., prevent) a pro-inflammatory immune response in the subject.

[0057] In one aspect, provided herein are methods of depleting a population of immune cells in a subject in need thereof, the method comprising administering to the subject a protein described herein (e.g., an ITP described herein), a conjugate described herein, a radioligand described herein, a fusion protein described herein, an immunogenic peptide or protein described herein, or anantibody described herein, a nucleic acid molecule described herein, an mRNA molecule described herein, a vector described herein, a viral particle described herein, a carrier described herein, a lipid nanoparticle described herein, a vaccine composition described herein, a cell described herein, or a pharmaceutical composition described herein, to thereby deplete a population of immune cells in the subject.

[0058] In some embodiments, the population of immune cells express CD30L on the surface. In some embodiments, the population of immune cells express one or more marker of activation. In some embodiments, the population of immune cells are activated. In some embodiments, the population of immune cells comprises T cells and / or B-cells.

[0059] In some embodiments, the subject has (e.g., has been diagnosed with) a pro- inflammatory (e.g., autoimmune) disease. In some embodiments, the pro-inflammatory disease is an autoimmune disease. In some embodiments, the pro-inflammatory (e.g., autoimmune) disease comprises flares (e.g., relapsing-remitting forms of pro-inflammatory (e.g., autoimmune) diseases).

[0060] In some embodiments, the protein, fusion protein, conjugate, radioligand, immunogenic peptide or protein, antibody, mRNA molecule, vector, viral particle, cell, carrier, lipid nanoparticle, vaccine composition, or pharmaceutical composition is administered to the subject prior to, at the start of, at initial signs of, or during a flare.

[0061] In some embodiments, the pro-inflammatory (e.g., autoimmune) disease is multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease, systemic lupus erythematosus, myasthenia gravis, or psoriasis. In some embodiments, the pro-inflammatory (e.g., autoimmune) disease is relapsing-remitting multiple sclerosis, relapsing-remitting rheumatoid arthritis, relapsing-remitting inflammatory bowel disease, relapsing-remitting systemic lupus erythematosus, relapsing-remitting myasthenia gravis, or relapsing-remitting psoriasis.

[0062] In one aspect, provided herein are methods of treating, ameliorating, or preventing a pro-inflammatory disease in a subject in need thereof, the method comprising administering to the subject a protein described herein (e.g., an ITP described herein), a conjugate described herein, a radioligand described herein, a fusion protein described herein, an immunogenic peptide or protein described herein, or an antibody described herein, a nucleic acid molecule described herein, an mRNA molecule described herein, a vector described herein, a viral particle described herein, a carrier described herein, a lipid nanoparticle described herein, a vaccine composition describedherein, a cell described herein, or a pharmaceutical composition described herein, to thereby treat, ameliorate, or prevent the pro-inflammatory immune response in the subject.

[0063] In some embodiments, the pro-inflammatory disease is an autoimmune disease. In some embodiments, the pro-inflammatory (e.g., autoimmune) disease comprises flares (e.g., relapsing-remitting forms of pro-inflammatory (e.g., autoimmune) diseases).

[0064] In some embodiments, the protein, fusion protein, conjugate, radioligand, immunogenic peptide or protein, antibody, mRNA molecule, vector, viral particle, cell, carrier, lipid nanoparticle, vaccine composition, or pharmaceutical composition is administered to the subject prior to, at the start of, at initial signs of, or during a flare.

[0065] In some embodiments, the pro-inflammatory (e.g., autoimmune) disease is multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease, systemic lupus erythematosus, myasthenia gravis, or psoriasis. In some embodiments, the pro-inflammatory (e.g., autoimmune) disease is relapsing-remitting multiple sclerosis, relapsing-remitting rheumatoid arthritis, relapsing-remitting inflammatory bowel disease, relapsing-remitting systemic lupus erythematosus, relapsing-remitting myasthenia gravis, or relapsing-remitting psoriasis.

[0066] In one aspect, provided herein are methods of inducing or enhancing an immune response in a subject in need thereof, the method comprising administering to the subject (i) an immunogenic peptide or protein described herein (or a conjugate or a fusion protein thereof); (ii) a nucleic acid molecule encoding (i); (iii) a vector comprising (ii); (iv) a carrier comprising (i), (ii), or (iii); a vaccine composition comprising (i), (ii), (iii), or (iv); or a pharmaceutical composition comprising (i), (ii), (iii), (iv), or (v), to thereby induce or enhance an immune response in the subject.

[0067] In one aspect, provided herein are methods of vaccinating a subject in need thereof (e.g., against a viral infection), the method comprising administering to the subject (i) an immunogenic peptide or protein described herein (or a conjugate or a fusion protein thereof); (ii) a nucleic acid molecule encoding (i); (iii) a vector comprising (ii); (iv) a carrier comprising (i), (ii), or (iii); a vaccine composition comprising (i), (ii), (iii), or (iv); or a pharmaceutical composition comprising (i), (ii), (iii), (iv), or (v), to thereby vaccinate the subject in need thereof (e.g., against a virus).

[0068] In one aspect, provided herein are methods of determining the presence of a virus in a subject, the method comprising (a) obtaining the sample from a subject or providing a sample thathas been obtained from a subject, and (b) determining the presence or absence of a protein described herein (e.g., an ITP described herein) (or a fragment or variant thereof) or a nucleic acid molecule encoding the protein described herein (e.g., an ITP described herein) (or the fragment or variant thereof) in the sample.

[0069] In one aspect, provided herein are methods of diagnosing a viral infection in a subject, the method comprising (a) obtaining a sample from a subject or providing a sample that has been obtained from a subject, (b) determining the presence or absence of a protein described herein (e.g., an ITP described herein) (or a fragment or variant thereof) or a nucleic acid molecule encoding the protein described herein (e.g., an ITP described herein) (or a fragment or variant thereof), and (c) diagnosing the subject as having the viral infection if the protein described herein (e.g., an ITP described herein) (or a fragment or variant thereof) or a nucleic acid molecule encoding the protein described herein (e.g., an ITP described herein) (or the fragment or variant thereof) is determined to be present in the sample in step (b).

[0070] In some embodiments, the method is an in vitro method.

[0071] In one aspect, provided herein are methods of treating a viral infection in a subject, the method comprising (a) receiving testing results that determined the presence of a protein described herein (e.g., an ITP described herein) (or a fragment or variant thereof) or a nucleic acid molecule encoding a protein described herein (e.g., an ITP described herein) (or the fragment or valiant 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.

[0072] In some embodiments, the sample is a blood, cell, tissue, or saliva, or nasal swab.

[0073] In some embodiments, an antibody described herein is utilized to determine the presence or absence of the protein described herein (e.g., an ITP described herein) (or the fragment or valiant thereof).

[0074] In some embodiments, the subject according to any one of the foregoing methods is a human.4. BRIEF DESCRIPTION OF THE FIGURES

[0075] FIG. 1 is a graph showing the % binding ranges of 149 ITP-Ig Fc proteins (identified as being less than 110 amino acids in length) (including fusion proteins comprising one of each of ITPs-1-58) to hCD30L (as determined by Luminex assay as described in Example 1). The graphis a representative of one experiment executed in duplicates.

[0076] FIG. 2 is a graph showing the % binding ranges of specific ITPs (as indicated) sampled for secondary validation based on their range of binding to CD30L in the Luminex assay (data shown in FIG. 1).

[0077] FIG. 3 is a line graph showing the percent binding of the indicated agent (ITFP-1-12, hIgG4 Fc Control, CD30R-hIgG4-Fc, and mock) to CD30L (as determined by ELISA). The graph represents a single experiment performed in duplicates.

[0078] FIG. 4 is a graph showing CD30L binding of the indicated agent (ITFP-1-12, anti- CD30 antibody (positive control), anti-CD30L antibody (positive control), isotype antibody (negative control), hIgG4 Fc Control (negative control), CD30R-hIgG4-Fc (positive control), and mock (negative control)) to CD30L (as determined by competitive ELISA). The graph represents a single experiment performed in duplicates. The graph shows the segregation of ITFPs in their ability to compete with biotinylated CD30R and bind CD30L either competitively or non- competitively.5. DETAILED DESCRIPTION

[0079] The inventors have, inter alia, identified and developed immunoreceptor targeting proteins that specifically bind to TNFSF ligand CD30L. Accordingly, the novel immunoreceptor targeting proteins disclosed herein may be useful for various methods, including, e.g., binding to (and thereby selecting) CD30L expressing cells, selectively inhibiting or reducing (e.g., preventing) binding of CD30L to CD30, inhibiting or reducing (e.g., preventing) signaling of CD30 (including, e.g., signaling mediated through the binding of CD30 to CD30L), and modulating (e.g., suppressing or reducing (e.g., preventing)) an immune response, as well as in diagnostic assays. As such, the current disclosure provides, inter alia, novel immunoreceptor targeting proteins, nucleic acid molecules encoding, the methods for utilizing the same.TABLE OF CONTENTS5.1 Definitions5.2 Immunoreceptor Targeting Proteins5.3 Exemplary Properties of Immunoreceptor Targeting Proteins5.4 Immunoreceptor Targeting Protein Fusions & Conjugates5.11.2 Carriers Conjugated to ITPs5.11.3 Lipid Based Carriers / Lipid Nanoformulations5.11.3.1 Cationic Lipids (Positively Charged) and Ionizable Lipids5.11.3.2 Non-Cationic Lipids (e.g., Phospholipids)5.11.3.3 Structural Lipids5.11.3.4 Polymers and Polyethylene Glycol (PEG) - Lipids5.11.3.5 Percentages of Lipid Nanoformulation Components5.12 Adjuvants5.13 Pharmaceutical Compositions5.14 Methods of Use5.14.1 Methods of Delivery5.14.2 Methods of Inhibiting or Reducing (e.g., Preventing) Binding of CD30L toCD305.14.3 Methods of Inhibiting of Reducing (e.g., Preventing) Signaling Mediated by CD30L Binding to CD305.14.4 Methods of Suppressing or Reducing (e.g., Preventing) a Pro-Inflammatory Immune Response5.14.5 Methods of Depleting a Population of Immune Cells5.14.6 Methods of Treating, Ameliorating, or Preventing a Pro-Inflammatory Disease5.14.7 Methods of Treating, Ameliorating, or Preventing a Metabolic Syndrome5.14.8 Methods of Inducing or Enhancing an Immune Response5.14.9 Methods of Vaccinating a Subject5.14.10 Diagnostic Methods5.15 Kits5.1 Definitions

[0080] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0081] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the ail to which the claimed subject matterbelongs. It is to be understood that the foregoing general description and the following detailed description arc exemplary and explanatory only and arc not restrictive of any subject matter claimed.

[0082] Use of the singular herein includes the plural unless specifically stated otherwise. For example, as used herein, the singular’ forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Furthermore, use of the term “including” as well as other forms, such as “include,” “includes,” and “included,” is not limiting.

[0083] It is understood that wherever aspects are described herein with the language “comprising,” otherwise analogous aspects described in terms of “consisting of’ and “consisting essentially of’ are also provided.

[0084] The term “and / or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and / or” as used in a phrase such as “A and / or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Likewise, the term “and / or” as used in a phrase such as “A, B, and / or C” is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0085] As described herein, any concentration range, percentage range, ratio range or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated.

[0086] The term “about” refers to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. When particular’ values or compositions are provided herein, unless otherwise stated, the meaning of “about” should be assumed to be within an acceptable error range for that particular value or composition.

[0087] Where proteins and / or polypeptides are described herein, it is understood that nucleic acid molecules (e.g., RNA (e.g., mRNA) or DNA molecules) encoding the protein are also provided herein.

[0088] Where proteins, peptides, nucleic acid molecules, vectors, carriers, etc. are described herein, it is understood that isolated forms of the proteins, peptides, nucleic acid molecules,vectors, carriers, etc. are also provided herein.

[0089] Where proteins, peptides, nucleic acid molecules, etc. arc described herein, it is understood that recombinant forms of the proteins, peptides, nucleic acid molecules, etc. are also provided herein.

[0090] Where polypeptides or sets of polypeptides are described herein, it is understood that proteins comprising the polypeptides or sets of polypeptides folded into their three-dimensional structure (i.e., tertiary or quaternary structure) are also provided herein and vice versa.

[0091] As used herein, the term “adjuvant” refers to a substance that causes stimulation of the immune system of a subject when administered to the subject.

[0092] As used herein, the term “administering” refers to the physical introduction of an agent, e.g., a therapeutic agent (or a precursor of the therapeutic agent that is metabolized or altered within the body of the subject to produce the therapeutic agent in vivo) or vaccine to a subject, using any of the various methods and delivery systems known to those skilled in the art. Administering can also be performed, for example, once, a plurality of times, and / or over one or more extended periods.

[0093] As used herein, the term “affinity” refers to the strength of the binding of one protein (e.g., a Ligand) to another protein (e.g., a Receptor). The affinity of a protein is measured by the dissociation constant Kd, defined as [Ligand] x [Receptor] / [Ligand-Receptor] where [Ligand- Receptor] is the molar concentration of the Ligand-Receptor complex, [Ligand] is the molar concentration of the unbound Ligand and [Receptor] is the molar concentration of the unbound Receptor. The affinity constant Ka is defined by 1 / Kd. Standard methods of measuring affinity are known to the person of ordinary skill in the art and described herein, see, e.g., § 5.3.

[0094] As used herein, the term “agent” is used generically to describe any macro or micro molecule. Exemplary agents include, but are not limited proteins, peptides, nucleic acid molecules (e.g., DNA molecules, RNA molecules), vectors, carriers, carbohydrates, lipids, synthetic polymers, etc.

[0095] As used herein, the term “antibody” or “antibodies” is used in the broadest sense and encompasses various immunoglobulin (Ig) (e.g., human Ig (hlg), murine Ig (mlg)) structures, including, but not limited to monoclonal antibodies, polyclonal antibodies, multispecific (e.g., bispecific, trispecific) antibodies, and antibody fragments so long as they exhibit the desired antigen-binding activity (i.e., antigen binding fragments or variants). The term antibody thusincludes, for example, full-length antibodies; antigen-binding fragments of full-length antibodies; molecules comprising antibody CDRs, VH regions, and / or VL regions; and antibody-like scaffolds (e.g., fibronectins). Examples of antibodies include, without limitation, monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, camelized antibodies, intrabodies, affybodies, diabodies, tribodies, heteroconjugate antibodies, antibody-drug conjugates, single domain antibodies (e.g.,VHH, (VHH)2), single chain antibodies, single-chain Fvs (scFv; (scFv)2), Fab fragments (e.g., Fab, single chain Fab (scFab), F(ab’)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, and conjugates or fusion proteins comprising any of the above. Antibodies can be of Ig isotype (e.g., IgG, IgE, IgM, IgD, or IgA), any class (e.g., IgGi, IgG , IgGa, IgG4, IgAi or IgA2), or any subclass (e.g., IgG2a or IgG2b) of Ig). In certain embodiments, antibodies described herein are IgG antibodies, or a class (e.g., human IgGi or IgG4) or subclass thereof. In certain embodiments, antibodies described herein are mlgG antibodies, or a class (e.g., mlgGl or mIgG2a) or subclass thereof. In some embodiments, the antibody is a human, humanized, or chimeric IgGi or IgG4 monoclonal antibody. In some embodiments, the term antibodies refers to a monoclonal or polyclonal antibody population. Antibodies described herein can be produced by any standard methods known in the art, e.g., recombinant production in host cells, see, e.g., § 5.6; or synthetic production.

[0096] As used herein, the term “antibody mimetic” refers to non-Ig based antigen binding domain. Various antibody-like scaffolds are known in the art. For example, 10th type III domain of fibronectin (e.g., AdNectins®) and designed ankyrin repeat proteins (e.g., DARPins®) have been used as alternative scaffolds for antigen-binding domains, see, e.g., Gebauer and Skerra, Engineered protein scaffolds as next-generation antibody therapeutics. Curr Opin Chem Biol 13:245-255 (2009) and Stumpp et al., Darpins: A new generation of protein therapeutics. Drug Discovery Today 13: 695-701 (2008), the full contents of each of which is incorporated by reference herein for all purposes. Exemplary antibody-like scaffolds include, but are not limited to, lipocalins (see, e.g., US7250297) (e.g., Anticalin®), protein A-derived molecules such as z- domains of protein a (see, e.g., US5831012) (e.g., Affibody®), A domains of membrane receptors stabilized by disulfide bonds and Ca2+ (see, e.g., US7803907) (e.g., Avimer / Maxibody®), a serum transferrin (see, e.g., US2004023334) (e.g., Transbody®); a designed ankyrin repeat protein (see,e.g., US7417130) (e.g., DARPin®), a fibronectin (see, e.g., US6818418) (e.g., AdNectin®), a C- typc lectin domain (see, e.g., US2004132094) (e.g., Tetranectin®); a human gamma-crystallin or ubiquitin (see, e.g., US7838629) (e.g., Affilin®); a kunitz type domain of human protease inhibitors (see, e.g., US2004209243), C-Type Lectins (see, e.g., US2004132094) (e.g., Tetranectins®), cysteine knots or knottins (see, e.g., US7186524) (e.g., Microbodies®), nucleic acid aptamers (see, e.g., US5475096), thioredoxin A scaffold (see, e.g., US6004746) (peptide aptamers), and 10th type III domain of fibronectin (see, e.g., US6818418) (e.g., AdNectins®), and cystine-dense peptides (see, e.g., W02023023031). Additional exemplary antibody-like scaffolds are known in the art and for example described in Storz U. Intellectual property protection: strategies for antibody inventions. MAbs. 2011;3(3):310-317. doi:10.4161 / mabs.3.3.15530. The entire contents of each of the foregoing references is incorporated herein by reference for all purposes. Antibody like scaffolds include e.g., naturally occurring antigen binders, variant (e.g., functional variants) of naturally occurring antigen binders, fragments (e.g., functional fragments) of naturally occurring antigen binders, and synthetic antigen binders (i.e., not naturally occurring antigen binders).

[0097] As used herein the term “CD30L” or “CD30 ligand” refers to the type II transmembrane protein of the TNFSF. The amino acid sequence of an exemplary reference membrane human CD30L (hCD30L) protein is set forth in SEQ ID NO: 1.

[0098] As used herein the teim “CD30” refers to the type I transmembrane receptor of the TNFSF that binds CD30L. The amino acid sequence of an exemplary reference immature human CD30 (hCD30) protein is set forth in SEQ ID NO: 2 and the amino acid sequence of an exemplary reference mature hCD30 protein is set forth in SEQ ID NO: 3.

[0099] The terms “CHI” and “CHI region” are used interchangeably herein and refer to the first constant region of an immunoglobulin heavy chain. The amino acid sequence of an exemplary reference hlgGl CHI region is set forth in SEQ ID NO: 9; and the amino acid sequence of an exemplary reference hIgG4 CHI region is set forth in SEQ ID NO: 22.

[0100] The terms “CH2” and “CH2 region” are used interchangeably herein and refer to the second constant region of an immunoglobulin heavy chain. The amino acid sequence of an exemplary reference hlgGl CH2 region is set forth in SEQ ID NO: 11; and the amino acid sequence of an exemplary reference hIgG4 CH2 region is set forth in SEQ ID NO: 25.

[0101] The terms “CH3” and “CH3 region” are used interchangeably herein and refer to thethird constant region of an immunoglobulin heavy chain. The amino acid sequence of an exemplary reference hlgGl CH3 region is set forth in SEQ ID NO: 12; and the amino acid sequence of an exemplary reference hIgG4 CH3 region is set forth in SEQ ID NO: 26.

[0102] As used herein, the term “circular RNA” refers to a translatable RNA molecule that forms a circular’ structure through covalent or non-covalent bonds. In some embodiments, the circular RNA is covalently closed.

[0103] As used herein, the term “conjugation” refers to chemical conjugation of a protein with a moiety (e.g., small molecule, polypeptide, nucleic acid molecule, carbohydrate, lipid, synthetic polymer (e.g., polymers of polyethylene glycol (PEG)), etc.). The moiety can be directly connected to the protein or indirectly connected through a linker, e.g., as described herein. Chemical conjugation methods are well known in the ait, as are commercially available conjugation reagents and kits, with detailed instructions for their use readily available from the commercial suppliers.

[0104] As used herein, the term “derived from,” with reference to a nucleic acid molecule refers to a nucleic acid molecule that has at least 70% sequence identity to a reference nucleic acid molecule (e.g., a naturally occurring nucleic acid molecule) or a fragment thereof. The term “derived from,” with reference to a protein refers to a protein that comprises an amino acid sequence that has at least 70% sequence identity to the amino acid sequence of a reference protein (e.g., a naturally occurring protein). The term “derived from” as used herein does not denote any specific process or method for obtaining the nucleic acid molecule, polypeptide, or protein. For example, the nucleic acid molecule, polypeptide, or protein can be recombinantly produced or chemically synthesized.

[0105] As used herein, the term “diagnosing” or “diagnosis” refers to a determination of the presence, absence, severity, or course of treatment of a disease (e.g., an infection, e.g., a viral infection). The term “diagnosing” encompasses an initial determination as well as subsequent determinations (e.g., monitoring) after the initial determination.

[0106] As used herein, the term “disease” refers to any abnormal condition that impairs physiological function. The term is used broadly to encompass any disorder, illness, abnormality, pathology, sickness, condition, or syndrome in which physiological function is impaired, irrespective of the nature of the etiology.

[0107] The terms “DNA” and “polydeoxyribonucleotide” are used interchangeably herein and refer to macromolecules that include multiple deoxyribonucleotides that are polymerized viaphosphodiester bonds. Deoxyribonucleotides are nucleotides in which the sugar is deoxyribose.

[0108] The term “effector function” when used in reference to an antibody refers to those biological activities attributable to the Fc region of an antibody, which therefore vary with the antibody isotype. Antibody effector functions include, but are not limited to, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement dependent cytotoxicity (CDC), Fc receptor binding (e.g., FcyRI, FcyRIIa, FcyRIIc, FcyRIIIa, and / or FcyRIIIb (e.g., FcyRI, FcyRIIa, and / or FcyRIIIa)), and Clq binding.

[0109] As used herein, the term “Fc region” refers to the C-terminal region of an Ig heavy chain that comprises from N- to C-terminus at least a CH2 region operably connected to a CH3 region. In some embodiments, the Fc region comprises an Ig hinge region or at least a portion of an Ig hinge region operably connected to the N-terminus of the CH2 region. In some embodiments, the Fc region is engineered relative to a reference Fc region, see, e.g., § 5.4.5.3. Additional examples of proteins with engineered Fc regions can be found in Saunders 2019 (K. O. Saunders, “Conceptual Approaches to Modulating Antibody Effector Functions and Circulation Half-Life,” 2019, Frontiers in Immunology, V. 10, Art. 1296, pp. 1-20, the entire contents of which is incorporated by reference herein for all purposes).

[0110] The term “functional variant” as used herein in reference to a protein refers to a protein that comprises at least one but no more than 15%, not more than 12%, no more than 10%, no more than 8% amino acid variation (e.g., substitution, deletion, addition) compared to the amino acid sequence of a reference protein, wherein the protein retains at least one particular function of the reference protein. Not all functions of the reference protein (e.g., wild type) need be retained by the functional variant of the protein. In some instances, one or more functions are selectively reduced or eliminated. In some embodiments, the reference protein is a wild type protein. For example, a functional variant of a TNF superfamily ligand binding protein can refer to a TNF superfamily ligand binding protein comprising one or more amino acid substitution as compared to a reference TNF superfamily ligand binding protein (e.g., a wild type protein) that retains the ability to specifically bind the TNF superfamily ligand.

[0111] The term “functional fragment” as used herein in reference to a protein refers to a fragment of a reference protein that retains at least one particular’ function. Not all functions of the reference protein need be retained by a functional fragment of the protein. In some instances, one or more functions are selectively reduced or eliminated. In some embodiments, the referenceprotein is a wild type protein. For example, a functional fragment of a TNF superfamily ligand binding protein can refer to a fragment of a TNF superfamily ligand binding protein that retains the ability to specifically bind the TNF superfamily ligand.

[0112] As used herein, the term “fuse” and grammatical equivalents thereof refer to the operable connection of at least a first polypeptide to a second polypeptide, wherein the first and second polypeptides are not naturally found operably connected together. For example, the first and second polypeptides are derived from different proteins. The term fuse encompasses both a direct connection of the at least two polypeptides through a peptide bond, and the indirect connection through a linker (e.g., a peptide linker).

[0113] As used herein, the term “fusion protein” and grammatical equivalents thereof refers to a protein that comprises at least one polypeptide operably connected to another polypeptide, wherein the first and second polypeptides are different and not naturally found operably connected together. For example, the first and second polypeptides of the fusion protein are each derived from different proteins. The at least two polypeptides of the fusion protein can be directly operably connected through a peptide bond; or can be indirectly operably connected through a linker (e.g., a peptide linker). Therefore, for example, the term fusion polypeptide encompasses embodiments, wherein Polypeptide A is directly operably connected to Polypeptide B through a peptide bond (Polypeptide A - Polypeptide B), and embodiments, wherein Polypeptide A is operably connected to Polypeptide B through a peptide linker (Polypeptide A - peptide linker - Polypeptide B).

[0114] As used herein, the term “half-life extension moiety” refers to a moiety (e.g., small molecule, polypeptide, nucleic acid molecule, carbohydrate, lipid, synthetic polymer e.g., polymers of PEG), etc.) that when conjugated or otherwise operably connected (e.g., fused) to a protein (the subject protein), increases the half-life of the subject protein in vivo when administered to a subject (e.g., a human subject). The pharmacokinetic properties of the protein can be evaluated utilizing in vivo models known in the art.

[0115] As used herein, the term “half-life extension polypeptide” or “half-life extension protein” refers to a protein that when operably connected to another protein (the subject protein), increases the half-life of the subject protein in vivo when administered to a subject (e.g., a human subject). The pharmacokinetic properties of the protein can be evaluated utilizing in vivo models known in the ait.

[0116] As used herein, the term “heterologous”, when used to describe a first element inreference to a second element means that the first element and second element do not exist in nature disposed as described. For example, a polypeptide comprising a “heterologous moiety” means a polypeptide that is joined to a moiety (e.g., small molecule, polypeptide, nucleic acid molecule, carbohydrate, lipid, synthetic polymer (e.g., polymers of PEG), etc.) that is not joined to the polypeptide in nature. In one embodiment, the heterologous moiety is not derived from a protein comprising or consisting of the amino acid sequence set forth in any one of SEQ ID NOS: 129-240. For example, a non-limiting example of a heterologous moiety is a heterologous polypeptide (as defined herein). In one embodiment, the heterologous polypeptide is a polypeptide derived from a protein other than a protein comprising or consisting of the amino acid sequence set forth in any one of SEQ ID NOS: 129-240. For example, a non-limiting example of a heterologous polypeptide, as described herein, is a human Ig Fc region.

[0117] As used, herein the term “heterologous signal peptide” refers to a signal peptide that is not operably connected to a subject protein in nature. For example, in reference to a polypeptide comprising a signal peptide from human IL-2 operably connected to human IL- 12, the human IL- 2 signal peptide would constitute a heterologous signal peptide. The terms “signal peptide” and “signal sequence” are used interchangeably herein.

[0118] The terms “hinge” or “hinge region” are used interchangeably herein and refer to the hinge region of an immunoglobulin heavy chain. The amino acid sequence of an exemplary reference hlgGl hinge region is set forth in SEQ ID NO: 10; and the amino acid sequence of an exemplary reference hIgG4 hinge region is set forth in SEQ ID NO: 23.

[0119] As used herein, the term “homologous signal peptide” refers to a signal peptide that is operably connected to a subject protein in nature. For example, in reference to a polypeptide comprising a signal peptide from human IL-2 operably connected to human IL-2, the human IL-2 signal peptide would constitute a homologous signal peptide.

[0120] As used herein, the term “immunogen” refers to a substance that is capable of inducing an immune response e.g., an adaptive immune response) in a subject e.g., a human subject). An immunogen may have one or more isoforms, sequence variants, or splice variants that have equivalent biological and immunological activity, and are thus also considered for the purposes of this disclosure to be immunogenic equivalents of the immunogen.

[0121] As used herein, the term “immunogenic peptide or protein” refers to a peptide or protein that comprises an immunogen.

[0122] As used herein, the term “in combination with” means that two (or more) different agents or treatments arc administered to a subject as part of a defined treatment regimen for a particular disease or condition. The treatment regimen defines the doses and periodicity of administration of each agent such that the effects of the separate agents on the subject overlap. In some embodiments, the delivery of the two or more agents is simultaneous or concurrent and the agents may be co-formulated. In other embodiments, the two or more agents are not co-formulated and are administered in a sequential manner as part of a prescribed. In some embodiments, administration of two or more agents or treatments in combination is such that the reduction in a symptom, or other parameter related to the condition is greater than what would be observed with one agent or treatment delivered alone or in the absence of the other. The effect of the two treatments can be partially additive, wholly additive, or greater than additive e.g., synergistic). Sequential or substantially simultaneous administration of each therapeutic agent can be effected by any appropriate route including, but not limited to, oral routes, intravenous routes, and intramuscular routes. The therapeutic agents can be administered by the same route or by different routes.

[0123] As used herein, the term “isolated” with reference to a polypeptide, protein, or nucleic acid molecule refers to a polypeptide, protein, or nucleic acid molecule that is substantially free of other cellular components with which it is associated in the natural state.

[0124] As used herein, the term “moiety” is used generically to describe any macro or micro molecule that can be operably connected to a protein described herein. Exemplary moieties include, but are not limited small molecules, polypeptides, nucleic acid molecules (e.g., DNA, RNA), carbohydrates, lipids, synthetic polymers (e.g., polymers of PEG).

[0125] As used herein, the term “modified nucleotide,” “nucleotide modification,” or use of the term “modification” and the like in reference to a nucleotide or nucleic acid sequence refers to a nucleotide comprising a chemical modification, e.g., a modified sugar moiety, a modified nucleobase, and / or a modified internucleoside linkage, or any combination thereof. Exemplary modifications are provided herein, see, e.g., § 5.5.4.2. In certain embodiments of the instant disclosure, inclusion of a deoxynucleotide - which is acknowledged as a naturally occurring form of nucleotide - if present within an RNA molecule is considered to constitute a modified nucleotide.

[0126] As used herein, the term “obtaining a sample” refers to the acquisition of a sample. Theterm includes the direct acquisition from a subject and the indirect acquisition through one or more third parties wherein one of the third parlies directly acquired the sample from the subject.

[0127] As used herein, the term “operably connected” refers to the linkage of two moieties in a functional relationship. For example, a polypeptide is operably connected to another polypeptide when they are linked (either directly or indirectly via a peptide linker) in frame such that both polypeptides are functional (e.g., a fusion protein described herein). Or for example, a transcription regulatory nucleic acid molecule e.g., a promoter, enhancer, or other expression control element is operably linked to a nucleic acid molecule that encodes a protein if it affects the transcription of the nucleic acid molecule that encodes the protein. The term “operably connected” can also refer to the conjugation of a moiety to e.g., a nucleic acid molecule or polypeptide (e.g., the conjugation of a PEG polymer to a protein).

[0128] The determination of “percent identity” between two sequences (e.g., peptide or protein (amino acid sequences) or polynucleotide (nucleic acid sequences)) can be accomplished using a mathematical algorithm. A specific, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin S & Altschul SF (1990) PNAS 87: 2264-2268, modified as in Karlin S & Altschul SF (1993) PNAS 90: 5873-5877, each of which is herein incorporated by reference in its entirety. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul SF et al., (1990) J Mol Biol 215: 403, which is herein incorporated by reference in its entirety. BLAST nucleotide searches can be performed with the NBLAST nucleotide program parameters set, e.g., for score=100, wordlength=12 to obtain nucleotide sequences homologous to a nucleic acid molecule described herein. BLAST protein searches can be performed with the XBLAST program parameters set, e.g., to score 50, wordlength=3 to obtain amino acid sequences homologous to a protein molecule described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul SF et al., (1997) Nuc Acids Res 25: 3389-3402, which is herein incorporated by reference in its entirety. Alternatively, PSI BLAST can be used to perform an iterated search which detects distant relationships between molecules (Id.). When utilizing BLAST, Gapped BLAST, and PSI Blast programs, the default parameters of the respective programs (e.g., of XBLAST and NBLAST) can be used (see, e.g., National Center for Biotechnology Information (NCBI) on the worldwide web, ncbi.nlm.nih.gov). Another specific, non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers andMiller, 1988, CABIOS 4:11-17, which is herein incorporated by reference in its entirety. Such an algorithm is incorporated in the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM 120 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.

[0129] As used herein, the term “pharmaceutical composition” means a composition that is suitable for administration to an animal, e.g., a human subject, and comprises a therapeutic agent and a pharmaceutically acceptable carrier or diluent. A “pharmaceutically acceptable earner or diluent” means a substance intended for use in contact with the tissues of human beings and / or non-human animals, and without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable therapeutic benefit / risk ratio.

[0130] As used herein, the term “plurality” means 2 or more (e.g., 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 9 or more, or 10 or more).

[0131] As used herein, the term “poly(A) sequence,” refers to a sequence of adenosine nucleotides, typically located at the 3'-end of a coding linear RNA, of up to about 1000 adenosine nucleotides. In some embodiments, the poly(A) sequence is essentially homopolymeric, e.g., a poly(A) sequence of e.g., 100 adenosine nucleotides having essentially the length of 100 nucleotides. In other embodiments, the poly(A) sequence may be interrupted by at least one nucleotide different from an adenosine nucleotide, e.g., a poly(A) sequence of e.g., 100 adenosine nucleotides may have a length of more than 100 nucleotides (comprising 100 adenosine nucleotides and in addition said at least one nucleotide - or a stretch of nucleotides - different from an adenosine nucleotide). It has to be understood that “poly(A) sequence” as defined herein typically relates to mRNA - however in the context of the invention, the term likewise relates to corresponding sequences in a DNA molecule (e.g., a “poly(T) sequence”).

[0132] The terms “polynucleotide” and “nucleic acid molecule” are used interchangeably herein and refer to a polymer of DNA or RNA. The nucleic acid molecule can be single-stranded or double-stranded; contain natural, non-natural, or altered nucleotides; and contain a natural, nonnatural, or altered intemucleotide linkage, such as a phosphoroamidate linkage or a phosphorothioate linkage, instead of the phosphodiester found between the nucleotides of anunmodified nucleic acid molecule. Nucleic acid molecules include, but are not limited to, all nucleic acid molecules which arc obtained by any means available in the art, including, without limitation, recombinant means, e.g., the cloning of nucleic acid molecules from a recombinant library or a cell genome, using ordinary cloning technology and polymerase chain reaction, and the like, and by synthetic means. The skilled artisan will appreciate that, except where otherwise noted, nucleic acid sequences set forth in the instant application will recite thymidine (T) in a representative DNA sequence but where the sequence represents RNA (e.g., mRNA), the thymidines (Ts) would be substituted for uracils (Us). Thus, any of the RNA molecules encoded by a DNA identified by a particular sequence identification number may also comprise the corresponding RNA (e.g., mRNA) sequence encoded by the DNA, where each thymidine (T) of the DNA sequence is substituted with uracil (U).

[0133] As used herein, the terms “protein” and “polypeptide” refers to a polymer of at least 2 (e.g., at least 5) amino acids linked by a peptide bond. The term “polypeptide” does not denote a specific length of the polymer chain of amino acids. It is common in the art to refer to shorter polymers of amino acids (e.g., approximately 2-50 amino acids) as peptides; and to refer to longer polymers of amino acids (e.g., approximately over 50 amino acids) as polypeptides. However, the terms “peptide” and “polypeptide” and “protein” are used interchangeably herein. In some embodiments, the protein is folded into its three-dimensional structure. Where linear polypeptides are contemplated herein (i.e., primary structure (amino acid sequence)), it should be understood that proteins folded into their three-dimensional structure are also provided herein. Where proteins are contemplated herein (i.e., folded into their three-dimensional structure) polypeptides in their primary structure (i.e., the amino acid sequence) are also provided herein.

[0134] A “prophylactic” treatment is a treatment administered to a subject who does not exhibit signs of a disease or exhibits only early signs for the purpose of decreasing the risk of developing pathology.

[0135] The terms “RNA” and “polyribonucleotide” are used interchangeably herein and refer to macromolecules that include multiple ribonucleotides that a e polymerized via phosphodiester bonds. Ribonucleotides are nucleotides in which the sugar is ribose. RNA may contain modified nucleotides; and contain natural, non-natural, or altered internucleotide linkages, such as a phosphoroamidate linkage or a phosphorothioate linkage, instead of the phosphodiester linkage found between the nucleotides of an unmodified nucleic acid molecule.

[0136] As used herein, the term “sample” encompass a variety of biological specimens obtained from a subject. Exemplary sample types include, e.g., blood and other liquid samples of biological origin (including, but not limited to, whole-blood, peripheral blood mononuclear cells (PBMCs), serum, plasma, urine, saliva, amniotic fluid, stool, synovial fluid, etc.), nasopharyngeal swabs, solid tissue samples such as biopsies (or cells derived therefrom and the progeny thereof), tissue cultures (or cells derived therefrom and the progeny thereof), and cell cultures (or cells derived therefrom and the progeny thereof). The term also includes samples that have been manipulated in any way after their procurement from a subject, such as by centrifugation, filtration, washing, precipitation, dialysis, chromatography, lysis, treatment with reagents, enriched for certain cell populations, refrigeration, freezing, staining, etc.

[0137] As used herein, the term “translatable RNA” refers to any RNA that encodes at least one polypeptide and can be translated to produce the encoded protein in vitro, in vivo, in situ or ex vivo. A translatable RNA may be an mRNA or a circular RNA encoding a polypeptide.

[0138] The term “(scFv ” as used herein refers to an antibody that comprises a first and a second scFv operably connected (e.g., via a peptide linker). The first and second scFv can specifically bind the same or different antigens. In some embodiments, the first and second scFv are operably connected by a peptide linker.

[0139] The term “scFv-Fc” as used herein refers to an antibody that comprises a scFv operably linked (e.g., via a peptide linker) to an Fc domain or subunit of an Fc domain. In some embodiments, a scFv is operably connected to only a first Fc domain of a first and a second Fc domain pair. In some embodiments, a first scFv is operably connected to a first Fc domain and a second scFv is operably connected to a second Fc domain of a first and second Fc domain pair.

[0140] The term “(scFv)2-Fc” as used herein refers to a (scFv)o operably linked (e.g., via a peptide linker) to an Fc domain or a subunit of an Fc domain. In some embodiments, a (scFv)2 is operably connected to only a first Fc domain of a first and a second Fc domain pair. In some embodiments, a first (scFv)2 is operably connected to a first Fc domain and a second (scFv)2 is operably connected to a second Fc domain of a first and second Fc domain pair.

[0141] As used herein, the term “single domain antibody” or “sdAb” refers to an antibody having a single monomeric variable antibody domain. A sdAb is able to specifically bind to a specific antigen. A VHH (as defined herein) is an example of a sdAb.

[0142] As used herein, the term “signal peptide” or “signal sequence” refers to a sequence(e.g., an amino acid sequence) that can direct the transport or localization of a protein to a certain organelle, cell compartment, or extracellular export. The term encompasses both the signal sequence peptide and the nucleic acid sequence encoding the signal peptide. Thus, references to a signal peptide in the context of a nucleic acid refers to the nucleic acid sequence encoding the signal peptide.

[0143] As used herein, the term “specifically binds” refers to preferential interaction, i.e., significantly higher binding affinity, between a first protein (e.g., a ligand) and a second protein (e.g., the ligand’s cognate receptor) relative to other amino acid sequences. Herein, when a first protein is said to “specifically bind” to a second protein, it is understood that the first protein specifically binds to an epitope of the second protein. The term “epitope” refers to the portion of the second protein that the first protein specifically recognizes. The term specifically binds includes molecules that are cross reactive with the same epitope of a different species. For example, an antibody that specifically binds human CD30L may be cross reactive with CD30L of another species (e.g., cynomolgus, murine, etc.), and still be considered herein to specifically bind human CD30L. A protein can specifically bind more than one different protein.

[0144] As used herein, the term “subject” includes any animal, such as a human or other animal. In some embodiments, the subject is a vertebrate animal (e.g., mammal, bird, fish, reptile, or amphibian). In some embodiments, the subject is a human. In some embodiments, the method subject is a non-human mammal. In some embodiments, the subject is a non-human mammal is such as a non-human primate (e.g., monkeys, apes), ungulate (e.g., cattle, buffalo, sheep, goat, pig, camel, llama, alpaca, deer, horses, donkeys), carnivore (e.g., dog, cat), rodent (e.g., rat, mouse), or lagomorph (e.g., rabbit). In some embodiments, the subject is a bird, such as a member of the avian taxa Galliformes (e.g., chickens, turkeys, pheasants, quail), Anseriformes (e.g., ducks, geese), Paleaognathae (e.g., ostriches, emus), Columbiformes (e.g., pigeons, doves), or Psittaciformes (e.g., parrots).

[0145] As used herein, the term “therapeutically effective amount” of a therapeutic agent refers to any amount of the therapeutic agent that, when used alone or in combination with another therapeutic agent, improves a disease condition, e.g., protects a subject against the onset of a disease (or infection); improves a symptom of disease or infection, e.g., decreases severity of disease or infection symptoms, decreases frequency or duration of disease or infection symptoms, increases disease or infection symptom-free periods; prevents or reduces impairment or disabilitydue to the disease or infection; or promotes disease (or infection) regression. The ability of a therapeutic agent to improve a disease condition can be evaluated using a variety of methods known to the skilled practitioner, such as in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by assaying the activity of the agent in in vitro assays.

[0146] As used herein, the terms “treat,” treating,” “treatment,” and the like refer to reducing or ameliorating a disease or infection and / or symptom(s) associated therewith or obtaining a desired pharmacologic and / or physiologic effect. It will be appreciated that, although not precluded, treating a disease or infection does not require that the disease or infection, or symptom(s) associated therewith be completely eliminated. In some embodiments, the effect is therapeutic, i.e., without limitation, the effect partially or completely reduces, diminishes, abrogates, abates, alleviates, decreases the intensity of, or cures a disease and / or adverse symptom attributable to the disease or infection. In some embodiments, the effect is preventative, i.e., the effect protects or prevents an occurrence or reoccurrence of a disease or infection. To this end, the presently disclosed methods comprise administering a therapeutically effective amount of a composition as described herein.

[0147] As used herein, the term “variant” or “variation” with reference to a nucleic acid molecule, refers to a nucleic acid molecule that comprises at least one substitution, alteration, inversion, addition, or deletion of nucleotide compared to a reference nucleic acid molecule. As used herein, the term “valiant” or “variation” with reference to a protein refers to a protein that comprises at least one substitution, alteration, inversion, addition, or deletion of an amino acid residue compared to a reference protein.

[0148] As used herein, the term “variant Ig Fc fusion protein” refers to a fusion protein comprising an immunoreceptor targeting protein described herein and an Ig Fc region, wherein the Ig Fc region comprises one or more variation e.g., one or more amino acid substitution, deletion, or addition)) that decreases or abolishes one or more Fc effector function, relative to a reference Ig Fc fusion protein that does not comprise the one or more variation.

[0149] The terms “VL” and “VL domain” are used interchangeably to refer to the light chain variable region of an antibody.

[0150] The terms “VH” and “VH domain” are used interchangeably to refer to the heavy chain variable region of an antibody.

[0151] The term “VHH” as used herein refers to a type of single domain antibody (sdAb) thathas a single monomeric heavy chain variable antibody domain (VH). Such antibodies can be found in or produced from camclid mammals (e.g., camels, llamas) which arc naturally devoid of light chains or synthetically produced.

[0152] As used herein, the term “5'-untranslated region” or “5'-UTR” refers to a part of a nucleic acid molecule located 5' (i.e., “upstream”) of a coding sequence and which is not translated into protein. Typically, a 5'-UTR stalls with the transcriptional start site and ends before the start codon of the coding sequence. A 5'-UTR may comprise elements for controlling gene expression, also called regulatory elements. Such regulatory elements may be, e.g., ribosomal binding sites, miRNA binding sites etc. The 5'-UTR may be post-transcriptionally modified, e.g., by enzymatic or post-transcriptional addition of a 5'-cap structure.

[0153] As used herein the term “3'-untranslated region” or “3'-UTR” refers to a part of a nucleic acid molecule located 3' (i.e., downstream) of a coding sequence and which is not translated into protein. A 3'-UTR may located between a coding sequence and an (optional) terminal poly(A) sequence of a nucleic acid sequence. A 3'-UTR may comprise elements for controlling gene expression, also called regulatory elements. Such regulatory elements may be, e.g., ribosomal binding sites, miRNA binding sites etc.5.2 Immunoreceptor Targeting Proteins

[0154] The present disclosure provides, inter alia, immunoreceptor targeting proteins (ITPs) (and functional fragments and variants thereof) that e.g., specifically bind to TNF superfamily (TNFSF) ligand CD30L. The TNF superfamily of ligands and receptors regulates multiple cellular functions, including e.g., immune responses, cell proliferation, cell survival, cell differentiation, and programmed cell death. The TNF superfamily comprises 19 ligands and 29 receptors. Exemplary TNF superfamily ligands and their cognate receptors, include, e.g., CD30L and CD30. CD30 is a type 1 transmembrane receptor expressed by, e.g., activated immune cells (e.g., activated T-cells, activated B-cells). CD30 engages in various biological processes through interaction with CD30L (also known as CD 153). CD30L is a type II transmembrane protein belonging to the TNFSF8. CD30L is expressed by, e.g., activated immune cells (e.g., activated T- cells, activated B-cells).

[0155] The amino acid sequence of CD30L is set forth in SEQ ID NOS: 1. The amino acid sequence of the immature and mature form of hCD30 is set forth in SEQ ID NOS: 2 and 3,respectively. See Table 1 , herein.Table 1. The Amino Acid Sequence of human CD30 Ligand CD30 Receptor.

[0156] The present disclosure provides, inter alia, immunoreceptor targeting proteins (and functional fragments and variants thereof) that specifically bind CD30L. The amino acid sequence of the immunoreceptor targeting proteins provided herein is set forth in Table 2. The amino acid sequence of the mature form of the immunoreceptor targeting proteins and polypeptides i.e., lacking the native signal peptide) is set forth in SEQ ID NOS: 129-186. The amino acid sequence of the immature form of the immunoreceptor targeting proteins (z.e., containing the native signalpeptide) is set forth in SEQ ID NOS: 187-240.

[0157] The signal peptides have been computationally predicted using standard methods (see, e.g., Teufel, F., Almagro Armenteros, J. J., Johansen, A.R. et al. SignalP 6.0 predicts all five types of signal peptides using protein language models. Nat Biotechnol (2022). https: / / doi.org / 10.1038 / s41587-021-01156-3, the entire contents of which is incorporated by reference herein for all purposes). A person of ordinary skill in the art would know how to experimentally identify and / or validate a computationally predicted signal peptide using standard methods known in the art, e.g., expression of the immunoreceptor targeting protein from a host cell and sequencing of the intracellular form and the extracellular form of the expressed protein (see, e.g., Zhang Z, Henzel WJ. Signal peptide prediction based on analysis of experimentally verified cleavage sites. Protein Sci. 2004;13(10):2819-2824. doi: 10.1110 / ps.04682504, the entire contents of which is incorporated by reference herein for all purposes).Table 2. The Amino Acid Sequence of Immunoreceptor Targeting Proteins.

[0158] In some embodiments, the amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises an amino acid sequence at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a protein set forth in Table 2. In some embodiments, the amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises an amino acid sequence at least about85% identical to the amino acid sequence of a protein set forth in Table 2. In some embodiments, the amino acid sequence of the protein (e.g., the immunorcccptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises an amino acid sequence at least about 90% identical to the amino acid sequence of a protein set forth in Table 2. In some embodiments, the amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises an amino acid sequence at least about 95% identical to the amino acid sequence of a protein set forth in Table 2. In some embodiments, the amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises an amino acid sequence at least about 99% identical to the amino acid sequence of a protein set forth in Table 2.

[0159] In some embodiments, the amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises an amino acid sequence about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a protein set forth in Table 2. In some embodiments, the amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises an amino acid sequence about 85% identical to the amino acid sequence of a protein set forth in Table 2. In some embodiments, the amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises an amino acid sequence about 90% identical to the amino acid sequence of a protein set forth in Table 2. In some embodiments, the amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises an amino acid sequence about 95% identical to the amino acid sequence of a protein set forth in Table 2. In some embodiments, the amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises an amino acid sequence about 99% identical to the amino acid sequence of a protein set forth in Table 2. In some embodiments, the amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises an amino acid sequence about 100% identical to the amino acid sequence of aprotein set forth in Table 2.

[0160] In some embodiments, the amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) consists of an amino acid sequence at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a protein set forth in Table 2. In some embodiments, the amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) consists of an amino acid sequence at least about 85% identical to the amino acid sequence of a protein set forth in Table 2. In some embodiments, the amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) consists of an amino acid sequence at least about 90% identical to the amino acid sequence of a protein set forth in Table 2. In some embodiments, the amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) consists of an amino acid sequence at least about 95% identical to the amino acid sequence of a protein set forth in Table 2. In some embodiments, the amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) consists of an amino acid sequence at least about 99% identical to the amino acid sequence of a protein set forth in Table 2.

[0161] In some embodiments, the amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) consists of an amino acid sequence about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a protein set forth in Table 2. In some embodiments, the amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) consists of an amino acid sequence about 85% identical to the amino acid sequence of a protein set forth in Table 2. In some embodiments, the amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) consists of an amino acid sequence about 90% identical to the amino acid sequence of a protein set forth in Table 2. In some embodiments, the amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) consists of anamino acid sequence about 95% identical to the amino acid sequence of a protein set forth in Table 2. In some embodiments, the amino acid sequence of the protein (e.g., the immunorcccptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) consists of an amino acid sequence about 99% identical to the amino acid sequence of a protein set forth in Table 2. In some embodiments, the amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) consists of an amino acid sequence about 100% identical to the amino acid sequence of a protein set forth in Table 2.

[0162] In some embodiments, the amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises the amino acid sequence of a protein set forth in Table 2, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises the amino acid sequence of a protein set forth in Table 2, and further comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises the amino acid sequence of a protein set forth in Table 2, and further consists of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises the amino acid sequence of a protein set forth in Table 2, and further comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises the amino acid sequence of a protein set forth in Table 2, and further consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises the amino acid sequence of aprotein set forth in Table 2, and further comprises or no more than about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.).

[0163] In some embodiments, the amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) consists of the amino acid sequence of a protein set forth in Table 2, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) consists of the amino acid sequence of a protein set forth in Table 2, and further comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) consists of the amino acid sequence of a protein set forth in Table 2, and further consists of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) consists of the amino acid sequence of a protein set forth in Table 2, and further comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) consists of the amino acid sequence of a protein set forth in Table 2, and further consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) consists of the amino acid sequence of a protein set forth in Table 2, and further comprises or no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.).

[0164] In some embodiments, the amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acidsequence set forth in any one of SEQ TD NOS: 129-240. For example, the amino acid sequence of the protein (e.g., the immunorcccptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) may comprise an amino acid sequence at least 85% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 129-240. The amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) may comprise an amino acid sequence at least 90% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 129-240. The amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) may comprise an amino acid sequence at least 95% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 129-240. In some embodiments, the amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) may comprise an amino acid sequence 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 129-240.

[0165] In some embodiments, the amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 129-240. For example, the amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) may consist of an amino acid sequence at least 85% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 129-240. The amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) may consist of an amino acid sequence at least 90% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 129-240. The amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) may consist of an amino acid sequence at least 95% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 129-240. In some embodiments, the amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) may consist of an amino acid sequence 100% identical to the amino acidsequence set forth in any one of SEQ ID NOS: 129-240.

[0166] In embodiments, the amino acid sequence of the protein (e.g., the immunorcccptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises the amino acid sequence set forth in any one of SEQ ID NOS: 129-240, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional valiant, or functional fragment / variant thereof) comprises the amino acid sequence set forth in any one of SEQ ID NOS: 129-240, and further comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises the amino acid sequence set forth in any one of SEQ ID NOS: 129-240, and further comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises the amino acid sequence set forth in any one of SEQ ID NOS: 129-240, and further consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises the amino acid sequence set forth in any one of SEQ ID NOS: 129-240, and further comprises or consists of no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.).

[0167] In embodiments, the amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) consists of the amino acid sequence set forth in any one of SEQ ID NOS: 129-240, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) consists of the amino acid sequence set forth in any one of SEQ ID NOS: 129-240, and further comprises at least about 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the protein (e.g., the immunorcccptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) consists of the amino acid sequence set forth in any one of SEQ ID NOS: 129-240, and further comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) consists of the amino acid sequence set forth in any one of SEQ ID NOS: 129-240, and further consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) consists of the amino acid sequence set forth in any one of SEQ ID NOS: 129-240, and further comprises or consists of no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.).

[0168] In some embodiments, the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises a homologous signal peptide operably connected to the protein (e.g., the immunoreceptor targeting protein). In some embodiments, the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises a homologous signal peptide operably connected to the N-terminus of the protein (e.g., the immunoreceptor targeting protein). In some embodiments, the amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises the amino acid sequence set forth in any one of SEQ ID NOS: 129-240 and comprises a homologous signal peptide operably connected to the N- terminus of the protein (e.g., the immunoreceptor targeting protein). In some embodiments, the amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional valiant, or functional fragment / variant thereof) consists of the amino acid sequence set forth in any one of SEQ ID NOS: 129-240 and comprises a homologous signal peptide operably connected to the N-terminus of the protein (e.g., the immunoreceptor targeting protein).

[0169] In some embodiments, the amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functionalfragment / variant thereof) comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 129-186. For example, the amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) may comprise an amino acid sequence at least 85% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 129-186. The amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) may comprise an amino acid sequence at least 90% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 129-186. The amino acid sequence of the protein e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) may comprise an amino acid sequence at least 95% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 129-186. In some embodiments, the amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) may comprise an amino acid sequence 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 129-186.

[0170] In some embodiments, the amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 129-186. For example, the amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) may consist of an amino acid sequence at least 85% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 129-186. The amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) may consist of an amino acid sequence at least 90% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 129-186. The amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) may consist of an amino acid sequence at least 95% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 129-186. In some embodiments, the amino acid sequence of the protein (e.g., theimmunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragmcnt / variant thereof) may consist of an amino acid sequence 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 129-186.

[0171] In embodiments, the amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises the amino acid sequence set forth in any one of SEQ ID NOS: 129-186, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises the amino acid sequence set forth in any one of SEQ ID NOS: 129-186, and further comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises the amino acid sequence set forth in any one of SEQ ID NOS: 129-186, and further comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises the amino acid sequence set forth in any one of SEQ ID NOS: 129-186, and further consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises the amino acid sequence set forth in any one of SEQ ID NOS: 129-186, and further comprises or consists of no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.).

[0172] In embodiments, the amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional valiant, or functional fragment / variant thereof) consists of the amino acid sequence set forth in any one of SEQ ID NOS: 129-186, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment,functional variant, or functional fragment / variant thereof) consists of the amino acid sequence set forth in any one of SEQ ID NOS: 129-186, and further comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) consists of the amino acid sequence set forth in any one of SEQ ID NOS: 129-186, and further comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) consists of the amino acid sequence set forth in any one of SEQ ID NOS: 129-186, and further consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional valiant, or functional fragment / variant thereof) consists of the amino acid sequence set forth in any one of SEQ ID NOS: 129-186, and further comprises or consists of no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.).

[0173] In some embodiments, the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional valiant, or functional fragment / variant thereof) comprises a homologous signal peptide operably connected to the protein (e.g., the immunoreceptor targeting protein). In some embodiments, the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional valiant, or functional fragment / variant thereof) comprises a homologous signal peptide operably connected to the N-terminus of the protein (e.g., the immunoreceptor targeting protein). In some embodiments, the amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional valiant, or functional fragment / variant thereof) comprises the amino acid sequence set forth in any one of SEQ ID NOS: 129-186 and comprises a homologous signal peptide operably connected to the N- terminus of the protein (e.g., the immunoreceptor targeting protein). In some embodiments, the amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) consists of the amino acid sequence set forth in any one of SEQ ID NOS: 129-186 and comprises a homologous signal peptide operably connected to the N-terminus of the protein (e.g., the immunoreceptor targeting protein).

[0174] In some embodiments, the amino acid sequence of the protein (e.g., the immunorcccptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 187-240. For example, the amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) may comprise an amino acid sequence at least 85% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 187-240. The amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) may comprise an amino acid sequence at least 90% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 187-240. The amino acid sequence of the protein e.g., the immunoreceptor targeting protein) (or a functional fragment, functional valiant, or functional fragment / variant thereof) may comprise an amino acid sequence at least 95% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 187-240. In some embodiments, the amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional valiant, or functional fragment / variant thereof) may comprise an amino acid sequence 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 187-240.

[0175] In some embodiments, the amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 187-240. For example, the amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) may consist of an amino acid sequence at least 85% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 187-240. The amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) may consist of an amino acid sequence at least 90% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 187-240. The amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) may consist ofan amino acid sequence at least 95% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 187-240. In some embodiments, the amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) may consist of an amino acid sequence 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 187-240.

[0176] In embodiments, the amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises the amino acid sequence set forth in any one of SEQ ID NOS: 187-240, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises the amino acid sequence set forth in any one of SEQ ID NOS: 187-240, and further comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises the amino acid sequence set forth in any one of SEQ ID NOS: 187-240, and further comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises the amino acid sequence set forth in any one of SEQ ID NOS: 187-240, and further consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises the amino acid sequence set forth in any one of SEQ ID NOS: 187-240, and further comprises or consists of no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.).

[0177] In embodiments, the amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) consists of the amino acid sequence set forth in any one of SEQ ID NOS: 187-240, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), aminoacid variations (e.g., substitutions, additions, deletions, etc.). Tn some embodiments, the amino acid sequence of the protein (e.g., the immunorcccptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) consists of the amino acid sequence set forth in any one of SEQ ID NOS: 187-240, and further comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) consists of the amino acid sequence set forth in any one of SEQ ID NOS: 187-240, and further comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) consists of the amino acid sequence set forth in any one of SEQ ID NOS: 187-240, and further consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) consists of the amino acid sequence set forth in any one of SEQ ID NOS: 187-240, and further comprises or consists of no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.).

[0178] In some embodiments, the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises a homologous signal peptide operably connected to the protein (e.g., the immunoreceptor targeting protein). In some embodiments, the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises a homologous signal peptide operably connected to the N-terminus of the protein (e.g., the immunoreceptor targeting protein). In some embodiments, the amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) comprises the amino acid sequence set forth in any one of SEQ ID NOS: 187-240 and comprises a homologous signal peptide operably connected to the N- terminus of the protein (e.g., the immunoreceptor targeting protein). In some embodiments, the amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) consists of the amino acidsequence set forth in any one of SEQ ID NOS: 187-240 and comprises a homologous signal peptide operably connected to the N-tcrminus of the protein (e.g., the immunorcccptor targeting protein).

[0179] In some embodiments, the amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) no more than 300, 290, 280, 270, 260, 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 115, 110, 100, 95, 90, 80, 70, 60, or 50 amino acids in length. In some embodiments, the amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) is less than 120, 115, 110, 100, 95, 90, 80, 70, 60, or 50 amino acids in length. In some embodiments, the amino acid sequence of the protein (e.g., the immunoreceptor targeting protein) (or a functional fragment, functional variant, or functional fragment / variant thereof) is from about 50-300, 50-250, 50-200, 50-150, 50-120, 50-110, 50-100, 50-90, 50-60, 60-120, 60-110, 60-100, 60-90, 60-80, 60- 70, 70-120, 70-110, 70-100, 70-90, 70-80, 80-120, 80-110, 80-100, 80-90, 90-120, 90-110, 90- 100, 100-120, or 100-110.5.3 Exemplary Properties of Immunoreceptor Targeting Proteins

[0180] In some embodiments, the immunoreceptor targeting proteins described herein are immunosuppressive (e.g., when administered to a subject). In some embodiments, the immunoreceptor targeting proteins described herein are anti-inflammatory (e.g., when administered to a subject). In some embodiments, the immunoreceptor targeting proteins described herein suppress pro-inflammatory response (e.g., when administered to a subject).

[0181] In some embodiments, the immunoreceptor targeting proteins described herein bind a subset (e.g., one or more) TNFSF ligand. In some embodiments, the immunoreceptor targeting protein specifically binds CD30E. In some embodiments, the immunoreceptor targeting protein specifically binds hCD30E.

[0182] In some embodiments, the immunoreceptor targeting protein specifically binds CD30E expressed on the surface of an immune cell. In some embodiments, the immunoreceptor targeting protein specifically binds CD30E expressed on the surface of an activated immune cell. In some embodiments, the immunoreceptor targeting protein specifically binds hCD30L expressed on the surface of an activated immune cell.

[0183] In some embodiments, the immunoreceptor targeting protein can act as a decoyreceptor for a TNFSF ligand described herein (e.g., CD30L). In some embodiments, the immunorcccptor targeting protein inhibits or reduces (e.g., prevents) binding of CD30L to CD30. In some embodiments, the immunoreceptor targeting protein specifically binds to CD30L and inhibits binding of CD30L to CD30. In some embodiments, the immunoreceptor targeting protein inhibits or reduces (e.g., prevents) binding of hCD30L to hCD30. In some embodiments, the immunoreceptor targeting protein specifically binds to hCD30 and inhibits or reduces (e.g., prevents) binding of hCD30 to hCD30L.

[0184] Binding affinity can be measured by standard assays known in the art. For example, binding affinity can be measured by surface plasmon resonance (SPR) (e.g., BIAcore®-based assay), a common method known in the art (see, e.g., Wilson, Science 295:2103, 2002; Wolff et al., Cancer Res. 55:2560, 1993; and U.S. Patent Nos. 5,283,173, 5,468,614, the full contents of each of which are incorporated by reference herein for all purposes). SPR measures changes in the concentration of molecules at a sensor surface as molecules bind to or dissociate from the surface. The change in the SPR signal is directly proportional to the change in mass concentration close to the surface, thereby allowing measurement of binding kinetics between two molecules (e.g., proteins). The dissociation constant for the complex can be determined by monitoring changes in the refractive index with respect to time as buffer is passed over the chip.

[0185] Other suitable assays for measuring the binding of one protein to another (e.g., binding of a protein described herein to a TNFSF ligand) include, for example, immunoassays such as enzyme linked immunosorbent assays (ELISA) and radioimmunoassays (RIA), or determination of binding by monitoring the change in the spectroscopic or optical properties of the proteins through fluorescence, UV absorption, circular dichroism, or nuclear magnetic resonance (NMR). Other exemplary assays include, but are not limited to, Western blot, analytical ultracentrifugation, spectroscopy, flow cytometry, sequencing and other methods for detection of binding of proteins.5.4 Immunoreceptor Targeting Protein Fusions & Conjugates

[0186] In some embodiments, the immunoreceptor targeting protein (e.g., described herein) is operably connected to a heterologous moiety (e.g., a heterologous polypeptide) forming a fusion or conjugate protein, respectively. As such, further provided herein are, inter alia, fusion proteins comprising an immunoreceptor targeting protein (e.g., described herein) and one or more heterologous proteins (or a functional fragment, functional variant, or domain thereof). Furtherprovided herein are, inter alia, conjugates comprising an immunoreceptor targeting protein (e.g., described herein) (or a nucleic acid molecule encoding an immunorcccptor targeting protein (e.g., described herein) and one or more heterologous moieties.

[0187] 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, and synthetic polymers (e.g., polymers of PEG). In some embodiments, the heterologous moiety is a detectable moiety (e.g., protein, e.g., a fluorescent protein). In some embodiments, the heterologous moiety is a therapeutic agent. In some embodiments, the heterologous moiety is an imaging agent. In some embodiments, the heterologous moiety is a radioligand. In some embodiments, the heterologous moiety is a diagnostic agent. In some embodiments, the heterologous moiety is non-effector moiety, e.g., a protein sequence that acts as a ’’handle” or linker but has otherwise no independent biological effect. In some embodiments, the heterologous moiety comprises an antibody, an antibody mimetic, or one or more Ig constant region (Fc region). The heterologous moiety can be any one or more of (any combination of) the foregoing.5.4.1 Radioligands

[0188] In some embodiments, the heterologous moiety comprises a radioisotope. As such, provided herein are radioligands comprising an immunoreceptor targeting protein (e.g., described herein) operably connected (e.g., through a linker) to one more radioisotope. In some embodiments, the radioisotope acts as a therapeutic agent. In some embodiments, the radioisotope acts as an imaging agent. In some embodiments, the immunoreceptor targeting protein (e.g., described herein) acts as a targeting moiety for the radioisotope. In some embodiments, the radioisotope and the immunoreceptor targeting protein (e.g., described herein) are operably connected through a linker.

[0189] Radioisotopes are known in the art. See, e.g., Sgouros, G., Bodei, L., McDevitt, M.R. et al. Radiopharmaceutical therapy in cancer: clinical advances and challenges. Nat Rev Drug Discov 19, 589-608 (2020). https: / / doi.org / 10.1038 / s41573-020-0073-9; and Zhang, Longjiang et al. “Delivery of therapeutic radioisotopes using nanoparticle platforms: potential benefit in systemic radiation therapy.” Nanotechnology, science and applications vol. 3 159-70. 3 Dec. 2010, doi: 10.2147 / NSA.S7462; the entire contents of each of which are incorporated herein by referencefor all purposes.

[0190] Exemplary radioisotopes include, but arc not limited to, Lutetium- 177, Radium-223, Iodine-131, Iodine-125, Fluorine-18, Ir-192, Xenon-133, Yttrium-90, Carbon-11, Idium-111, Strontium-89, Copper-67, Copper-64, Rhenium-186, Actinium-225, Astatine-211, Bismuth-213, Bismuth-212, Samarium- 153, Holmium-166, Thorium-227, and Lead-212.

[0191] Methods of operably connecting proteins to radionuclides (e.g., through one or more linkers) are known in the art. See, e.g., Gupta, Suprit et al. “Antibody labeling with radioiodine and radiometals.” Methods in molecular- biology (Clifton, N.J.) vol. 1141 (2014): 147-57. doi: 10.1007 / 978- 1-4939-0363-4_9; Marion Chomet, State of the Art in Radiolabeling of Antibodies with Common and Uncommon Radiometals for Preclinical and Clinical Immuno-PET, Bioconjugate Chem. 2021, 32, 7, 1315-1330; Martina Steiner, Dario Neri; Antibody-Radionuclide Conjugates for Cancer Therapy: Historical Considerations and New Trends. Clin Cancer Res 15 October 2011; 17 (20): 6406-6416. https: / / doi.org / 10.1158 / 1078-0432.CCR-l l-0483; the entire contents of each of which are incorporated herein by reference for all purposes.5.4.2 Chimeric Antigen Receptors

[0192] In some embodiments, an immunoreceptor targeting protein described herein is part of a chimeric antigen receptor (CAR). In some embodiments, an immunoreceptor targeting protein described herein is the extracellular antigen-binding domain of a CAR. Standard CAR domains are known in art, including, e.g., transmembrane domains and intracellular signaling domains. See, e.g., W02024056809, W02023240064A1, and WO2023205148A1, WO2023133092A1, the entire contents of each of which is incorporated herein by reference for all purposes.

[0193] Exemplary transmembrane domains include, e.g., the alpha, beta or zeta chain of T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8 (for example, CD8 alpha, CD8 beta), CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. In some embodiments, a transmembrane domain may include at least the transmembrane region(s) of a costimulatory molecule, for example, MHC class I molecule, TNF receptor proteins, Immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), activating NK cell receptors, BTLA, a Toll ligand receptor, 0X40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CDlla / CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7,NKp80 (KLRF1 ), NKp44, NKp30, NKp46, CD 19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDlld, ITGAE, CD 103, ITGAL, CDlla, LFA-1, ITGAM, CDUb, ITGAX, CDUc, ITGB1, CD29, ITGB2, CD 18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, LylO8), SLAM (SLAMF1, CD 150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, LAT, GADS, SLP- 76, PAG / Cbp, CD 19a, and a ligand that specifically binds with CD83. In some instances, the transmembrane domain can be attached to the extracellular region of the CAR, for example, the antigen-binding domain of the CAR, via a hinge, for example, a hinge from a human protein. For example, in some embodiments, the hinge can be a human Ig (immunoglobulin) hinge, for example, an IgG4 hinge, or a CD8a hinge.

[0194] Exemplary intracellular' signaling domains include, e.g., the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivative or variant of these sequences and any recombinant sequence that has the same functional capability. In some embodiments, the intracellular signaling domain comprises a primary signaling domain and one or more costimulatory signaling domain. Exemplary primary signaling domains, include, e.g., intracellular signaling domains of TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278 (also known as “ICOS”), FccRI, DAP10, DAP12, CD32, and CD66d. Exemplary of proteins with costimulatory domains suitable for use in CAR described herein include, e.g., MHC class I molecule, TNF receptor proteins, Immunoglobulin- like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), activating NK cell receptors, BTLA, a Toll ligand receptor, 0X40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD1 la / CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD 19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD lid, ITGAE, CD 103, ITGAL, CDlla, LFA-1, ITGAM, CDUb, ITGAX, CDUc, ITGB1, CD29, ITGB2, CD 18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229),CD 160 (BY55), PSGL1 , CD 100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, LAT, GADS, SLP- 76, PAG / Cbp, CD 19a, and a ligand that specifically binds with CD83, and the like.5.4.3 Signal Peptides

[0195] In some embodiments, the heterologous polypeptide is a heterologous signal peptide. Heterologous signal peptides are known in the art. In some embodiments, the immunoreceptor targeting protein comprises a heterologous signal peptide operably connected to the immunoreceptor targeting protein. In some embodiments, the immunoreceptor targeting protein comprises a heterologous signal peptide operably connected to the N-terminus of the immunoreceptor targeting protein. In some embodiments, the amino acid sequence of the immunoreceptor targeting protein comprises the amino acid sequence set forth in any one of SEQ ID NOS: 129-240 and comprises a heterologous signal peptide operably connected to the N- tcrminus of the immunorcccptor targeting protein. In some embodiments, the amino acid sequence of the immunoreceptor targeting protein consists of the amino acid sequence set forth in any one of SEQ ID NOS: 129-240 and comprises a heterologous signal peptide operably connected to the N-terminus of the immunoreceptor targeting protein. In some embodiments, the amino acid sequence of the immunoreceptor targeting protein comprises the amino acid sequence set forth in any one of SEQ ID NOS: 129-186 and comprises a heterologous signal peptide operably connected to the N-terminus of the immunoreceptor targeting protein. In some embodiments, the amino acid sequence of the immunoreceptor targeting protein consists of the amino acid sequence set forth in any one of SEQ ID NOS: 129-186 and comprises a heterologous signal peptide operably connected to the N-terminus of the immunoreceptor targeting protein.

[0196] Commonly used heterologous signal peptides are known in the art, for example, the native signal peptide of human interleukin 2 (hIL-2), human oncostatin M (hOSM), human chymotrypsinogen (hCTRBl), human trypsinogen 2 (hTRY2), and human insulin (hINS). A person of ordinary skill can determine the appropriate signal peptide using standard methodology known in the art. The amino acid sequence of exemplary signal peptides is provided in Table 3.Table 3. The Amino Acid Sequence of Exemplary Signal Peptides.[001971 In some embodiments, the amino acid sequence of the signal peptide comprises the amino acid sequence of any one of the signal peptides set forth in Table 3. In some embodiments, the amino acid sequence of the signal peptide comprises the amino acid sequence of any one of the signal peptides set forth in Table 3, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the signal peptide comprises the amino acid sequence of any one of the signal peptides set forth in Table 3, comprising 1, 2, or 3 amino acid variations (e.g., substitutions, deletions, additions). In some embodiments, the amino acid sequence of the signal peptide comprises the amino acid sequence of any one of the signal peptides set forth in Table 3, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid substitutions. In some embodiments, the amino acid sequence of the signal peptide comprises the amino acid sequence of any one of the signal peptides set forth in Table 3, comprising 1, 2, or 3 amino acid substitutions.

[0198] In some embodiments, the amino acid sequence of the signal peptide consists of the amino acid sequence of any one of the signal peptides set forth in Table 3. In some embodiments, the amino acid sequence of the signal peptide consists of the amino acid sequence of any one of the signal peptides set forth in Table 3, and further consists of 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the signal peptide consists of the amino acid sequence of any one of the signal peptides set forth in Table 3, comprising 1, 2, or 3 amino acid variations (e.g., substitutions, deletions, additions). In some embodiments, the amino acid sequence of the signal peptide consists of the amino acid sequence of any one of the signal peptides set forth in Table 3, and further consists of 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid substitutions. In some embodiments, the amino acid sequence of the signal peptide consists of the amino acid sequence of any one of the signal peptides set forth in Table 3, comprising 1, 2, or 3 amino acid substitutions.

[0199] In some embodiments, the amino acid sequence of the signal peptide comprises theamino acid sequence set forth in any one of SEQ ID NOS: 4-8. In some embodiments, the amino acid sequence of the signal peptide comprises the amino acid sequence set forth in any one of SEQ ID NOS: 4-8, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the signal peptide comprises the amino acid sequence set forth in any one of SEQ ID NOS: 4-8, comprising 1, 2, or 3 amino acid variations (e.g., substitutions, deletions, additions). In some embodiments, the amino acid sequence of the signal peptide comprises the amino acid sequence set forth in any one of SEQ ID NOS: 4-8, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid substitutions. In some embodiments, the amino acid sequence of the signal peptide comprises the amino acid sequence set forth in any one of SEQ ID NOS: 4-8, comprising 1, 2, or 3 amino acid substitutions.

[0200] In some embodiments, the amino acid sequence of the signal peptide consists of the amino acid sequence set forth in any one of SEQ ID NOS: 4-8. In some embodiments, the amino acid sequence of the signal peptide consists of the amino acid sequence set forth in any one of SEQ ID NOS: 4-8, and further consists of 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the signal peptide consists of the amino acid sequence set forth in any one of SEQ ID NOS: 4-8, comprising 1, 2, or 3 amino acid variations (e.g., substitutions, deletions, additions). In some embodiments, the amino acid sequence of the signal peptide consists of the amino acid sequence set forth in any one of SEQ ID NOS: 4-8, and further consists of 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid substitutions. In some embodiments, the amino acid sequence of the signal peptide consists of the amino acid sequence set forth in any one of SEQ ID NOS: 4-8, comprising 1, 2, or 3 amino acid substitutions.5.4.4 Half-Life Extension Moieties

[0201] In some embodiments, the heterologous moiety is a half-life extension moiety. Exemplary half-life extension moieties include, but are not limited to, an immunoglobulin (e.g., human Ig (hlg), murine Ig (mlg)), a fragment of an Ig (e.g., hlg, mlg), an Ig (e.g., hlg, mlg) constant region, a fragment of an Ig (e.g., hlg, mlg) constant region, an Ig (e.g., hlg, mlg) Fc regionhuman transferrin, human serum albumin (HSA), an HSA binding protein or peptide, and polyethylene glycol (PEG) (and polymers thereof). In some embodiments, the heterologous polypeptide is a half-life extension polypeptide. Exemplary half-life extension polypeptides include, but are not limited to, an Ig, a fragment of an Ig, one or more Ig heavy chain constant region, a fragment of an Ig constant region, an Ig Fc region, a hlg, a fragment of a hlg, one or more hlg heavy chain constant region, a fragment of a hlg constant region, a hlg Fc region, a mlg, a fragment of a mlg, one or more mlg heavy chain constant region, a fragment of a mlg constant region, a mlg Fc region, human transferrin, human serum albumin (HSA), and an HSA binding protein or peptide. The immunoreceptor targeting protein described herein fused or conjugated to a half-life extending moiety or a half-life extending moiety can be evaluated for their pharmacokinetic properties utilizing standard in vivo methods known in the art.5.4.5 Ig Fusion Proteins5.4.5.1 Antibody Fusion Proteins

[0202] In some embodiments, the heterologous protein comprises an antibody. The antibody can act to further target the immunoreceptor targeting protein e.g., to a specified cell type expressing a specific cell surface protein. Exemplary antibodies include, full-length antibodies, scFv, Fab, single domain antibodies (e.g., VHH), scFv-Fc, Fab-Fc, and single domain antibody- Fc (e.g., VHH-Fc).5.4.5.2 Ig Fusion Proteins

[0203] In some embodiments, the heterologous protein comprises one or more Ig heavy chain constant regions (e.g., a CH2 region, a CH3 region, a hinge region, an Fc region (e.g., in some embodiments, preferably an Fc region). In some embodiments, the Ig is an IgG. In some embodiments, the IgG is IgGl, IgG2, IgG3, or IgG4 (e.g., in some embodiments preferably an IgG4).

[0204] In some embodiments, the heterologous protein comprises an IgG CH2 region and an IgG CH3 region. In some embodiments, the heterologous protein comprises a partial IgG hinge region, IgG CH2 region, and IgG CH3 region. In some embodiments, the heterologous protein comprises an IgG hinge region, IgG CH2 region, and IgG CH3 region. In some embodiments, the heterologous protein comprises an IgGl CH2 region and an IgGl CH3 region. In some embodiments, the heterologous protein comprises a partial IgGl hinge region, IgGl CH2 region,and IgG1 CH3 region. In some embodiments, the heterologous protein comprises an IgG1 hinge region, IgGl CH2 region, and IgGl CH3 region. In some embodiments, the heterologous protein comprises an IgG4 CH2 region and an IgG4 CH3 region. In some embodiments, the heterologous protein comprises a partial IgG4 hinge region, IgG4 CH2 region, and IgG4 CH3 region. In some embodiments, the heterologous protein comprises an IgG4 hinge region, IgG4 CH2 region, and IgG4 CH3 region.

[0205] In some embodiments, the heterologous protein consists of an IgG CH2 region and an IgG CH3 region. In some embodiments, the heterologous protein consists of a partial IgG hinge region, IgG CH2 region, and IgG CH3 region. In some embodiments, the heterologous protein consists of an IgG hinge region, IgG CH2 region, and IgG CH3 region. In some embodiments, the heterologous protein consists of an IgGl CH2 region and an IgGl CH3 region. In some embodiments, the heterologous protein consists of a partial IgGl hinge region, IgGl CH2 region, and IgGl CH3 region. In some embodiments, the heterologous protein consists of an IgGl hinge region, IgGl CH2 region, and IgGl CH3 region. In some embodiments, the heterologous protein consists of an IgG4 CH2 region and an IgG4 CH3 region. In some embodiments, the heterologous protein consists of a partial IgG4 hinge region, IgG4 CH2 region, and IgG4 CH3 region. In some embodiments, the heterologous protein consists of an IgG4 hinge region, IgG4 CH2 region, and IgG4 CH3 region.

[0206] In some embodiments, the heterologous protein comprises an Ig Fc region. In some embodiments, the Ig Fc region comprises at least a portion of a hinge region, a CH2 region, and a CH3 region. In some embodiments, the Ig Fc region comprises a hinge region, a CH2 region, and a CH3 region. In some embodiments, the Ig Fc region comprises at least a portion of an IgG hinge region, an IgG CH2 region, and an IgG CH3 region. In some embodiments, the Ig Fc region comprises an IgG hinge region, an IgG CH2 region, and an IgG CH3 region. In some embodiments, the Ig Fc region comprises at least a portion of an IgGl hinge region, an IgGl CH2 region, and an IgGl CH3 region. In some embodiments, the Ig Fc region comprises an IgGl hinge region, an IgGl CH2 region, and an IgGl CH3 region. In some embodiments, the Ig Fc region comprises at least a portion of an IgG4 hinge region, an IgG4 CH2 region, and an IgG4 CH3 region. In some embodiments, the Ig Fc region comprises an IgG4 hinge region, an IgG4 CH2 region, and an IgG4 CH3 region.

[0207] In some embodiments, the heterologous protein consists of an Ig Fc region. In someembodiments, the Ig Fc region consists of at least a portion of a hinge region, a CH2 region, and a CH3 region. In some embodiments, the Ig Fc region consists of a hinge region, a CH2 region, and a CH3 region. In some embodiments, the Ig Fc region consists of at least a portion of an IgG hinge region, an IgG CH2 region, and an IgG CH3 region. In some embodiments, the Ig Fc region consists of an IgG hinge region, an IgG CH2 region, and an IgG CH3 region. In some embodiments, the Ig Fc region consists of at least a portion of an IgGl hinge region, an IgGl CH2 region, and an IgGl CH3 region. In some embodiments, the Ig Fc region consists of an IgGl hinge region, an IgGl CH2 region, and an IgGl CH3 region. In some embodiments, the Ig Fc region consists of at least a portion of an IgG4 hinge region, an IgG4 CH2 region, and an IgG4 CH3 region. In some embodiments, the Ig Fc region consists of an IgG4 hinge region, an IgG4 CH2 region, and an IgG4 CH3 region.

[0208] In some embodiments, the heterologous protein comprises one or more hlg heavy chain constant regions (e.g., a CH2 region, a CH3 region, a hinge region, an Fc region). In some embodiments, the hlg is a human IgG (hlgG). In some embodiments, the hlgG is hlgGl, IgG2, IgG3, or IgG4. In some embodiments, the hlgG is IgGl or IgG4. In some embodiments, the hlgG is hlgGl. In some embodiments, the hlgG is hIgG4.

[0209] In some embodiments, the heterologous protein comprises a hlgG CH2 region and a hlgG CH3 region. In some embodiments, the heterologous protein comprises a partial hlgG hinge region, hlgG CH2 region, and hlgG CH3 region. In some embodiments, the heterologous protein comprises a hlgG hinge region, hlgG CH2 region, and hlgG CH3 region. In some embodiments, the heterologous protein comprises a hlgGl CH2 region and a hlgGl CH3 region. In some embodiments, the heterologous protein comprises a partial hlgGl hinge region, hlgGl CH2 region, and hlgGl CH3 region. In some embodiments, the heterologous protein comprises a hlgGl hinge region, hlgGl CH2 region, and hlgGl CH3 region. In some embodiments, the heterologous protein comprises a hIgG4 CH2 region and a hIgG4 CH3 region. In some embodiments, the heterologous protein comprises a partial hIgG4 hinge region, hIgG4 CH2 region, and hIgG4 CH3 region. In some embodiments, the heterologous protein comprises a hIgG4 hinge region, hIgG4 CH2 region, and hIgG4 CH3 region.

[0210] In some embodiments, the heterologous protein consists of a hlgG CH2 region and a hlgG CH3 region. In some embodiments, the heterologous protein consists of a partial hlgG hinge region, hlgG CH2 region, and hlgG CH3 region. In some embodiments, the heterologous proteinconsists of a hlgG hinge region, hTgG CH2 region, and hlgG CH3 region. In some embodiments, the heterologous protein consists of a hlgGl CH2 region and a hlgGl CH3 region. In some embodiments, the heterologous protein consists of a partial hlgGl hinge region, hlgGl CH2 region, and hlgGl CH3 region. In some embodiments, the heterologous protein consists of a hlgGl hinge region, hlgGl CH2 region, and hlgGl CH3 region. In some embodiments, the heterologous protein consists of a h!gG4 CH2 region and a h!gG4 CH3 region. In some embodiments, the heterologous protein consists of a partial h!gG4 hinge region, h!gG4 CH2 region, and h!gG4 CH3 region. In some embodiments, the heterologous protein consists of a h!gG4 hinge region, h!gG4 CH2 region, and h!gG4 CH3 region.

[0211] In some embodiments, the heterologous protein comprises a hlg Fc region. In some embodiments, the hlg Fc region comprises at least a portion of a hinge region, a CH2 region, and a CH3 region. In some embodiments, the hlg Fc region comprises a hinge region, a CH2 region, and a CH3 region. In some embodiments, the hlg Fc region comprises at least a portion of a hlgG hinge region, a hlgG CH2 region, and a hlgG CH3 region. In some embodiments, the hlg Fc region comprises a hlgG hinge region, a hlgG CH2 region, and a hlgG CH3 region. In some embodiments, the hlg Fc region comprises at least a portion of a hlgGl hinge region, a hlgGl CH2 region, and a hlgGl CH3 region. In some embodiments, the hlg Fc region comprises a hlgGl hinge region, a hlgGl CH2 region, and a hlgGl CH3 region. In some embodiments, the hlg Fc region comprises at least a portion of a h!gG4 hinge region, a h!gG4 CH2 region, and a h!gG4 CH3 region. In some embodiments, the hlg Fc region comprises a h!gG4 hinge region, a h!gG4 CH2 region, and a h!gG4 CH3 region.

[0212] In some embodiments, the heterologous protein consists of a hlg Fc region. In some embodiments, the hlg Fc region consists of at least a portion of a hinge region, a CH2 region, and a CH3 region. In some embodiments, the hlg Fc region consists of a hinge region, a CH2 region, and a CH3 region. In some embodiments, the hlg Fc region consists of at least a portion of a hlgG hinge region, a hlgG CH2 region, and a hlgG CH3 region. In some embodiments, the hlg Fc region consists of a hlgG hinge region, a hlgG CH2 region, and a hlgG CH3 region. In some embodiments, the hlg Fc region consists of at least a portion of a hlgGl hinge region, a hlgGl CH2 region, and a hlgGl CH3 region. In some embodiments, the hlg Fc region consists of a hlgGl hinge region, a hlgGl CH2 region, and a hlgGl CH3 region. In some embodiments, the hlg Fc region consists of at least a portion of a hIgG4 hinge region, a hIgG4 CH2 region, and a hIgG4CH3 region. In some embodiments, the hlg Fc region consists of a hIgG4 hinge region, a h!gG4 CH2 region, and a hIgG4 CH3 region.

[0213] The amino acid sequence of exemplary reference hlgGl and hIgG4 heavy chain constant regions and hlg light chain constant regions, which can be incorporated in one or more of the embodiments described herein (e.g., fusion proteins and polypeptide), is provided in Table 4.Table 4. The Amino Acid Sequence of Exemplary hlg heavy chain constant region components and hlg light chain constant regions.

[0214] In some embodiments, the amino acid sequence of the heterologous protein comprises an amino acid sequence set forth in Table 4. In some embodiments, the amino acid sequence of the heterologous protein comprises an amino acid sequence set forth in Table 4, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the heterologous protein comprises an amino acid sequence set forth in Table 4, comprising at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the heterologous protein comprises an amino acid sequence set forth in Table 4, comprising about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the heterologous protein comprises an amino acid sequence set forth in Table 4, comprising no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid variations (e.g., amino acid substitutions, deletions, or additions).

[0215] In some embodiments, the amino acid sequence of the heterologous protein consists of an amino acid sequence set forth in Table 4. In some embodiments, the amino acid sequence of the heterologous protein consists of an amino acid sequence set forth in Table 4, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acidvariations (e.g., amino acid substitutions, deletions, or additions). Tn some embodiments, the amino acid sequence of the heterologous protein consists of an amino acid sequence set forth in Table 4, comprising at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the heterologous protein consists of an amino acid sequence set forth in Table 4, comprising about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the heterologous protein consists of an amino acid sequence set forth in Table 4, comprising no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid variations (e.g., amino acid substitutions, deletions, or additions).

[0216] In some embodiments, the amino acid sequence of the heterologous protein comprises the amino acid sequence set forth in any one of SEQ ID NOS: 9-39. In some embodiments, the amino acid sequence of the heterologous protein comprises the amino acid sequence set forth in any one of SEQ ID NOS: 9-39, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the heterologous protein comprises the amino acid sequence set forth in any one of SEQ ID NOS: 9-39, comprising at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the heterologous protein comprises the amino acid sequence set forth in any one of SEQ ID NOS: 9-39, comprising about 1, 2, 3, 4,5, 6, 7, 8, 9, or 10 amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the heterologous protein comprises the amino acid sequence set forth in any one of SEQ ID NOS: 9-39, comprising no more than about 1, 2, 3, 4, 5,6, 7, 8, 9, or 10 amino acid variations (e.g., amino acid substitutions, deletions, or additions).

[0217] In some embodiments, the amino acid sequence of the heterologous protein consists of the amino acid sequence set forth in any one of SEQ ID NOS: 9-39. In some embodiments, the amino acid sequence of the heterologous protein consists of the amino acid sequence set forth in any one of SEQ ID NOS: 9-39, and further comprising 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the heterologous protein consists of the amino acid sequence set forth in any one of SEQ ID NOS: 9-39, comprising at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid variations (e.g., amino acid substitutions, deletions, oradditions). In some embodiments, the amino acid sequence of the heterologous protein consists of the amino acid sequence set forth in any one of SEQ ID NOS: 9-39, comprising about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the heterologous protein consists of the amino acid sequence set forth in any one of SEQ ID NOS: 90-12, comprising no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., amino acid substitutions, deletions, or additions).

[0218] In some embodiments, wherein the heterologous protein comprises a CH3 region (e.g., comprises an Fc region; a hinge region, CH2 region, and CH3 region, etc.), the CH3 region lacks the C-terminal lysine (e.g., residue 232 of SEQ ID NO: 18, numbering according to SEQ ID NO: 18; or e.g., residue 229 of SEQ ID NO: 32, numbering according to SEQ ID NO: 32). In some embodiments, the CH3 region further lacks the C-terminal glycine (e.g., residue 231 of SEQ ID NO: 18, numbering according to SEQ ID NO: 18; or e.g., residue 228 of SEQ ID NO: 32, numbering according to SEQ ID NO: 32).

[0219] In some embodiments, the heterologous protein comprises one or more mlg heavy chain constant regions (e.g., a CH2 region, a CH3 region, a hinge region, an Fc region). In some embodiments, the mlg is mlgG (mlgG). In some embodiments, the mlgG is mlgGl, mIgG2a, mIgG2c, mIgG2b, or mIgG3. In some embodiments, the mlgG is mlgGl or mIgG2a. In some embodiments, the mlgG is mlgGl. In some embodiments, the mlgG is mIgG2a.

[0220] In some embodiments, the heterologous protein comprises a mlgG CH2 region and a mlgG CH3 region. In some embodiments, the heterologous protein comprises a partial mlgG hinge region, mlgG CH2 region, and mlgG CH3 region. In some embodiments, the heterologous protein comprises a mlgG hinge region, mlgG CH2 region, and mlgG CH3 region. In some embodiments, the heterologous protein comprises a mlgGl CH2 region and a mlgGl CH3 region. In some embodiments, the heterologous protein comprises a partial mlgGl hinge region, mlgGl CH2 region, and mlgGl CH3 region. In some embodiments, the heterologous protein comprises a mlgGl hinge region, mlgGl CH2 region, and mlgGl CH3 region. In some embodiments, the heterologous protein comprises a mIgG2a CH2 region and a m!gG2a CH3 region. In some embodiments, the heterologous protein comprises a partial mIgG2a hinge region, mlg2a CH2 region, and mIgG2a CH3 region. In some embodiments, the heterologous protein comprises a m!gG2a hinge region, mIgG2a CH2 region, and mIgG2a CH3 region.

[0221] In some embodiments, the heterologous protein comprises a mlg Fc region. In someembodiments, the mlg Fc region comprises at least a portion of a hinge region, a CH2 region, and a CH3 region. In some embodiments, the mlg Fc region comprises a hinge region, a CH2 region, and a CH3 region. In some embodiments, the mlg Fc region comprises at least a portion of a mlgG hinge region, a mlgG CH2 region, and a mlgG CH3 region. In some embodiments, the mlg Fc region comprises a mlgG hinge region, a mlgG CH2 region, and a mlgG CH3 region. In some embodiments, the mlg Fc region comprises at least a portion of a mlgGl hinge region, a mlgGl CH2 region, and a mlgGl CH3 region. In some embodiments, the mlg Fc region comprises a mlgGl hinge region, a mlgGl CH2 region, and a mlgGl CH3 region. In some embodiments, the mlg Fc region comprises at least a portion of a mIgG2a hinge region, a mIgG2a CH2 region, and a mIgG2a CH3 region. In some embodiments, the mlg Fc region comprises a mIgG2a hinge region, a mIgG2a CH2 region, and a mIgG2a CH3 region.

[0222] In some embodiments, the heterologous protein consists of a mlgG CH2 region and a mlgG CH3 region. In some embodiments, the heterologous protein consists of a partial mlgG hinge region, mlgG CH2 region, and mlgG CH3 region. In some embodiments, the heterologous protein consists of a mlgG hinge region, mlgG CH2 region, and mlgG CH3 region. In some embodiments, the heterologous protein consists of a mlgGl CH2 region and a mlgGl CH3 region. In some embodiments, the heterologous protein consists of a partial mlgGl hinge region, mlgGl CH2 region, and mlgGl CH3 region. In some embodiments, the heterologous protein consists of a mlgGl hinge region, mlgGl CH2 region, and mlgGl CH3 region. In some embodiments, the heterologous protein consists of a mIgG2a CH2 region and a mIgG2a CH3 region. In some embodiments, the heterologous protein consists of a partial mIgG2a hinge region, mlg2a CH2 region, and mIgG2a CH3 region. In some embodiments, the heterologous protein consists of a mIgG2a hinge region, mIgG2a CH2 region, and mIgG2a CH3 region.

[0223] In some embodiments, the heterologous protein consists of a mlg Fc region. In some embodiments, the mlg Fc region consists of at least a portion of a hinge region, a CH2 region, and a CH3 region. In some embodiments, the mlg Fc region consists of a hinge region, a CH2 region, and a CH3 region. In some embodiments, the mlg Fc region consists of at least a portion of a mlgG hinge region, a mlgG CH2 region, and a mlgG CH3 region. In some embodiments, the mlg Fc region consists of a mlgG hinge region, a mlgG CH2 region, and a mlgG CH3 region. In some embodiments, the mlg Fc region consists of at least a portion of a mlgGl hinge region, a mlgGl CH2 region, and a mlgGl CH3 region. In some embodiments, the mlg Fc region consists of amlgGl hinge region, a mlgGl CH2 region, and a mTgGl CH3 region. In some embodiments, the mlg Fc region consists of at least a portion of a mIgG2a hinge region, a mIgG2a CH2 region, and a mIgG2a CH3 region. In some embodiments, the mlg Fc region consists of a mIgG2a hinge region, a mIgG2a CH2 region, and a mIgG2a CH3 region.

[0224] The amino acid sequence of exemplary reference mlgGl and mIgG2a heavy chain constant regions, which can be incorporated in one or more of the embodiments described herein (e.g., fusion proteins and polypeptide), is provided in Table 5.Table 5. The Amino Acid Sequence of Exemplary mlg heavy chain constant region components.

[0225] In some embodiments, the amino acid sequence of the heterologous protein comprises an amino acid sequence set forth in Table 5. In some embodiments, the amino acid sequence of the heterologous protein comprises an amino acid sequence set forth in Table 5, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the heterologous protein comprises an amino acid sequence set forth in Table 5, comprising at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the heterologous protein comprises an amino acid sequence set forth in Table 5, comprising about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the heterologous protein comprises an amino acid sequence set forth in Table 5, comprising no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid variations (e.g., amino acid substitutions, deletions, or additions).

[0226] In some embodiments, the amino acid sequence of the heterologous protein consists of an amino acid sequence set forth in Table 5. In some embodiments, the amino acid sequence of the heterologous protein consists of an amino acid sequence set forth in Table 5, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the heterologous protein consists of an amino acid sequence set forth in Table 5, comprising at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the heterologous protein consists of an amino acid sequence set forth in Table 5, comprising about 1,2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the heterologous protein consists of an amino acid sequence set forth in Table 5, comprising no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid variations (e.g., amino acid substitutions, deletions, or additions).

[0227] In some embodiments, the amino acid sequence of the heterologous protein comprises the amino acid sequence set forth in any one of SEQ ID NOS: 40-60. In some embodiments, the amino acid sequence of the heterologous protein comprises the amino acid sequence set forth in any one of SEQ ID NOS: 40-60, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the heterologous protein comprises the amino acid sequence set forth in any one of SEQ ID NOS: 40-60, comprising at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the heterologous protein comprises the amino acid sequence set forth in any one of SEQ ID NOS: 40-60, comprising about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the heterologous protein comprises the amino acid sequence set forth in any one of SEQ ID NOS: 40-60, comprising no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., amino acid substitutions, deletions, or additions).

[0228] In some embodiments, the amino acid sequence of the heterologous protein consists of the amino acid sequence set forth in any one of SEQ ID NOS: 40-60. In some embodiments, the amino acid sequence of the heterologous protein consists of the amino acid sequence set forth in any one of SEQ ID NOS: 40-60, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the heterologous protein consists of the amino acid sequence set forth in any one of SEQ ID NOS: 40-60, comprising at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the heterologous protein consists of the amino acid sequence set forth in any one of SEQ ID NOS: 40-60, comprising about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the heterologous protein consists of the aminoacid sequence set forth in any one of SEQ ID NOS: 40-60, comprising no more than about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., amino acid substitutions, deletions, or additions).

[0229] In some embodiments, wherein the heterologous protein comprises a CH3 region (e.g., comprises an Fc region; a hinge region, CH2 region, and CH3 region, etc.), the CH3 region lacks the C-terminal lysine (e.g., residue 227 of SEQ ID NO: 48, numbering according to SEQ ID NO: 48; or e.g., residue 223 of SEQ ID NO: 55, numbering according to SEQ ID NO: 55). In some embodiments, the CH3 region further lacks the C-terminal glycine (e.g., residue 226 of SEQ ID NO: 48, numbering according to SEQ ID NO: 48; or e.g., residue 222 of SEQ ID NO: 55, numbering according to SEQ ID NO: 55).5.4.53 Ig Effector Function

[0230] In some embodiments, the Ig (e.g., hlg, mlg) Fc region of a fusion protein described herein exhibits a decrease in one or more Fc effector function relative to a reference (e.g., wild type) Ig (e.g., hlg, mlg) Fc region. Exemplary Ig (e.g., hlg, mlg) Fc effector functions include, but are not limited to, antibody dependent cellular cytotoxicity (ADCC), antibody dependent cellular phagocytosis (ADCP), complement dependent cytotoxicity (CDC), and binding affinity to one or more human Fc receptor (e.g., an Fey receptor (e.g., FcyRI, FcyRIIa, FcyRIIb, FcyRIIc, FcyRIIIa, and / or FcyRIIIb)).

[0231] Standard in vitro and / or in vivo assays known in the art can be conducted to evaluate Fc effector function, including, any one or more of ADCC, CDC, ADCP, Fc receptor (e.g., Fey receptor) binding affinity, and Clq binding affinity.

[0232] For example, ADCC activity can be assessed utilizing standard (radioactive and nonradioactive) methods known in the art (see, e.g., W02006 / 082515, W02012 / 130831), the entire contents of each of which is incorporated by reference herein for all purposes). For example, ADCC activity can be assessed using a chromium-5 (51Cr) assay. Briefly,51Cr is pre-loaded into target cells expressing CD20, NK cells are added to the culture, and radioactivity in the cell culture supernatant is assessed (indicative of lysis of the target cells by the NK cells). Similar nonradioactive assays can also be utilized that employ a similar method, but the target cells are pre- loaded with fluorescent dyes, such as calcein-AM, CFSE, BCECF, or lanthanide flurophore (Europium). See, e.g., Parekh, Bhavin S et al. “Development and validation of an antibodydependent cell-mediated cytotoxicity-reporter gene assay.” mAbs vol. 4,3 (2012): 310-8. Doi: 10.4161 / mabs.19873, the entire contents of which is incorporated by reference herein for allpurposes. Exemplary commercially available non-radioactive assays include, for example, ACTI™ non-radioactivc cytotoxicity assay for flow cytometry (Cell Technology, Inc. Mountain View, Calif.; and CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, Wis.). Additional non-limiting examples of in vitro assays that can be used to assess ADCC activity of a fusion protein described herein include those described in US5500362; US5821337; Hellstrom, I., et al., Proc. Nat’l Acad. Sci. USA 83 (1986) 7059-7063; Hellstrom, I., et al., Proc. Nat’l Acad. Sci. USA 82 (1985) 1499-1502; and Bruggemann, M., et al., J. Exp. Med. 166 (1987) 1351-1361, the entire contents of each of which is incorporated by reference herein. Alternatively, or additionally, ADCC activity of a fusion protein described herein may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes, et al., Proc. Nat’l Acad. Sci. USA 95 (1998) 652-656, the entire contents of which is incorporated by reference herein for all purposes.

[0233] C Iq binding assays can be utilized to assess the ability of a hlg fusion protein described herein to bind Clq (or bind with less affinity than a reference fusion protein) and hence lack (or have decreased) CDC activity. The binding of a hlg fusion protein described herein to Clq can be determined by a variety of in vitro assays (e.g., biochemical or immunological based assays) known in the art for determining Fc-Clq interactions, including e.g., equilibrium methods (e.g., enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA)), or kinetic methods (e.g., surface plasmon resonance (SPR) analysis), and other methods such as indirect binding assays, competitive inhibition assays, fluorescence resonance energy transfer (FRET), gel electrophoresis, and chromatography (e.g., gel filtration). These and other methods may utilize a label on one or more of the components being examined and / or employ a variety of detection methods including but not limited to chromogenic, fluorescent, luminescent, or isotopic labels. A detailed description of binding affinities and kinetics can be found in e.g., Paul, W. E., ed., Fundamental Immunology, 4thEd., Lippincott-Raven, Philadelphia (1999), the entire contents of which is incorporated by reference herein. For example, see, e.g., Clq and C3c binding ELISAs described in W02006 / 029879 and W02005 / 100402, the entire contents of each of which is incorporated by reference herein for all purposes. Additional CDC activity assays include those described in e.g., Gazzano-Santoro, et al., J. Immunol. Methods 202 (1996) 163; Cragg, M. S., et al., Blood 101 (2003) 1045-1052; and Cragg, M. S., and Glennie, M. J., Blood 103 (2004) 2738- 2743), the entire contents of each of which is incorporated by reference herein for all purposes.

[0234] ADCP activity can be measured by in vitro or in vivo methods known in the art andalso commercially available assays (see, e.g., van de Donk NW, Moreau P, Plesner T, et al. “Clinical efficacy and management of monoclonal antibodies targeting CD38 and SLAMF7 in multiple myeloma,” Blood, 127(6):681-695 (2016), the entire contents of each of which is incorporated by reference herein for all purposes). For example, a primary cell based ADCP assay can be used in which fresh human peripheral blood mononuclear' cells (PBMCs) are isolated, monocytes isolated and differentiated in culture to macrophages using standard procedures. The macrophages are fluorescently labeled added to cultures containing fluorescently labeled target cells expressing CD20 and a fusion protein described herein. Phagocytosis events can be analyzed using FACS screening and / or microscopy. A modified reporter version of the above described assay can also be used that employs an engineered cell line that stably expresses FcyRIIa (CD32a) as the effector cell line (e.g., an engineered T cell line, e.g., THP-1), removing the requirement for primary cells. Exemplary ADCP assays are described in e.g., Ackerman, M. E. et al. A robust, high-throughput assay to determine the phagocytic activity of clinical antibody samples. J. Immunol. Methods 366, 8-19 (2011); and Mcandrew, E. G. et al. Determining the phagocytic activity of clinical antibody samples. J. Vis. Exp. 3588 (2011). Doi: 10.3791 / 3588; the entire contents of each of which is incorporated by reference herein.

[0235] Binding of a hlg fusion protein described herein to an Ig (e.g., hlg, mlg) Fc receptor can be determined by a variety of in vitro assays (e.g., biochemical or immunological based assays) known in the ail for determining Fc-Fc receptor interactions, i.e., specific binding of an Fc region to an Fc receptor. Common assays include equilibrium methods (e.g., enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA)), or kinetic methods (e.g., surface plasmon resonance (SPR) analysis), and other methods such as indirect binding assays, competitive inhibition assays, fluorescence resonance energy transfer (FRET), gel electrophoresis, and chromatography (e.g., gel filtration). These and other methods may utilize a label on one or more of the components being examined and / or employ a variety of detection methods including but not limited to chromogenic, fluorescent, luminescent, or isotopic labels. A detailed description of binding affinities and kinetics can be found in e.g., Paul, W. E., ed., Fundamental Immunology, 4” Ed., Lippincott-Raven, Philadelphia (1999), the entire contents of which is incorporated by reference herein for all purposes.(i) Reduced Ig Effector Function

[0236] In some embodiments, the Ig Fc region exhibits a decrease in or no detectable activityof one or more Fc effector. As described above, exemplary Ig Fc effector functions include, but arc not limited to, ADCC, ADCP, CDC, binding affinity to Clq, and binding affinity to one or more human Fc receptor (e. ., an Fey receptor (e.g., FcyRI, FcyRIIa, FcyRIIb, FcyRIIc, FcyRIIIa, and / or FcyRIIIb)).

[0237] In some embodiments, the hlg Fc region is modified (e.g., comprises one or more variation (e.g., one or more amino acid substitution, deletion, addition, etc.); altered glycosylation)) (referred to herein as a “modified hlg Fc”). In some embodiments, the modification (e.g., the variation (e.g., one or more amino acid substitution, deletion, addition, etc.); altered glycosylation decreases or abolishes one or more Fc effector function, relative to a reference hlg Fc that does not comprise the modification (e.g., the one or more variation (e.g., the one or more amino acid substitution, deletion, addition, etc.; the altered glycosylation)).

[0238] In some embodiments, the modified Ig (e.g., hlg, mlg) Fc fusion protein exhibits no detectable or decreased ADCC compared to a reference fusion protein that does not comprise the Ig (e.g., hlg, mlg) Fc modification (e.g., the one or more variation (e.g., one or more amino acid substitution, deletion, or addition)). In some embodiments, the modified Ig (e.g., hlg, mlg) Fc fusion protein exhibits no detectable or decreased CDC compared to a reference fusion protein that does not comprise the Ig (e.g., hlg, mlg) Fc modification (e.g., the one or more variation (e.g., one or more amino acid substitution, deletion, or addition)). In some embodiments, the modified Ig (e.g., hlg, mlg) Fc fusion protein exhibits no detectable or decreased ADCP compared to a reference fusion protein that does not comprise the Ig (e.g., hlg, mlg) Fc modification (e.g., the one or more variation (e.g., one or more amino acid substitution, deletion, or addition)).

[0239] In some embodiments, the modified Ig (e.g., hlg, mlg) Fc fusion protein exhibits decreased or no binding affinity to one or more Fc receptor (e.g., human Fc receptor) (e.g., an Fey receptor (e.g., an Fey receptor (e.g., FcyRI, FcyRIIa, FcyRIIb, FcyRIIc, FcyRIIIa, and / or FcyRIIIb)) compared to a reference fusion protein that does not comprise the Ig (e.g., hlg, mlg) Fc modification (e.g., the one or more variation (e.g., one or more amino acid substitution, deletion, or addition)).

[0240] In some embodiments, the modified Ig (e.g., hlg, mlg) Fc fusion protein exhibits decreased or no binding affinity to FcyRI, FcyRIIa, FcyRIIIa, and / or FcyRIIIb compared to a reference fusion protein that does not comprise the hlg Fc modification (e.g., the one or more variation (e.g., one or more amino acid substitution, deletion, or addition)). In some embodiments,the modified Ig (e.g., hlg, mlg) Fc fusion protein exhibits decreased or no binding affinity to FcyRI compared to a reference fusion protein that docs not comprise the Ig (e.g., hlg, mlg) Fc modification (e.g., the one or more variation (e.g., one or more amino acid substitution, deletion, or addition)). In some embodiments, the modified Ig (e.g., hlg, mlg) Fc fusion protein exhibits decreased or no binding affinity to FcyRIIa compared to a reference fusion protein that does not comprise the Ig (e.g., hlg, mlg) Fc modification (e.g., the one or more variation (e.g., one or more amino acid substitution, deletion, or addition)). In some embodiments, the modified Ig (e.g., hlg, mlg) Fc fusion protein exhibits decreased or no binding affinity to FcyRIIIa compared to a reference fusion protein that does not comprise the Ig (e.g., hlg, mlg) Fc modification (e.g., the one or more variation (e.g., one or more amino acid substitution, deletion, or addition)). In some embodiments, the modified Ig (e.g., hlg, mlg) Fc fusion protein exhibits decreased or no binding affinity to FcyRIIIb compared to a reference fusion protein that does not comprise the Ig (e.g., hlg, mlg) Fc modification (e.g., the one or more variation (e.g., one or more amino acid substitution, deletion, or addition)).

[0241] In some embodiments, the modified Ig (e.g., hlg, mlg) Fc fusion protein exhibits increased binding affinity to one or more Fc receptor (e.g., human Fc receptor) (e.g., an Fey receptor (e.g., FcyRIIb)) compared to a reference fusion protein that does not comprise the hlg Fc modification (e.g., the one or more variation (e.g., one or more amino acid substitution, deletion, or addition)). In some embodiments, the modified Ig (e.g., hlg, mlg) Fc fusion protein exhibits increased binding affinity to FcyRIIb compared to a reference fusion protein that does not comprise the Ig (e.g., hlg, mlg) Fc modification (e.g., the one or more variation (e.g., one or more amino acid substitution, deletion, or addition)).

[0242] In some embodiments, the modified Ig (e.g., hlg, mlg) Fc fusion protein exhibits decreased or no binding affinity to Clq compared to a reference fusion protein that does not comprise the Ig (e.g., hlg, mlg) Fc modification (e.g., the one or more variation (e.g., one or more amino acid substitution, deletion, or addition)).

[0243] Amino acid substitutions that decrease or abolish one or more Ig (e.g., hlg, mlg) Fc effector function are known in the art. See for example, Saunders Kevin, “Conceptual Approaches to Modulating Antibody Effector Functions and Circulation Half-Life,” Frontiers in Immunology, vlO (June 7, 2019) DOI=10.3389 / fimmu.2019.01296, the full contents of which is incorporated by reference herein for all purposes, see more particularly for example, e.g., Table 3 of Saunders.

[0244] In some embodiments, the modified Ig Fc fusion protein comprises a hlg Fc region comprising one or more amino acid variation. In some embodiments, the modified hlg Fc fusion protein comprises a hlg4 Fc region comprising one or more amino acid variation. In some embodiments, the hIgG4 Fc region comprises an amino acid substitution at amino acid positions S228, F234, and / or L235, EU numbering according to Kabat. In some embodiments, the hIgG4 Fc region comprises the following amino acid substitutions S228P, F234A, and / or L235A, 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 comprises the following amino acid substitutions S228P and / or L235E, EU numbering according to Kabat.

[0245] In some embodiments, the modified hlg Fc fusion protein comprises a hlgGl Fc region comprising one or more amino acid variations. In some embodiments, the hlgGl Fc region comprises an amino acid substitution at amino acid positions L234, L235, and / or P329, EU numbering according to Kabat. In some embodiments, the hlgGl Fc region comprises the following amino acid substitutions L234A and / or L235A, EU numbering according to Kabat. In some embodiments, the hlgGl Fc region comprises the following amino acid substitutions L234A, L235A, and P329G, EU numbering according to Kabat. In some embodiments, the hlgGl Fc region comprises the following amino acid substitutions L234A, L235A, and P329A, EU numbering according to Kabat.

[0246] The amino acid sequence of exemplary variant hlg Fc regions that are known in the art to exhibit a decrease in one more effector function is provided in Table 6.Table 6. The amino acid sequence of exemplary variant hlg Fc Regions.

[0247] In some embodiments, the modified hlg Fc fusion protein comprises a hlg Fc region comprising an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a polypeptide set forth in Table 6. For example, the modified hlg Fc fusion protein may comprise a hlg Fc region comprising an amino acid sequence at least 85% identical to the amino acid sequence of apolypeptide set forth in Table 6. The modified hlg Fc fusion protein may comprise a hlg Fc region comprising an amino acid sequence at least 90% identical to the amino acid sequence of a polypeptide set forth in Table 6. The modified hlg Fc fusion protein may comprise a hlg Fc region comprising an amino acid sequence at least 95% identical to the amino acid sequence of a polypeptide set forth in Table 6. In some embodiments, the modified hlg Fc fusion protein preferably may comprise a hlg Fc region comprising an amino acid sequence 100% identical to the amino acid sequence of a polypeptide set forth in Table 6.

[0248] In some embodiments, the modified hlg Fc fusion protein comprises a hlg Fc region consisting of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a polypeptide set forth in Table 6. For example, the modified hlg Fc fusion protein may comprise a hlg Fc region consisting of an amino acid sequence at least 85% identical to the amino acid sequence of a polypeptide set forth in Table 6. The modified hlg Fc fusion protein may comprise a hlg Fc region consisting of an amino acid sequence at least 90% identical to the amino acid sequence of a polypeptide set forth in Table 6. The modified hlg Fc fusion protein may comprise a hlg Fc region consisting of an amino acid sequence at least 95% identical to the amino acid sequence of a polypeptide set forth in Table 6. In some embodiments, the modified hlg Fc fusion protein preferably may comprise a hlg Fc region consisting of an amino acid sequence 100% identical to the amino acid sequence of a polypeptide set forth in Table 6.

[0249] In some embodiments, the amino acid sequence of the modified hlg Fc fusion protein comprises a hlg Fc region that comprises the amino acid sequence of a polypeptide set forth in Table 6, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the modified hlg Fc fusion protein comprises a hlg Fc region that comprises the amino acid sequence of a polypeptide set forth in Table 6, and further comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the modified hlg Fc fusion protein comprises a hlg Fc region that comprises the amino acid sequence of a polypeptide set forth in Table 6, and further comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the modified hlg Fc fusion protein comprises a hlg Fc region that comprises the amino acidsequence of a polypeptide set forth in Table 6, and further consists of about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the modified hlg Fc fusion protein comprises a hlg Fc region that comprises the amino acid sequence of a polypeptide set forth in Table 6, and further comprises no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.).

[0250] In some embodiments, the amino acid sequence of the modified hlg Fc fusion protein comprises a hlg Fc region that consists of the amino acid sequence of a polypeptide set forth in Table 6, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the modified hlg Fc fusion protein comprises a hlg Fc region that consists of the amino acid sequence of a polypeptide set forth in Table 6, and further comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the modified hlg Fc fusion protein comprises a hlg Fc region that consists of the amino acid sequence of a polypeptide set forth in Table 6, and further comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the modified hlg Fc fusion protein comprises a hlg Fc region that consists of the amino acid sequence of a polypeptide set forth in Table 6, and further consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the modified hlg Fc fusion protein comprises a hlg Fc region that consists of the amino acid sequence of a polypeptide set forth in Table 6, and further comprises no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.).

[0251] In some embodiments, the amino acid sequence of the modified hlg Fc fusion protein comprises a hlg Fc region that comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 61-74. For example, the amino acid sequence of the modified hlg Fc fusion protein may comprise a hlg Fc region that comprises an amino acid sequence at least 85% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 61-74. The amino acid sequence of the modified hlg Fc fusion protein may comprise a hlg Fcregion that comprises an amino acid sequence at least 90% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 61-74. The amino acid sequence of the modified hlg Fc fusion protein may comprise a hlg Fc region that comprises an amino acid sequence at least 95% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 61-74. In some embodiments, the amino acid sequence of the modified hlg Fc fusion protein preferably may comprise a hlg Fc region that comprises an amino acid sequence 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 61-74.

[0252] In some embodiments, the amino acid sequence of the modified hlg Fc fusion protein comprises a hlg Fc region that consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 61-74. For example, the amino acid sequence of the modified hlg Fc fusion protein may comprise a hlg Fc region that consists of an amino acid sequence at least 85% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 61-74. The amino acid sequence of the modified hlg Fc fusion protein may comprise a hlg Fc region that consists of an amino acid sequence at least 90% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 61-74. The amino acid sequence of the modified hlg Fc fusion protein may comprise a hlg Fc region that consists of an amino acid sequence at least 95% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 61-74. In some embodiments, the amino acid sequence of the modified hlg Fc fusion protein preferably may comprise a hlg Fc region that consists of an amino acid sequence 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 61-74.

[0253] In some embodiments, the amino acid sequence of the modified hlg Fc fusion protein comprises a hlg Fc region that comprises the amino acid sequence set forth in any one of SEQ ID NOS: 61-74, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the modified hlg Fc fusion protein comprises a hlg Fc region that comprises the amino acid sequence set forth in any one of SEQ ID NOS: 61-74, 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 modified hlg Fc fusion protein comprises a hlg Fc region that comprises the amino acid sequence set forth in any one of SEQ ID NOS: 61-74, and further comprises about 1, 2, 3, 4, 5, 6,7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the modified hlg Fc fusion protein comprises a hlg Fc region that comprises the amino acid sequence set forth in any one of SEQ ID NOS: 61-74, and further consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the modified hlg Fc fusion protein comprises a hlg Fc region that comprises the amino acid sequence set forth in any one of SEQ ID NOS: 61-74, and further comprises no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.).

[0254] In some embodiments, the amino acid sequence of the modified hlg Fc fusion protein comprises a hlg Fc region that consists of the amino acid sequence set forth in any one of SEQ ID NOS: 61-74, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the modified hlg Fc fusion protein comprises a hlg Fc region that consists of the amino acid sequence set forth in any one of SEQ ID NOS: 61- 74, 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 modif ied hlg Fc fusion protein comprises a hlg Fc region that consists of the amino acid sequence set forth in any one of SEQ ID NOS: 61-74, and further comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the modified hlg Fc fusion protein comprises a hlg Fc region that consists of the amino acid sequence set forth in any one of SEQ ID NOS: 61-74, and further consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the modified hlg Fc fusion protein comprises a hlg Fc region that consists of the amino acid sequence set forth in any one of SEQ ID NOS: 61-74, and further comprises no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.).

[0255] In some embodiments, the modified mlg Fc fusion protein comprises a mIgG2a Fc region comprising one or more amino acid variations. In some embodiments, the mIgG2a Fc region comprises an amino acid substitution at amino acid positions L234, L235, and / or P329, EU numbering according to Kabat. In some embodiments, the mIgG2a Fc region comprises the following amino acid substitutions L234P and / or L235P, EU numbering according to Kabat. Insome 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.

[0256] In some embodiments, the modified mlg Fc fusion protein comprises a mIgG2a Fc region comprising one or more amino acid variations. In some embodiments, the mIgG2a Fc region comprises an amino acid substitution at amino acid positions L234, L235, and / or P329, EU numbering according to Kabat. In some embodiments, the mIgG2a Fc region comprises the following amino acid substitutions L234A and / or L235A, EU numbering according to Kabat. In some embodiments, the mIgG2a Fc region comprises the following amino acid substitutions L234A, L235A, and P329G, EU numbering according to Kabat. In some embodiments, the mIgG2a Fc region comprises the following amino acid substitutions L234A, L235A, and P329A, EU numbering according to Kabat.

[0257] The amino acid sequence of exemplary variant hlg Fc regions that are known in the art to exhibit a decrease in one more effector function is provided in Table 7.Table 7. The amino acid sequence of exemplary variant mlg Fc Regions.

[0258] In some embodiments, the modified mlg Fc fusion protein comprises a mlg Fc region comprising an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a polypeptide set forth in Table 7. For example, the modified mlg Fc fusion protein may comprise a mlg Fc region comprising an amino acid sequence at least 85% identical to the amino acid sequence of a polypeptide set forth in Table 7. The modified mlg Fc fusion protein may comprise a mlg Fc region comprising an amino acid sequence at least 90% identical to the amino acid sequence of a polypeptide set forth in Table 7. The modified mlg Fc fusion protein may comprise a mlg Fc regioncomprising an amino acid sequence at least 95% identical to the amino acid sequence of a polypeptide set forth in Table 7. In some embodiments, the modified mlg Fc fusion protein preferably may comprise a mlg Fc region comprising an amino acid sequence 100% identical to the amino acid sequence of a polypeptide set forth in Table 7.

[0259] In some embodiments, the amino acid sequence of the modified mlg Fc fusion protein comprises a mlg Fc region that comprises or consists of the amino acid sequence of a polypeptide set forth in Table 7, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the modified mlg Fc fusion protein comprises a mlg Fc region that comprises or consists of the amino acid sequence of a polypeptide set forth in Table 7, and further comprises or consists of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the modified mlg Fc fusion protein comprises a mlg Fc region that comprises or consists of the amino acid sequence of a polypeptide set forth in Table 7, and further comprises or consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the modified mlg Fc fusion protein comprises a mlg Fc region that comprises or consists of the amino acid sequence of a polypeptide set forth in Table 7, and further comprises or consists of no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.).

[0260] In some embodiments, the amino acid sequence of the modified mlg Fc fusion protein comprises a mlg Fc region that comprises or consists of the amino acid sequence of a polypeptide set forth in Table 7, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid substitutions. In some embodiments, the amino acid sequence of the modified mlg Fc fusion protein comprises a mlg Fc region that comprises or consists of the amino acid sequence of a polypeptide set forth in Table 7, and further comprises or consists of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions. In some embodiments, the amino acid sequence of the modified mlg Fc fusion protein comprises a mlg Fc region that comprises or consists of the amino acid sequence of a polypeptide set forth in Table 7, and further comprises or consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions. In some embodiments, the amino acid sequence of the modified mlg Fc fusion protein comprises a mlg Fc region that comprises or consists of the amino acid sequence of a polypeptide set forth in Table 7, and furthercomprises 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 modified mlg Fc fusion protein comprises a mlg Fc region that comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 75-82. For example, the amino acid sequence of the modified mlg Fc fusion protein may comprise a mlg Fc region that comprises an amino acid sequence at least 85% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 75-82. The amino acid sequence of the modified mlg Fc fusion protein may comprise a mlg Fc region that comprises an amino acid sequence at least 90% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 75-82. The amino acid sequence of the modified mlg Fc fusion protein may comprise a mlg Fc region that comprises an amino acid sequence at least 95% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 75-82. In some embodiments, the amino acid sequence of the modified mlg Fc fusion protein preferably may comprise a mlg Fc region that comprises an amino acid sequence 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 75-82. In some embodiments, the amino acid sequence of the modified mlg Fc fusion protein comprises a mlg Fc region that consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 75- 82. For example, the amino acid sequence of the modified mlg Fc fusion protein may comprise a mlg Fc region that consists of an amino acid sequence at least 85% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 75-82. The amino acid sequence of the modified mlg Fc fusion protein may comprise a hlg Fc region that consists of an amino acid sequence at least 90% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 75-82. The amino acid sequence of the modified mlg Fc fusion protein may comprise a mlg Fc region that consists of an amino acid sequence at least 95% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 75-82. In some embodiments, the amino acid sequence of the modified mlg Fc fusion protein preferably may comprise a mlg Fc region that consists of an amino acid sequence 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 75-82.

[0262] In some embodiments, the amino acid sequence of the modified mlg Fc fusion protein comprises a mlg Fc region that comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOS: 75-82, and further comprises 1 or more but less than 15% (less than 12%,less than 10%, less than 8%), amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the modified mlg Fc fusion protein comprises a mlg Fc region that comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOS: 75-82, 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 modified mlg Fc fusion protein comprises a mlg Fc region that comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOS: 75-82, 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 modified mlg Fc fusion protein comprises a mlg Fc region that comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOS: 75-82, and further comprises or consists of no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.).

[0263] In some embodiments, the amino acid sequence of the modified mlg Fc fusion protein comprises a mlg Fc region that comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOS: 75-82, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid substitutions. In some embodiments, the amino acid sequence of the modified mlg Fc fusion protein comprises a mlg Fc region that comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOS: 75-82, 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 modified mlg Fc fusion protein comprises a mlg Fc region that comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOS: 75-82, 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 modified mlg Fc fusion protein comprises a mlg Fc region that comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOS: 75-82, and further comprises or consists of no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions.(ii) Enhanced Ig Effector Function

[0264] In some embodiments, the 1g Fc region exhibits an enhancement (e.g., an increase) in one or more Fc effector function relative to a reference (e.g., wild type) Ig Fc region. Exemplary Ig Fc effector functions include, but are not limited to, ADCC, ADCP, CDC, binding affinity toClq, and binding affinity to one or more human Fc receptor (e.g., an Fey receptor (e.g., (e.g., FcyRI, FcyRIIa, FcyRIIIa, and / or FcyRIIIb). In some embodiments, the Fc region exhibits one or more enhanced Fc effector function, relative to a reference Ig (e.g., hlg, mlg).

[0265] In some embodiments, the hlg Fc region is modified e.g., comprises one or more variation (e.g., one or more amino acid substitution, deletion, addition, etc.); altered glycosylation (e.g., afucosylation))) (referred to herein as a “modified hlg Fc”). In some embodiments, the modification (e.g., the variation (e.g., one or more amino acid substitution, deletion, addition, etc.); altered glycosylation (e.g., afucosylation))) enhances (e.g., increases) one or more Fc effector function, relative to a reference hlg Fc that does not comprise the modification (e.g., the one or more variation (e.g., the one or more amino acid substitution, deletion, addition, etc.; the altered glycosylation (e.g., afucosylation))).

[0266] In some embodiments, the modified Ig (e.g., hlg, mlg) Fc fusion protein exhibits enhanced ADCC compared to a reference fusion protein that does not comprise the Ig (e.g., hlg, mlg) Fc modification (e.g., the one or more variation (e.g., one or more amino acid substitution, deletion, or addition)). In some embodiments, the modified Ig (e.g., hlg, mlg) Fc fusion protein exhibits enhanced CDC compared to a reference fusion protein that does not comprise the Ig (e.g., hlg, mlg) Fc modification (e.g., the one or more variation (e.g., one or more amino acid substitution, deletion, or addition)). In some embodiments, the modified Ig (e.g., hlg, mlg) Fc fusion protein exhibits enhanced ADCP compared to a reference fusion protein that does not comprise the Ig (e.g., hlg, mlg) Fc modification (e.g., the one or more variation (e.g., one or more amino acid substitution, deletion, or addition)).

[0267] In some embodiments, the modified Ig (e.g., hlg, mlg) Fc fusion protein exhibits enhanced binding affinity to one or more Fc receptor (e.g., human Fc receptor) (e.g., an Fey receptor (e.g., FcyRI, FcyRIIa, FcyRIIIa, and / or FcyRIIIb) compared to a reference fusion protein that does not comprise the Ig (e.g., hlg, mlg) Fc modification (e.g., the one or more variation (e.g., one or more amino acid substitution, deletion, or addition)). In some embodiments, the modified Ig (e.g., hlg, mlg) Fc fusion protein exhibits enhanced binding affinity to FcyRI, FcyRIIa, FcyRIIIa, and / or FcyRIIIb compared to a reference fusion protein that does not comprise the hlg Fc modification (e.g., the one or more variation (e.g., one or more amino acid substitution, deletion, or addition)). In some embodiments, the modified Ig (e.g., hlg, mlg) Fc fusion protein exhibits enhanced binding affinity to FcyRI compared to a reference fusion protein that does not comprisethe Ig (e.g., hTg, mTg) Fc modification (e.g., the one or more variation (e.g., one or more amino acid substitution, deletion, or addition)). In some embodiments, the modified Ig (e.g., hlg, mlg) Fc fusion protein exhibits enhanced binding affinity to FcyRIIa compared to a reference fusion protein that does not comprise the Ig e.g., hlg, mlg) Fc modification (e.g., the one or more variation (e.g., one or more amino acid substitution, deletion, or addition)). In some embodiments, the modified Ig (e.g., hlg, mlg) Fc fusion protein exhibits enhanced binding affinity to FcyRIIIa compared to a reference fusion protein that does not comprise the Ig (e.g., hlg, mlg) Fc modification (e.g., the one or more variation (e.g., one or more amino acid substitution, deletion, or addition)). In some embodiments, the modified Ig (e.g., hlg, mlg) Fc fusion protein exhibits enhanced binding affinity to FcyRIIIb compared to a reference fusion protein that does not comprise the Ig (e.g., hlg, mlg) Fc modification (e.g., the one or more variation (e.g., one or more amino acid substitution, deletion, or addition)).

[0268] In some embodiments, the modified Ig (e.g., hlg, mlg) Fc fusion protein exhibits reduced binding affinity to one or more Fc receptor (e.g., human Fc receptor) ((e.g., an Fey receptor e.g., FcyRIIb)) compared to a reference fusion protein that does not comprise the Ig (e.g., hlg, mlg) Fc modification (e.g., the one or more variation (e.g., one or more amino acid substitution, deletion, or addition)). In some embodiments, the modified Ig (e.g., hlg, mlg) Fc fusion protein exhibits reduced binding affinity to FcyRIIb compared to a reference fusion protein that does not comprise the Ig (e.g., hlg, mlg) Fc modification (e.g., the one or more variation (e.g., one or more amino acid substitution, deletion, or addition)).

[0269] In some embodiments, the modified Ig (e.g., hlg, mlg) Fc fusion protein exhibits enhanced binding affinity to Clq compared to a reference fusion protein that does not comprise the Ig (e.g., hlg, mlg) Fc modification (e.g., the one or more variation (e.g., one or more amino acid substitution, deletion, or addition)).

[0270] Amino acid substitutions and glycoengineering that enhance (e.g., increase) one or more hlg Fc effector function are known in the art. See for example, Liu R, Oldham RJ, Teal E, Beers SA, Cragg MS. Fc-Engineering for Modulated Effector Functions-Improving Antibodies for Cancer Treatment. Antibodies (Basel). 2020;9(4):64. Published 2020 Nov 17. doi:10.3390 / antib9040064; van der Horst HJ, Nijhof IS, Mutis T, Chamuleau MED. Fc- Engineered Antibodies with Enhanced Fc-Effector Function for the Treatment of B-Cell Malignancies. Cancers (Basel). 2020;12(10):3041. Published 2020 Oct 19.Doi : 10.3390 / cancers 12103041 ; and Saunders Kevin, “Conceptual Approaches to Modulating Antibody Effector Functions and Circulation Half-Life,” Frontiers in Immunology, vlO (June 7, 2019) DOI=10.3389 / fimmu.2019.01296, the full contents of each of which is incorporated by reference herein for all purposes.

[0271] Table 8 below, provides exemplary amino acid substitutions (and combinations thereof) and glycoengineering that can be utilized to increase one or more hlg Fc effector function. Amino acids in Table 8 are numbered according to the EU numbering scheme. The effects on effector function set forth in Table 8 are exemplar y only and not intended to be limiting. The amino acid substitutions set forth in Table 8 are with reference to an IgGl Fc region (except where noted). However, a person of ordinary skill in the could identify the corresponding amino acid in a non- IgGl Fc region, for example in an IgG2 or IgG4 Fc region, should the base amino acid be different between the IgGl and non-IgGl Fc region.Table 8. Exemplary hlg Fc Variations and Glycoengineering to Increase Effector Function.

[0272] In some embodiments, the Ig Fc region is a hlg Fc region. In some embodiments, the hlg Fc (e.g., IgGl Fc) region comprises any one or more of the amino acid substitutions set forth in Table 8 (i.e., any one or more amino acid substitution set forth in any set of amino acid substitutions set forth in Table 8). In some embodiments, the hlg Fc e.g., IgGl Fc) comprises any one or more of the sets of amino acid substitutions set forth in Table 8. In some embodiments, the hlg Fc (e.g., IgGl Fc) region comprises any one or more of the glycosylation changes set forth in Table 8.

[0273] In some embodiments, the hlg Fc (e.g., IgGl Fc) region comprises an amino acid substitution at any one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or more) of amino acid positions S298, E333, K334, S239, 1332, P247, A339, A330, G236, F243, R292, Y300, V305, P396, L235, F243, R292, Y300, P396, F243, R292, Y300, V3O5, P396, K326, E333, S267E, H268, S324, S298, E333, K334, L234, L235, G236, S239, H268, D270, S298 D270, K326, A330, and / or K334. In some embodiments, the hlg Fc (e.g., IgGl Fc) region comprises an amino acid substitution at from about 1-10 (e.g., 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2) of the following amino acid positions S298, E333, K334, S239, 1332, P247, A339, A330, G236, F243, R292, Y300, V305, P396, L235, F243, R292, Y300, P396, F243, R292, Y300, V3O5, P396, K326, E333, S267E, H268, S324, S298, E333, K334, L234, L235, G236, S239, H268, D270, S298 D270, K326, A330, and / or K334.

[0274] In some embodiments, the hlg Fc (e.g., IgGl Fc) region comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or more) of the following amino acid substitutions S298A, E333A, K334A, S239D, I332E, P247I, A339Q, A330L, G236A, F243L, R292P, Y300L, V305I, P396L, L235V, F243L, R292P, Y300L, P396L, F243L, R292P, Y300L, V3O5I, P396L, K326W, E333S, S267E, H268E, S324T, S298A, E333A, K334A, L234Y, L235Q, G236W, S239M, H268D, D270E, S298A D270E, K326D, A33OM, and / or K334E.

[0275] In some embodiments, the hlg Fc (e.g., IgGl Fc) region comprises from about 1-10 (e.g., 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2) of the following amino acid substitutions S298A, E333A, K334A, S239D, I332E, P247I, A339Q, A330L, G236A, F243L, R292P, Y300L, V305I,P396L, L235V, F243L, R292P, Y300L, P396L, F243L, R292P, Y300L, V305I, P396L, K326W, E333S, S267E, H268E, S324T, S298A, E333A, K334A, L234Y, L235Q, G236W, S239M, H268D, D270E, S298A D270E, K326D, A330M, and / or K334E.

[0276] In some embodiments, the hlg Fc region comprises a hlgGl Fc region comprising one or more amino acid variation relative to a reference hlgGl Fc region.

[0277] In some embodiments, the hlgGl Fc region comprises an amino acid substitution at one or more (e.g., 1, 2, or 3) of amino acid positions S298, E333, K334, EU numbering according to Kabat. In some embodiments, the hlgGl Fc region comprises one or more (e.g., 1, 2, or 3) of the following amino acid substitutions S298A, E333A, and / or K334A, EU numbering according to Kabat.

[0278] In some embodiments, the hlgGl Fc region comprises an amino acid substitution at one or more (e.g., 1 or 2) of amino acid positions S239 and / or 1332, EU numbering according to Kabat. In some embodiments, the hlgGl Fc region comprises one or more (e.g., 1 or 2) of the following amino acid substitutions S239D and / or I332E, EU numbering according to Kabat.

[0279] In some embodiments, the hlgGl Fc region comprises an amino acid substitution at one or more (e.g., 1 or 2) of amino acid positions P247 and / or A339, EU numbering according to Kabat. In some embodiments, the hlgGl Fc region comprises one or more (e.g., 1 or 2) of the following amino acid substitutions P247I and / or A339Q, EU numbering according to Kabat.

[0280] In some embodiments, the hlgGl Fc region comprises an amino acid substitution at one or more (e.g., 1, 2, or 3) of amino acid positions S239, A33O, and / or 1332, EU numbering according to Kabat. In some embodiments, the hlgGl Fc region comprises one or more (e.g., 1, 2, or 3) of the following amino acid substitutions S239D, A33OL, and / or I332E, EU numbering according to Kabat.

[0281] In some embodiments, the hlgGl Fc region comprises an amino acid substitution at one or more (e.g., 1, 2, or 3) of amino acid positions G236, S239, and / or 1332, EU numbering according to Kabat. In some embodiments, the hlgGl Fc region comprises one or more (e.g., 1, 2, or 3) of the following amino acid substitutions G236A, S239D, and / or I332E, EU numbering according to Kabat.

[0282] In some embodiments, the hlgGl Fc region comprises an amino acid substitution at one or more (e.g., 1, 2, 3, 4, or 5) of amino acid positions F243, R292, Y300, V3O5, and / or P396, EU numbering according to Kabat. In some embodiments, the hlgGl Fc region comprises one ormore (e.g., 1 , 2, 3, 4, or 5) of the following amino acid substitutions F243L, R292P, Y300L, V305I, and / or P396L, EU numbering according to Kabat.

[0283] In some embodiments, the hlgGl Fc region comprises an amino acid substitution at one or more (e.g., 1, 2, 3, 4, or 5) of amino acid positions L235, F243, R292, Y300, and P396, EU numbering according to Kabat. In some embodiments, the hlgGl Fc region comprises one or more (e.g., 1, 2, 3, 4, or 5) of the following amino acid substitutions L235V, F243L, R292P, Y300L, and / or P396L, EU numbering according to Kabat.

[0284] In some embodiments, the hlgGl Fc region comprises an amino acid substitution at one or more (e.g., 1, 2, 3, 4, 5, 6, or 7) of amino acid positions L234, L235, G236, S239, H268, D270, and / or S298, EU numbering according to Kabat. In some embodiments, the hlgGl Fc region comprises one or more (e.g., 1, 2, 3, 4, 5, 6, or 7) of the following amino acid substitutions L234Y, L235Q, G236W, S239M, H268D, D270E, and / or S298A, EU numbering according to Kabat.

[0285] In some embodiments, the hlgGl Fc region comprises an amino acid substitution at one or more (e.g., 1, 2, 3, or 4) of amino acid positions D270, K326, A330, and / or K334, EU numbering according to Kabat. In some embodiments, the hlgGl Fc region comprises one or more (e.g., 1, 2, 3, or 4) of the following amino acid substitutions D270E, K326D, A330M, and / or K334E, EU numbering according to Kabat.

[0286] In some embodiments, the hlgGl Fc region comprises an amino acid substitution at one or more (e.g., 1, 2, 3, 4, or 5) of amino acid positions F243, R292, Y300, V3O5, and / or P396, EU numbering according to Kabat. In some embodiments, the hlgGl Fc region comprises one or more (e.g., 1, 2, 3, 4, or 5) of the following amino acid substitutions F243L, R292P, Y300L, V305I, and / or P396L, EU numbering according to Kabat.

[0287] In some embodiments, the hlgGl Fc region comprises an amino acid substitution at one or more (e.g., 1, 2, or 3) of amino acid positions S239, 1332, and / or A330, EU numbering according to Kabat. In some embodiments, the hlgGl Fc region comprises one or more (e.g., I, 2, or 3) of the following amino acid substitutions S239D, I332E, and / or A330L, EU numbering according to Kabat.

[0288] In some embodiments, the hlgGl Fc region comprises an amino acid substitution at one or more (e.g., 1, 2, 3, or 4) of amino acid positions S239, 1332, A330, and / or G236, EU numbering according to Kabat. In some embodiments, the hlgGl Fc region comprises one or more (e.g., 1, 2, 3, or 4) of the following amino acid substitutions S239D, I332E, A33OL and / or G236A,EU numbering according to Kabat.

[0289] In some embodiments, the hlgGl Fc region comprises an amino acid substitution at one or more (e.g., 1, 2, or 3) of amino acid positions S239, 1332, and / or G326, EU numbering according to Kabat. In some embodiments, the hlgGl Fc region comprises one or more (e.g., 1, 2, or 3) of the following amino acid substitutions S239D, I332E, and / or G326A, EU numbering according to Kabat.

[0290] In some embodiments, the hlgGl Fc region comprises an amino acid substitution at amino acid position G326, EU numbering according to Kabat. In some embodiments, the hlgGl Fc region comprises a G326A amino acid substitution, EU numbering according to Kabat.

[0291] In some embodiments, the hlgGl Fc region comprises an amino acid substitution at one or more (e.g., 1, 2, or 3) of amino acid positions G236, S239, and / or 1332, EU numbering according to Kabat. In some embodiments, the hlgGl Fc region comprises one or more (e.g., 1, 2, or 3) of the following amino acid substitutions G236A, S239D, and / or I332E, EU numbering according to Kabat.

[0292] In some embodiments, the hlgGl Fc region comprises an amino acid substitution at one or more (e.g., 1 or 2) of amino acid positions S239 and / or 1332, EU numbering according to Kabat. In some embodiments, the hlgGl Fc region comprises one or more (e.g., 1 or 2) of the following amino acid substitutions S239D and / or I332E, EU numbering according to Kabat.

[0293] In some embodiments, the hlgGl Fc region comprises an amino acid substitution at one or more (e.g., 1 or 2) of amino acid positions K326 and / or E333, EU numbering according to Kabat. In some embodiments, the hlgGl Fc region comprises one or more (e.g., 1 or 2) of the following amino acid substitutions K326W and / or E333S, EU numbering according to Kabat.

[0294] In some embodiments, the hlgGl Fc region comprises an amino acid substitution at one or more (e.g., 1, 2, or 3) of amino acid positions S267, H268, and / or S324, EU numbering according to Kabat. In some embodiments, the hlgGl Fc region comprises one or more (e.g., 1, 2, or 3) of the following amino acid substitutions S267E, H268E, and / or S324T, EU numbering according to Kabat.

[0295] In some embodiments, the hlgGl Fc region comprises an amino acid substitution at one or more (e.g., 1, 2, or 3) of amino acid positions S298, E333, and / or K334, EU numbering according to Kabat. In some embodiments, the hlgGl Fc region comprises one or more (e.g., 1, 2, or 3) of the following amino acid substitutions S298A, E333A, and / or K334A, EU numberingaccording to Kabat.

[0296] In some embodiments, the hlgGl Fc region comprises an amino acid substitution at one or more (e.g., 1 or 2) of amino acid positions S239 and / or 1332, EU numbering according to Kabat. In some embodiments, the hlgGl Fc region comprises one or more (e.g., 1 or 2) of the following amino acid substitutions S239D and / or I332E, EU numbering according to Kabat.

[0297] In some embodiments, the hlgGl Fc region comprises an amino acid substitution at one or more (e.g., 1 or 2) of amino acid positions P247 and / or A339, EU numbering according to Kabat. In some embodiments, the hlgGl Fc region comprises one or more (e.g., 1 or 2) of the following amino acid substitutions P247I and / or A339Q, EU numbering according to Kabat.

[0298] In some embodiments, the hlg Fc region comprises one or more changes to the glycosylation. In some embodiments, the hlg Fc region is afucosylated. In some embodiments, the hlg Fc region is afucosylated and exhibits enhanced (e.g., increased) ADCC compared to a reference hlg Fc region that is not afucosylated.5.4.6 Linkers

[0299] As described herein, the heterologous moiety (e.g., heterologous protein) can be directly operably connected or indirectly operably connected to the immunoreceptor targeting protein (e.g., described herein). In some embodiments, the heterologous protein is directly operably connected to the immunoreceptor targeting protein (e.g., described herein) via a peptide bond. In some embodiment, the heterologous protein is indirectly operably connected to the immunoreceptor targeting protein (e.g., described herein) via a peptide linker.

[0300] In some embodiments, the peptide linker is one or any combination of a cleavable linker, a non-cleavable linker, a flexible linker, a rigid linker, a helical linker, and / or a non-helical linker.

[0301] In some embodiments, the amino acid sequence of the peptide linker comprises from or from about 2-30, 5-30, 10-30, 15-30, 20-30, 25-30, 2-25, 5-25, 10-25, 15-25, 20-25, 2-20, 5-20, 10-20, 15-20, 2-15, 5-15, 10-15, 2-10, or 5-10 amino acid residues. In some embodiments, the amino acid sequence of the peptide linker comprises at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid residues. In some embodiments, the amino acid sequence of the peptide linker comprises about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acidresidues. In some embodiments, the amino acid sequence of the peptide linker consists of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid residues. In some embodiments, the amino acid sequence of the peptide linker comprises no more than about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid residues. In some embodiments, the amino acid sequence of the peptide linker consists of no more than about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid residues.

[0302] In some embodiments, the amino acid sequence of the peptide linker comprises glycine, serine, or both glycine and serine amino acid residues. In some embodiments, the amino acid sequence of the peptide linker comprises glycine, serine, and proline amino acid residues. In some embodiments, the amino acid sequence of the peptide linker consists of glycine, serine, or both glycine and serine amino acid residues. In some embodiments, the amino acid sequence of the peptide linker consists of glycine, serine, and proline amino acid residues.

[0303] The amino acid sequence of exemplary peptide linkers, which can be incorporated in one or more of the embodiments described herein (e.g., fusion proteins), is set provided in Table 9.Table 9. The Amino Acid Sequence of Exemplary Peptide Linkers.

[0304] In some embodiments, the amino acid sequence of the peptide linker comprises the amino acid sequence of any one of the linkers set forth in Table 9. In some embodiments, the amino acid sequence of the peptide linker consists of the amino acid sequence of any one of the linkers set forth in Table 9. In some embodiments, the amino acid sequence of the peptide linker comprises the amino acid sequence of any one of the linkers set forth in Table 9, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acidvariations (e.g., amino acid substitutions, deletions, or additions). Tn some embodiments, the amino acid sequence of the peptide linker consists of the amino acid sequence of any one of the linkers set forth in Table 9, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the peptide linker comprises the amino acid sequence of any one of the linkers set forth in Table 9, comprising 1, 2, or 3 amino acid variations (e.g., substitutions, deletions, additions). In some embodiments, the amino acid sequence of the peptide linker consists of the amino acid sequence of any one of the linkers set forth in Table 9, comprising 1, 2, or 3 amino acid variations (e.g., substitutions, deletions, additions). In some embodiments, the amino acid sequence of the peptide linker comprises the amino acid sequence of any one of the linkers set forth in Table 9, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid substitutions. In some embodiments, the amino acid sequence of the peptide linker consists of the amino acid sequence of any one of the linkers set forth in Table 9, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid substitutions. In some embodiments, the amino acid sequence of the peptide linker comprises the amino acid sequence of any one of the linkers set forth in Table 9, comprising 1, 2, or 3 amino acid substitutions. In some embodiments, the amino acid sequence of the peptide linker consists of the amino acid sequence of any one of the linkers set forth in Table 9, comprising 1, 2, or 3 amino acid substitutions.

[0305] In some embodiments, the amino acid sequence of the peptide linker comprises the amino acid sequence set forth in any one of SEQ ID NOS: 83-92. In some embodiments, the amino acid sequence of the peptide linker consists of the amino acid sequence set forth in any one of SEQ ID NOS: 83-92. In some embodiments, the amino acid sequence of the peptide linker comprises the amino acid sequence set forth in any one of SEQ ID NOS: 83-92, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the peptide linker consists of the amino acid sequence set forth in any one of SEQ ID NOS: 83- 92, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the peptide linker comprises the amino acid sequence set forth in any one of SEQ ID NOS: 83-92, comprising 1, 2, or 3 amino acid variations (e.g.,substitutions, deletions, additions). In some embodiments, the amino acid sequence of the peptide linker consists of the amino acid sequence set forth in any one of SEQ ID NOS: 83-92, comprising 1, 2, or 3 amino acid variations (e.g., substitutions, deletions, additions). In some embodiments, the amino acid sequence of the peptide linker comprises the amino acid sequence set forth in any one of SEQ ID NOS: 83-92, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid substitutions. In some embodiments, the amino acid sequence of the peptide linker consists of the amino acid sequence set forth in any one of SEQ ID NOS: 83-92, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid substitutions. In some embodiments, the amino acid sequence of the peptide linker comprises the amino acid sequence set forth in any one of SEQ ID NOS: 83-92, comprising 1, 2, or 3 amino acid substitutions. In some embodiments, the amino acid sequence of the peptide linker consists of the amino acid sequence set forth in any one of SEQ ID NOS: 83-92, comprising 1, 2, or 3 amino acid substitutions.

[0306] In some embodiments, the amino acid sequence of the peptide linker comprises the amino acid sequence set forth in SEQ ID NO: 92. In some embodiments, the amino acid sequence of the peptide linker consists of the amino acid sequence set forth in SEQ ID NO: 92. In some embodiments, the amino acid sequence of the peptide linker comprises the amino acid sequence set forth in SEQ ID NO: 92, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the peptide linker consists of the amino acid sequence set forth in SEQ ID NO: 92, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the peptide linker comprises the amino acid sequence set forth in SEQ ID NO: 92, comprising 1, 2, or 3 amino acid variations (e.g., substitutions, deletions, additions). In some embodiments, the amino acid sequence of the peptide linker consists of the amino acid sequence set forth in SEQ ID NO: 92, comprising 1, 2, or 3 amino acid variations (e.g., substitutions, deletions, additions). In some embodiments, the amino acid sequence of the peptide linker comprises the amino acid sequence set forth in SEQ ID NO: 92, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid substitutions. In some embodiments, the amino acid sequence of the peptide linker consists of the amino acid sequence set forth in SEQ ID NO: 92,and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid substitutions. In some embodiments, the amino acid sequence of the peptide linker comprises the amino acid sequence set forth in SEQ ID NO: 92, comprising 1, 2, or 3 amino acid substitutions. In some embodiments, the amino acid sequence of the peptide linker consists of the amino acid sequence set forth in SEQ ID NO: 92, comprising 1, 2, or 3 amino acid substitutions.5.4.7 Orientation

[0307] The heterologous moiety (e.g., heterologous protein) and the immunoreceptor targeting protein (e.g., described herein) can be arranged in any configuration or order as long as the immunoreceptor targeting protein (e.g., described herein) maintains the ability to mediate its function (e.g., bind to its cognate partner) and in the embodiments wherein the heterologous moiety (e.g., heterologous protein) has a specific function, the heterologous moiety (e.g., heterologous protein) can mediate its function.

[0308] In some embodiments, the heterologous moiety is a heterologous protein (e.g., an Ig (e.g., hlg, mlg) Fc region (e.g., an Ig (e.g., hlg, mlg) Fc region described herein)) forming a fusion protein. In some embodiments, the fusion protein comprises from N- to C- terminus: an immunoreceptor targeting protein (e.g., described herein) and a heterologous protein (e.g., an Ig (e.g., hlg, mlg) Fc region (e.g., an Ig (e.g., hlg, mlg) Fc region described herein)). In some embodiments, the fusion protein comprises from N- to C- terminus: an immunoreceptor targeting protein (e.g., described herein), a peptide linker (e.g., described herein), and a heterologous protein (e.g., an Ig (e.g., hlg, mlg) Fc region (e.g., an Ig (e.g., hlg, mlg) Fc region described herein)). In this specific orientation, the N-terminus of the immunoreceptor targeting protein (e.g., described herein) is operably connected to the C-terminus of the heterologous protein (e.g., an Ig (e.g., hlg, mlg) Fc region (e.g., an Ig (e.g., hlg, mlg) Fc region described herein)) either directly or indirectly through the peptide linker (e.g., described herein).

[0309] In some embodiments, the fusion protein comprises from N- to C- terminus: a heterologous protein (e.g., an Ig (e.g., hlg, mlg) Fc region (e.g., an Ig (e.g., hlg, mlg) Fc region described herein)) and an immunoreceptor targeting protein (e.g., described herein). In some embodiments, the fusion protein comprises from N- to C- terminus: a heterologous protein (e.g., an Ig (e.g., hlg, mlg) Fc region (e.g., an Ig (e.g., hlg, mlg) Fc region described herein)), a peptide linker (e.g., described herein), and an immunoreceptor targeting protein (e.g., described herein).In this specific orientation, the C-terminus of the immunoreceptor targeting protein (e.g., described herein) is operably connected to the N-tcrminus of the heterologous protein (e.g., an Ig (e.g., hlg, mlg) Fc region (e.g., an Ig (e.g., hlg, mlg) Fc region described herein)) either directly or indirectly through the peptide linker (e.g., described herein).5.4.8 Multimeric Fusion Proteins

[0310] Provided herein are multimeric (e.g., dimeric) proteins comprising at least two fusion proteins or conjugates described herein (e.g., Ig (e.g., hlg, mlg) Fc fusion proteins described herein). In some embodiments, the protein is dimeric. In some embodiments, the protein is homodimeric. In some embodiments, the protein is heterodimeric. In some embodiments, the at least two fusion proteins described herein (e.g., Ig (e.g., hlg, mlg) Fc fusion proteins described herein) or conjugates associate via covalent or non-covalent interactions. In some embodiments, the at least two fusion proteins described herein (e.g., Ig (e.g., hlg, mlg) Fc fusion proteins described herein) or conjugates associate via at least one covalent interaction. In some embodiments, the at least two fusion proteins (e.g., Ig (e.g., hlg, mlg) Fc fusion proteins) or conjugates associate via one or more disulfide bond. In some embodiments, the at least two fusion proteins (e.g., Ig (e.g., hlg, mlg) Fc fusion proteins) or conjugates associate via 1, 2, 3, 4, or more disulfide bonds.

[0311] In some embodiments, the protein is dimeric comprising a first fusion protein (e.g., a hlg Fc fusion protein) or conjugate described herein and a second fusion protein (e.g., an Ig (e.g., hlg, mlg) Fc fusion protein) or conjugate described herein, wherein the amino acid sequence of the first protein comprises an amino acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of the second protein. For example, the amino acid sequence of the first protein may comprise an amino acid sequence at least about 85% identical to the amino acid sequence of the second protein. For example, the amino acid sequence of the first protein may comprise an amino acid sequence at least about 90% identical to the amino acid sequence of the second protein. For example, the amino acid sequence of the first protein may comprise an amino acid sequence at least about 95% identical to the amino acid sequence of the second protein. In some embodiments, the amino acid sequence of the first protein may preferably comprise an amino acid sequence 100% identical to the amino acid sequence of the second protein.

[0312] In some embodiments, the protein is dimeric comprising a first Ig (e.g., hlg, mlg) Fc fusion protein and a second Ig e.g., hlg, mlg) Fc fusion protein. In some embodiments, the dimeric protein is homodimeric. In some embodiments, the dimeric protein is heterodimeric. In some embodiments, the amino acid sequence of the first Ig (e.g., hlg, mlg) Fc fusion protein comprises an amino acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of the second Ig (e.g., hlg, mlg) Fc fusion protein.

[0313] An exemplary dimeric Ig (e.g., hlg, mlg) Fc fusion protein includes, for example, a protein comprising (i) a first Ig (e.g., hlg, mlg) Fc fusion protein comprising from N- to C- terminus: a first Ig (e.g., hlg, mlg) Fc region (e.g., described herein), a first peptide linker (e.g., described herein), and a first immunoreceptor targeting protein (e.g., described herein); and (ii) a second Ig (e.g., hlg, mlg) Fc fusion protein comprising from N- to C- terminus: a second Ig (e.g., hlg, mlg) Fc region (e.g., described herein), a second peptide linker (e.g., described herein), and a second immunoreceptor targeting protein (e.g., described herein). In some embodiments, the amino acid sequence of the first Ig (e.g., hlg, mlg) Fc fusion protein is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of the second Ig (e.g., hlg, mlg) Fc fusion protein. In this specific embodiment, the N-terminus of the immunoreceptor targeting protein (e.g., described herein) is operably connected to the C-terminus of the Ig (e.g., hlg, mlg) Fc region through the peptide linker (e.g., described herein).

[0314] Another exemplary dimeric Ig (e.g., hlg, mlg) Fc fusion protein includes, for example, a protein comprising (i) a first Ig (e.g., hlg, mlg) Fc fusion protein comprising from N- to C- terminus: a first immunoreceptor targeting protein (e.g., described herein), a first peptide linker (e.g., described herein), and a first Ig (e.g., hlg, mlg) Fc region (e.g., described herein); and (ii) a second immunoreceptor targeting protein (e.g., described herein), a second peptide linker (e.g., described herein), and a second Ig (e.g., hlg, mlg) Fc region (e.g., described herein). In some embodiments, the amino acid sequence of the first Ig (e.g., hlg, mlg) Fc fusion protein is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of the second Ig (e.g., hlg, mlg) Fc fusion protein. In this specific embodiment, the C-terminus of the immunoreceptor targeting protein (e.g., described herein) is operably connected to the N-terminus of the Ig (e.g.,hlg, mlg) Fc region either directly or indirectly through the peptide linker (e.g., described herein).5.4.9 Exemplary Ig Fusion Proteins

[0315] The amino acid sequence of exemplary immunoreceptor targeting fusion proteins (ITFPs) described herein is provided in Table 10. Each of the ITFPs 1-12 comprising the amnio acid sequence set forth in any one of SEQ ID NOS: 93, 96, 99, 102, 105, 108, 111, 114, 117, 120, 123, and 126comprises from N- to C-terminus the hIL-2 signal sequence (hIL-2ss), an effector function reduced hIgG4 Fc region, a peptide linker, and an immunoreceptor targeting protein identified herein (ITPs 1-12) (e.g., see Table 2, SEQ ID NOS: 129-140). Each of the ITFPs 1-12 comprising the amnio acid sequence set forth in any one of SEQ ID NOS: 94-95, 97-98, 100-101, 103-104, 106-107, 109-110, 112-113, 115-116, 118-119, 121-122, 124-125, and 127- 128comprises from N- to C-terminus an effector function reduced h!gG4 Fc region, a peptide linker, and an immunoreceptor targeting protein identified herein (ITPs 1-12) e.g., see Table 2, SEQ ID NOS: 129-140). The fusion proteins provided in Table 10 arc exemplary only, and not intended to be limiting. Similar fusion proteins can be made utilizing the additional ITPs listed in Table 2, e.g., any one of ITPs 1-58.Table 10. The Amino Acid Sequence of Exemplary Ig Fusion Proteins.

[0316] In some embodiments, the amino acid sequence of the immunoreceptor targeting fusion protein comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a polypeptide set forth in Table 10. In some embodiments, the amino acid sequence of the immunoreceptor targeting fusion protein comprises an amino acid sequence at least 85% identical to the amino acid sequence of a polypeptide set forth in Table 10. In some embodiments, the amino acid sequence of the immunorcccptor targeting fusion protein comprises an amino acid sequence at least 90% identical to the amino acid sequence of a polypeptide set forth in Table 10. In some embodiments, the amino acid sequence of the immunoreceptor targeting fusion protein comprises an amino acid sequence at least 95% identical to the amino acid sequence of a polypeptide set forth in Table 10. In some embodiments, the amino acid sequence of the immunoreceptor targeting fusion protein comprises an amino acid sequence at least 100% identical to the amino acid sequence of a polypeptide set forth in Table 10.

[0317] In some embodiments, the amino acid sequence of the immunoreceptor targeting fusion protein consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a polypeptide set forth in Table 10. In some embodiments, the amino acid sequence of the immunoreceptor targeting fusion protein consists of an amino acid sequence at least 85% identical to the amino acid sequence of a polypeptide set forth in Table 10. In some embodiments, the amino acid sequence of the immunoreceptor targeting fusion protein consists of an amino acid sequence at least 90% identical to the amino acid sequence of a polypeptide set forth in Table 10. In some embodiments, the amino acid sequence of the immunoreceptor targeting fusion protein consists of an amino acid sequence at least 95% identical to the amino acid sequence of a polypeptide set forth in Table 10. In some embodiments, the amino acid sequence of the immunoreceptor targeting fusion protein consists of an amino acid sequence at least 100% identical to the amino acid sequence of a polypeptide set forth in Table 10.

[0318] In some embodiments, the amino acid sequence of the immunoreceptor targeting fusion protein comprises the amino acid sequence of a polypeptide set forth in Table 10, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acidvariations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the immunorcccptor targeting fusion protein comprises the amino acid sequence of a polypeptide set forth in Table 10, 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 immunoreceptor targeting fusion protein comprises the amino acid sequence of a polypeptide set forth in Table 10, and further comprises about 1, 2,3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the immunoreceptor targeting fusion protein comprises the amino acid sequence of a polypeptide set forth in Table 10, and further consists of about 1, 2,3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the immunoreceptor targeting fusion protein comprises the amino acid sequence of a polypeptide set forth in Table 10, and further comprises no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.).

[0319] In some embodiments, the amino acid sequence of the immunoreceptor targeting fusion protein consists of the amino acid sequence of a polypeptide set forth in Table 10, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the immunoreceptor targeting fusion protein consists of the amino acid sequence of a polypeptide set forth in Table 10, 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 immunoreceptor targeting fusion protein consists of the amino acid sequence of a polypeptide set forth in Table 10, and further comprises about 1, 2,3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the immunoreceptor targeting fusion protein consists of the amino acid sequence of a polypeptide set forth in Table 10, and further consists of about 1, 2,3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the immunoreceptor targeting fusion protein consists of the amino acid sequence of a polypeptide set forth in Table 10, and further comprises no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.).

[0320] In some embodiments, the amino acid sequence of the immunoreceptor targeting fusion protein comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 93-128. In some embodiments, the amino acid sequence of the immunoreceptor targeting fusion protein comprises an amino acid sequence at least 85% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 93-128. In some embodiments, the amino acid sequence of the immunoreceptor targeting fusion protein comprises an amino acid sequence at least 90% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 93-128. In some embodiments, the amino acid sequence of the immunoreceptor targeting fusion protein comprises an amino acid sequence at least 95% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 93-128. In some embodiments, the amino acid sequence of the immunoreceptor targeting fusion protein comprises an amino acid sequence at least 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 93-128.

[0321] In some embodiments, the amino acid sequence of the immunoreceptor targeting fusion protein consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 93-128. In some embodiments, the amino acid sequence of the immunoreceptor targeting fusion protein consists of an amino acid sequence at least 85% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 93-128. In some embodiments, the amino acid sequence of the immunoreceptor targeting fusion protein consists of an amino acid sequence at least 90% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 93-128. In some embodiments, the amino acid sequence of the immunoreceptor targeting fusion protein consists of an amino acid sequence at least 95% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 93-128. In some embodiments, the amino acid sequence of the immunoreceptor targeting fusion protein consists of an amino acid sequence at least 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 93-128.

[0322] In some embodiments, the amino acid sequence of the immunoreceptor targeting fusion protein comprises the amino acid sequence set forth in any one of SEQ ID NOS: 93-128, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the immunoreceptor targeting fusion protein comprises the amino acid sequenceset forth in any one of SEQ ID NOS: 93-128, 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 immunoreceptor targeting fusion protein comprises the amino acid sequence set forth in any one of SEQ ID NOS: 93-128, and further comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the immunoreceptor targeting fusion protein comprises the amino acid sequence set forth in any one of SEQ ID NOS: 93-128, and further consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the immunoreceptor targeting fusion protein comprises the amino acid sequence set forth in any one of SEQ ID NOS: 93-128, and further comprises no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.).

[0323] In some embodiments, the amino acid sequence of the immunoreceptor targeting fusion protein consists of the amino acid sequence set forth in any one of SEQ ID NOS: 93-128, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the immunoreceptor targeting fusion protein consists of the amino acid sequence set forth in any one of SEQ ID NOS: 93-128, 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 immunoreceptor targeting fusion protein consists of the amino acid sequence set forth in any one of SEQ ID NOS: 93-128, and further comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the immunoreceptor targeting fusion protein consists of the amino acid sequence set forth in any one of SEQ ID NOS: 93-128, and further consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the immunoreceptor targeting fusion protein consists of the amino acid sequence set forth in any one of SEQ ID NOS: 93-128, and further comprises no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.).5.5 Immunogenic Peptides & Proteins

[0324] In one aspect, provided herein are immunogenic peptides or proteins comprising at least an immunogenic fragment of an immunorcccptor targeting protein described herein.

[0325] In some embodiments, the immunogenic peptide or protein does not specifically bind a TNFSF ligand (e.g., hCD30L) or binds a TNFSF ligand (e.g., hCD30L) with lower affinity relative to a reference immunoreceptor targeting protein described herein. Binding to and / or affinity for the TNF superfamily member (e.g., described herein, e.g., CD30L) can be assessed using standard methods known in the art and described herein, see, e.g., § 5.3.

[0326] In some embodiments, the immunogenic peptide or protein further comprises one or more heterologous peptide or protein element, or a nucleic acid molecule (e.g., described herein) that encodes at least one heterologous peptide or protein element. In some embodiments, the at least one heterologous peptide or protein element may impart an additional function to the immunogenic peptide or protein, e.g., to promote or improve secretion of the encoded immunogenic peptide or protein (e.g., a signal peptide (e.g., described herein), promote or improve anchoring of the encoded immunogenic peptide or protein described herein in the plasma membrane (e.g., via transmembrane elements), promote or improve formation of immunogen complexes (e.g., via multimerization domains or immunogen clustering elements), or promote or improve virus-like particle formation (VLP forming sequence).

[0327] In some embodiments, the immunogenic peptide or protein is formulated with an adjuvant.5.5.1 Fragments of Immunoreceptor Targeting Proteins

[0328] In some embodiments, the immunogenic peptide or protein comprises an immunogenic fragment of an immunoreceptor targeting protein described herein. In some embodiments, the immunogenic peptide or protein consists of an immunogenic fragment of an immunoreceptor targeting protein described herein. In some embodiments, the immunogenic peptide or protein comprises an immunoreceptor targeting protein described herein. In some embodiments, the immunogenic peptide or protein consists of an immunoreceptor targeting protein described herein. In some embodiments, the immunogenic peptide or protein comprises a full-length immunoreceptor targeting protein described herein. In some embodiments, the immunogenic peptide or protein consists of a full-length immunoreceptor targeting protein described herein.

[0329] In some embodiments, the immunogenic peptide or protein comprises at least about 10,15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, or 130 amino acids. In some embodiments, the immunogenic peptide or protein comprises from about 10-130, 10-120, 10-110, 10-100, 10-90, 10-80, 10-70, 10-60, 10-50, 10-40, 10-30, or 10-20 amino acids. In some embodiments, the immunogenic peptide or protein comprises at least about 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, or 130 amino acids. In some embodiments, the immunogenic peptide or protein comprises about 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, or 130 amino acids. In some embodiments, the immunogenic peptide or protein consists of about 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, or 130 amino acids. In some embodiments, the immunogenic peptide or protein comprises or consists of no more than about 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, or 130 amino acids.

[0330] In some embodiments, the immunogenic peptide or protein comprises at least a portion of an immunoreceptor targeting protein described herein. In some embodiments, the immunogenic peptide or protein comprises or consists of at least about 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, or 130 contiguous amino acids of an immunoreceptor targeting protein described herein. In some embodiments, the immunogenic peptide or protein comprises or consists of about 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, or 130 contiguous amino acids of an immunoreceptor targeting protein described herein. In some embodiments, the immunogenic peptide or protein comprises or consists of no more than about 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, or 130 contiguous amino acids of an immunoreceptor targeting protein described herein. In some embodiments, the immunogenic peptide or protein comprises or consists of from about 10-130, 10-120, 10-110, 10-100, 10-90, 10-80, 10-70, 10-60, 10-50, 10-40, 10-30, or 10-20 contiguous amino acids of an immunoreceptor targeting protein described herein.

[0331] In some embodiments, immunogenic peptide or protein comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a portion of the amino acid sequence of an immunoreceptor targeting protein described herein. In some embodiments, the immunogenic peptide or protein comprises an amino acid sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a contiguous stretch of at least about 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, or 130 amino acids of an immunoreceptor targeting protein described herein. In some embodiments, the immunogenic peptide or protein comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identicalto a contiguous stretch of at least about 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1 10, 120, or 130 amino acids of an immunorcccptor targeting protein described herein. In some embodiments, the immunogenic peptide or protein comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a contiguous stretch of about 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, or 130 amino acids of an immunoreceptor targeting protein described herein. In some embodiments, the immunogenic peptide or protein comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a contiguous stretch of no more than about 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, or 130 amino acids of an immunoreceptor targeting protein described herein.

[0332] In some embodiments, the amino acid sequence of the immunogenic peptide or protein comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid variations (e.g., substitution, addition, deletion) relative to the amino acid sequence of an immunoreceptor targeting protein described herein. In some embodiments, the amino acid sequence of an immunogenic peptide or protein comprises at least one amino acid variation (e.g., substitution, addition, deletion) relative to the amino acid sequence of an immunoreceptor targeting protein described herein. In some embodiments, the amino acid sequence of the immunogenic peptide or protein comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid variations (e.g., substitution, addition, deletion) relative to the amino acid sequence of an immunoreceptor targeting protein described herein. In some embodiments, the amino acid sequence of the immunogenic peptide or protein comprises or consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid variations (e.g., substitution, addition, deletion) relative to the amino acid sequence of an immunoreceptor targeting protein described herein. In some embodiments, the amino acid sequence of the immunogenic peptide or protein comprises or consists of no more than about I, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acid variations (e.g., substitution, addition, deletion) relative to the amino acid sequence of an immunoreceptor targeting protein described herein.

[0333] In some embodiments, the immunogenic peptide or protein comprises at least a portion of an immunoreceptor targeting protein described herein. In some embodiments, the immunogenic peptide or protein comprises or consists of at least about 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, or 130 contiguous amino acids of an immunoreceptor targeting protein set forth in Table 2. In some embodiments, the immunogenic peptide or protein comprises or consists of about 10,15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120 or 130 contiguous amino acids of an immunorcccptor targeting protein set forth in Table 2. In some embodiments, the immunogenic peptide or protein comprises or consists of no more than about 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130 contiguous amino acids of an immunoreceptor targeting protein set forth in Table 2. In some embodiments, the immunogenic peptide or protein comprises or consists of from about 10-130, 10-120, 10-110, 10-100, 10-90, 10-80, 10-70, 10-60, 10-50, 10-40, 10-30, or 10-20 contiguous amino acids of an immunoreceptor targeting protein set forth in Table 2.

[0334] In some embodiments, immunogenic peptide or protein comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a portion of the amino acid sequence of an immunoreceptor targeting protein set forth in Table 2. In some embodiments, the immunogenic peptide or protein comprises an amino acid sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a contiguous stretch of at least about 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, or 130 amino acids of an immunoreceptor targeting protein set forth in Table 2. In some embodiments, the immunogenic peptide or protein comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a contiguous stretch of at least about 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, or 130 amino acids of an immunoreceptor targeting protein set forth in Table 2. In some embodiments, the immunogenic peptide or protein comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a contiguous stretch of about 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, or 130 amino acids of an immunoreceptor targeting protein set forth in Table 2. In some embodiments, the immunogenic peptide or protein comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a contiguous stretch of no more than about 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, or 130 amino acids of an immunoreceptor targeting protein set forth in Table 2.

[0335] In some embodiments, the amino acid sequence of the immunogenic peptide or protein comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid variations (e.g., substitution, addition, deletion) relative to the amino acid sequence of an immunoreceptor targeting protein set forth in Table 2. In some embodiments, the amino acid sequence of the immunogenic peptide or protein comprises at least one amino acid variation (e.g.,substitution, addition, deletion) relative to the amino acid sequence of an immunoreceptor targeting protein set forth in Table 2. In some embodiments, the amino acid sequence of the immunogenic peptide or protein comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid variations (e.g., substitution, addition, deletion) relative to the amino acid sequence of an immunoreceptor targeting protein set forth in Table 2. In some embodiments, the amino acid sequence of the immunogenic peptide or protein comprises or consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid variations (e.g., substitution, addition, deletion) relative to the amino acid sequence of an immunoreceptor targeting protein set forth in Table 2. In some embodiments, the amino acid sequence of the immunogenic peptide or protein comprises or consists of no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acid variations e.g., substitution, addition, deletion) relative to the amino acid sequence of an immunoreceptor targeting protein set forth in Table 2.

[0336] In some embodiments, the immunogenic peptide or protein comprises at least a portion of an immunoreceptor targeting protein described herein. In some embodiments, the immunogenic peptide or protein comprises or consists of at least about 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, or 130 contiguous amino acids of an immunoreceptor targeting protein set forth the in any one of SEQ ID NOS: 129-240. In some embodiments, the immunogenic peptide or protein comprises or consists of about 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, or 200 contiguous amino acids of an immunoreceptor targeting protein set forth the in any one of SEQ ID NOS: 129- 240. In some embodiments, the immunogenic peptide or protein comprises or consists of no more than about 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, or 130 contiguous amino acids of an immunoreceptor targeting protein set forth the in any one of SEQ ID NOS: 129-240. In some embodiments, the immunogenic peptide or protein comprises or consists of from about 10-130, 10-120, 10-110, 10-100, 10-90, 10-80, 10-70, 10-60, 10-50, 10-40, 10-30, or 10-20 contiguous amino acids of an immunoreceptor targeting protein set forth the in any one of SEQ ID NOS: 129- 240.

[0337] In some embodiments, immunogenic peptide or protein comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a portion of the amino acid sequence of an immunoreceptor targeting protein set forth the in any one of SEQ ID NOS: 129-240. 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 about10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, or 130 amino acids of an immunoreceptor targeting protein set forth in any one of SEQ ID NOS: 129-240. In some embodiments, the immunogenic peptide or protein comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a contiguous stretch of at least about 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, or 130 amino acids of an immunoreceptor targeting protein set forth the in any one of SEQ ID NOS: 129-240. In some embodiments, the immunogenic peptide or protein comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a contiguous stretch of about 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, or 200 amino acids of an immunoreceptor targeting protein set forth the in any one of SEQ ID NOS: 129-240. In some embodiments, the immunogenic peptide or protein comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a contiguous stretch of no more than about 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, or 130 amino acids of an immunoreceptor targeting protein set forth the in any one of SEQ ID NOS: 129-240.

[0338] In some embodiments, the amino acid sequence of the immunogenic peptide or protein comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid variations (e.g., substitution, addition, deletion) relative to the amino acid sequence of an immunoreceptor targeting protein set forth the in any one of SEQ ID NOS: 129-240. In some embodiments, the amino acid sequence of the immunogenic peptide or protein comprises at least one amino acid variation (e.g., substitution, addition, deletion) relative to the amino acid sequence of an immunoreceptor targeting protein set forth the in any one of SEQ ID NOS: 129-240. In some embodiments, the amino acid sequence of the immunogenic peptide or protein comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid variations (e.g., substitution, addition, deletion) relative to the amino acid sequence of an immunoreceptor targeting protein set forth the in any one of SEQ ID NOS: 129-240. In some embodiments, the amino acid sequence of the immunogenic peptide or protein comprises or consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid variations (e.g., substitution, addition, deletion) relative to the amino acid sequence of an immunoreceptor targeting protein set forth the in any one of SEQ ID NOS: 129-240. In some embodiments, the amino acid sequence of the immunogenic peptide or protein comprises or consists of no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acid variations (e.g., substitution,addition, deletion) relative to the amino acid sequence of an immunoreceptor targeting protein set forth the in any one of SEQ ID NOS: 129-240.5.5.2 Variants of Immunoreceptor Targeting Proteins

[0339] In some embodiments, the immunogenic peptide or protein comprises at least one amino acid variation (e.g., substitution, addition, deletion) relative to the amino acid sequence of an immunoreceptor targeting protein described herein. In some embodiments, the amino acid sequence of the immunogenic peptide or protein comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid variations (e.g., substitutions, additions, deletions) relative to the amino acid sequence of an immunoreceptor targeting protein described herein. In some embodiments, the amino acid sequence of the immunogenic peptide or protein comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid variations (e.g., substitution, addition, deletion) relative to the amino acid sequence of an immunoreceptor targeting protein described herein.

[0340] In some embodiments, the amino acid sequence of the immunogenic peptide or protein comprises or consists of the amino acid sequence of an immunoreceptor targeting protein described herein comprising 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid variations (e.g., substitution, addition, deletion) relative to the amino acid sequence of the immunoreceptor targeting protein described herein.

[0341] In some embodiments, the amino acid sequence of the immunogenic peptide or protein comprises or consists of the amino acid sequence of an immunoreceptor targeting protein described herein comprising at least one amino acid variation (e.g., substitution, addition, deletion). In some embodiments, the amnio acid sequence of the immunogenic peptide or protein comprises or consists of the amino acid sequence of an immunoreceptor targeting protein described herein comprising at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid variations (e.g., substitution, addition, deletion). In some embodiments, the amino acid sequence of the immunogenic peptide or protein comprises or consists of the amino acid sequence of an immunoreceptor targeting protein described herein comprising no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acid variations (e.g., substitution, addition, deletion).

[0342] In some embodiments, the amino acid sequence of the immunogenic peptide or protein comprises or consists of the amino acid sequence of an immunoreceptor targeting protein described herein comprising one or more amino acid variation (e.g., substitution, addition, deletion). In someembodiments, the immunogenic peptide or protein comprises an amino acid sequence that, other than the one or more amino acid variation (e.g., substitution, addition, deletion), is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, or 130 amino acids of an immunoreceptor targeting protein described herein. In some embodiments, immunogenic peptide or protein comprises an amino acid sequence that, other than the one or more amino acid substitution, is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of an immunoreceptor targeting protein described herein.

[0343] In some embodiments, the immunogenic peptide or protein comprises at least one amino acid variation (e.g., substitution, addition, deletion) relative to the amino acid sequence of an immunoreceptor targeting protein set forth in Table 2. In some embodiments, the amino acid sequence of the immunogenic peptide or protein comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid variations (e.g., substitutions, additions, deletions) relative to the amino acid sequence of a reference immunoreceptor targeting protein set forth in Table 2. In some embodiments, the amino acid sequence of the immunogenic peptide or protein comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid variations (e.g., substitution, addition, deletion) relative to the amino acid sequence of an immunoreceptor targeting protein set forth in Table 2.

[0344] In some embodiments, the amino acid sequence of the immunogenic peptide or protein comprises or consists of the amino acid sequence of an immunoreceptor targeting protein set forth in Table 2 comprising 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid variations (e.g., substitution, addition, deletion) relative to the amino acid sequence of the immunoreceptor targeting protein set forth in Table 2.

[0345] In some embodiments, the amino acid sequence of the immunogenic peptide or protein comprises or consists of the amino acid sequence of an immunoreceptor targeting protein set forth in Table 2 comprising at least one amino acid variation (e.g., substitution, addition, deletion). In some embodiments, the amnio acid sequence of the immunogenic peptide or protein comprises or consists of the amino acid sequence of an immunoreceptor targeting protein set forth in Table 2 comprising at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid variations (e.g., substitution, addition, deletion). In some embodiments, the amino acid sequence of the immunogenic peptideor protein comprises or consists of the amino acid sequence of an immunoreceptor targeting protein set forth in Tabic 2 comprising no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acid variations (e.g., substitution, addition, deletion).

[0346] In some embodiments, the amino acid sequence of the immunogenic peptide or protein comprises or consists of the amino acid sequence of an immunoreceptor targeting protein set forth in Table 2 comprising one or more amino acid variation (e.g., substitution, addition, deletion). In some embodiments, the immunogenic peptide or protein comprises an amino acid sequence that, other than the one or more amino acid variation (e.g., substitution, addition, deletion), is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, or 130 amino acids of an immunoreceptor targeting protein set forth in Table 2. In some embodiments, immunogenic peptide or protein comprises an amino acid sequence that, other than the one or more amino acid substitution, is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of an immunoreceptor targeting protein described herein.

[0347] In some embodiments, the immunogenic peptide or protein comprises at least one amino acid variation (e.g., substitution, addition, deletion) relative to the amino acid sequence of an immunoreceptor targeting protein set forth in any one of SEQ ID NOS: 129-240. In some embodiments, the amino acid sequence of the immunogenic peptide or protein comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid variations (e.g., substitutions, additions, deletions) relative to a reference immunogenic protein set forth in any one of SEQ ID NOS: 129-240. In some embodiments, the amino acid sequence of the immunogenic peptide or protein comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid variations (e.g., substitution, addition, deletion) relative to the amino acid sequence of the immunoreceptor targeting protein set forth in any one of SEQ ID NOS: 129-240.

[0348] In some embodiments, the amino acid sequence of the immunogenic peptide or protein comprises or consists of the amino acid sequence of an immunoreceptor targeting protein set forth in any one of SEQ ID NOS: 129-240 comprising 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid variations (e.g., substitution, addition, deletion) relative to the amino acid sequence of the immunoreceptor targeting protein set forth in any one of SEQ ID NOS: 129-240.

[0349] In some embodiments, the amino acid sequence of the immunogenic peptide or protein comprises or consists of the amino acid sequence of an immunorcccptor targeting protein set forth in any one of SEQ ID NOS: 129-240 comprising at least one amino acid variation (e. ., substitution, addition, deletion). In some embodiments, the amnio acid sequence of the immunogenic peptide or protein comprises or consists of the amino acid sequence of an immunoreceptor targeting protein set forth in any one of SEQ ID NOS: 129-240 comprising at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid variations (e.g., substitution, addition, deletion). In some embodiments, the amino acid sequence of the immunogenic peptide or protein comprises or consists of the amino acid sequence of an immunoreceptor targeting protein set forth in any one of SEQ ID NOS: 129-240 comprising no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acid variations (e.g., substitution, addition, deletion).

[0350] In some embodiments, the amino acid sequence of the immunogenic peptide or protein comprises or consists of the amino acid sequence of an immunoreceptor targeting protein set forth in any one of SEQ ID NOS: 129-240 comprising one or more amino acid variation (e.g., substitution, addition, deletion). In some embodiments, the immunogenic peptide or protein comprises an amino acid sequence that, other than the one or more amino acid variation (e.g., substitution, addition, deletion), is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, or 130 amino acids of an immunoreceptor targeting protein set forth in any one of SEQ ID NOS: 129-240. In some embodiments, immunogenic peptide or protein comprises an amino acid sequence that, other than the one or more amino acid substitution, is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of an immunoreceptor targeting protein set forth in any one of SEQ ID NOS: 129- 240.5.5.3 Peptide and Protein-Based Vaccines

[0351] In some embodiments, an immunogenic peptide or protein described herein forms the basis for a pharmaceutical composition (e.g., a peptide or protein-based vaccine composition). Therefore, provided herein are pharmaceutical compositions (e.g., vaccine compositions) comprising an immunogenic peptide or protein described herein.

[0352] In some embodiments, the vaccine composition comprises a plurality of theimmunogenic peptides or proteins (e.g., described herein). In some embodiments, the pharmaceutical composition (e.g., vaccine composition) comprises a plurality of substantially the same immunogenic peptide or protein (e.g., described herein). In some embodiments, the pharmaceutical composition (e.g., vaccine composition) comprises a plurality of different immunogenic peptide or protein (e.g., described herein). In some embodiments, the pharmaceutical composition (e.g., vaccine composition) comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 different immunogenic peptides or proteins (e.g., described herein). In some embodiments, the pharmaceutical composition (e.g., vaccine composition) comprises or consists of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 different immunogenic peptides or proteins (e.g., described herein). In some embodiments, the pharmaceutical composition (e.g., vaccine composition) comprises at least one (e.g., 2, 3, 4, 5, 6) immunogenic peptide or protein described herein and at least one immunogenic peptide or protein from a different virus.5.5.4 Nucleic Acid-Based Vaccines

[0353] In some embodiments, a nucleic acid molecule encoding an immunogenic peptide or protein described herein (also referred to herein as an “immunogenic nucleic acid molecule”) forms the basis for a pharmaceutical composition (e.g., a vaccine composition (e.g., a nucleic acidbased vaccine)). In some embodiments, the nucleic acid molecule is RNA (e.g., mRNA or circular RNA) or DNA. In some embodiments, the nucleic acid molecule is mRNA. In some embodiments, the nucleic acid molecule is circular RNA (see, e.g., WO2019118919, the full contents of which are incorporated by reference herein for all purposes).

[0354] In some embodiments, the segment of the nucleic acid molecule encoding the immunogenic peptide or protein (e.g., described herein) comprises from about 30 to about 20000 nucleotides, about 50 to about 20000 nucleotides, about 500 to about 10000 nucleotides, about 1000 to about 10000 nucleotides, about 1000 to about 5000 nucleotides, or about 2000 to about 5000 nucleotides. In some embodiments, the segment of the nucleic acid molecule encoding the immunogenic peptide or protein (e.g., described herein) comprises at least 30 nucleotides, 50 nucleotides, 100 nucleotides, 200 nucleotides, 300 nucleotides, 400 nucleotides, 500 nucleotides, 1000 nucleotides, 2000 nucleotides, 3000 nucleotides, or 5000 nucleotides.

[0355] In some embodiments, the nucleic acid molecule is modified or varied (compared to a reference nucleic acid sequence), e.g., to impart one or more of (a) improved resistance to in vivodegradation, (b) improved stability in vivo, (c) reduced secondary structures, and / or (d) improved translatability in vivo, compared to the reference nucleic acid sequence. Alterations include, without limitation, e.g., codon optimization, nucleotide modification (see, e.g., description below), etc.

[0356] In some embodiments, the nucleic acid sequence is codon optimized, e.g., for expression in humans. Codon optimization, in some embodiments, may be used to match codon frequencies in target and host organisms to ensure proper folding; bias guanosine (G) and / or cytosine (C) content to increase nucleic acid stability; minimize tandem repeat codons or base runs that may impair gene construction or expression; customize transcriptional and translational control regions; insert or remove protein trafficking sequences; remove / add post translation alteration sites in encoded protein (e.g., glycosylation sites); add, remove, or shuffle protein domains; insert or delete restriction sites; modify ribosome binding sites and mRNA degradation sites; adjust translational rates to allow the various domains of the protein to fold properly; or to reduce or eliminate problem secondary structures within the nucleic acid molecule. In some embodiments, the codon optimized nucleic acid sequence shows one or more of the above (compared to a reference nucleic acid sequence). In some embodiments, the codon optimized nucleic acid sequence shows one or more of improved resistance to in vivo degradation, improved stability in vivo, reduced secondary structures, and / or improved translatability in vivo, compared to a reference nucleic acid sequence. Codon optimization methods, tools, algorithms, and services are known in the art, non-limiting examples include services from GeneArt (Life Technologies) and DNA2.0 (Menlo Park Calif.). In some embodiments, the open reading frame (ORF) sequence is optimized using optimization algorithms. In some embodiments, the nucleic acid sequence is modified or varied to optimize the number of G and / or C nucleotides as compared to a reference nucleic acid sequence. An increase in the number of G and C nucleotides may be generated by substitution of codons containing adenosine (T) or thymidine (T) (or uracil (U)) nucleotides by codons containing G or C nucleotides.

[0357] In some embodiments, the pharmaceutical composition comprises a plurality of substantially the same nucleic acid molecules encoding a plurality of immunogenic peptides or proteins (e.g., described herein). In some embodiments, the pharmaceutical composition comprises a plurality of different nucleic acid molecules encoding a plurality of different immunogenic peptides or proteins (e.g., described herein). In some embodiments, the pharmaceuticalcomposition comprises at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 different nucleic acid molecules encoding at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 different immunogenic peptides or proteins (e.g., described herein). In some embodiments, the pharmaceutical composition comprises or consists of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 different nucleic acid molecules encoding 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 different immunogenic peptides or proteins (e.g., described herein).5.5.4.1 DNA Molecules

[0358] In some embodiments, the nucleic acid molecule is a DNA molecule. In some embodiments, the DNA molecule is a linear coding DNA construct, contained within a plasmid, or contained within a viral vector. In some embodiments, the DNA molecule is a lineal' coding DNA construct. In some embodiments, the DNA molecule is contained within a plasmid. In some embodiments, the DNA molecule is contained with a viral vector. A more detailed description of viral vectors for both RNA and DNA molecules is provided in § 5.8.2.

[0359] The coding DNA may also comprise one or more heterologous nucleic acid elements to mediate expression of the coding region. These include, e.g., promoter(s), enhancer(s), polyadenylation signal(s), synthetic introns, transcriptional termination signals, polyadenylation sequences, and other transcription regulatory elements. A person of ordinary skill in the art is familiar’ with the transcriptional regulatory elements needed for expression of the coding DNA and can optimize the expression construct (e.g., linear DNA or a plasmid) accordingly.

[0360] In some embodiments, a promoter is operably linked to the respective coding nucleic acid sequence encoding the immunogenic peptide or protein. The person of ordinary skill in the art is aware of various promoters that can be employed, for example, a promoter from simian virus 40 (SV40), a mouse mammary tumor virus (MMTV) promoter, a human immunodeficiency virus (HIV) promoter, bovine immunodeficiency virus (BIV) long terminal repeat (LTR) promoter, a Moloney virus promoter, an avian leukosis virus (ALV) promoter, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter, Epstein Barr virus (EBV) promoter, or a Rous sarcoma virus (RSV) promoter. The promoter can also be a promoter from a human gene, for example, from human actin, human myosin, human hemoglobin, human muscle creatine, or human metalo thionein. The promoter can also be a tissue specific promoter, such as a muscle or skin specific promoter, natural or synthetic. Examples of such promoters are described in US patent application publication no. US20040175727, the entire contents of which is incorporated by reference herein for all purposes. Exemplary polyadenylation signals, include, but are notlimited to the bovine growth hormone (BGH) polyadenylation site, SV40 polyadenylation signals, and LTR polyadcnylation signals.

[0361] In some embodiments, the DNA is contained within a plasmid. A person of ordinary skill in the art is aware of suitable plasmids for expression of the DNA of interest. For example, Suitable plasmid DNA may be generated to allow efficient production of the encoded immunogens in cell lines, e.g., in insect cell lines, for example using vectors as described in W02009150222A2 and as defined in PCT claims 1 to 33, the disclosure relating to claim 1 to 33 of W02009150222A2 the entire contents of which is incorporated by reference herein for all purposes.5.5.4.2 RNA Molecules

[0362] In some embodiments, the nucleic acid molecule is an RNA molecule. In some embodiments, the RNA molecule is a translatable RNA molecule. In some embodiments, the RNA is selected from an mRNA, a self-replicating RNA, a circular RNA (e.g., a covalently closed RNA), a viral RNA, or a replicon RNA.

[0363] In some embodiments, the RNA molecule a circular RNA. Exemplary circular RNAs are described in e.g., US11458156, US20220143062, US20230212629, US20230072532, US11203767, US11352641, US20210371494, US11766449, US20230226096, WO2021189059, US20190345503, US20220288176, US11560567, WO2022271965, WO2022037692, W02023024500, WO2023115732, WO2023133684, WO2023143541, WO2023134611, and WO2022247943, the entire contents of each of which are incorporated herein by reference for all purposes.

[0364] In some embodiments, the RNA is a mRNA. The basic components of an mRNA molecule typically include at least one coding region (herein a coding region encoding at least one immunogenic peptide or protein described herein), a 5'-untranslated region (UTR), a 3'-UTR, a 5' cap, and a poly-A tail.

[0365] In some embodiments, the RNA (e.g., mRNA) comprises at least one heterologous UTR. The UTRs may harbor regulatory sequence elements that determine the RNA (e.g., mRNA) turnover, stability, localization, and / or expression of operably linked coding sequence(s). The heterologous UTRs may be derived from a naturally occurring genes or may be synthetically engineered. In some embodiments, the 5'-UTR comprises elements for controlling gene expression, e.g., ribosomal binding sites, miRNA binding sites. The 5’-UTR may be post- transcriptionally modified or varied, e.g., by enzymatic or post-transcriptional addition of a 5’capstructure. In some embodiments, the 3'-UTR comprises a polyadenylation signal. In some embodiments, the RNA (e.g., mRNA) comprises at least one coding region encoding the immunogenic peptide or protein described herein and 5'-UTR and / or a 3'-UTR. In some embodiments, the RNA (e.g., mRNA) comprises at least one coding sequence encoding an immunogenic peptide or protein described herein operably connected to at least one heterologous 5'-UTR and at least one 3'-UTR.

[0366] In some embodiments, the RNA e.g., mRNA, circular RNA) comprises a poly(A) sequence. The poly(A) sequence may comprise from about 10 to 500 adenosine nucleotides, 10 to 200 adenosine nucleotides, 20 to 200 adenosine nucleotides, 30 to 200 adenosine nucleotides, 40 to 200 adenosine nucleotides, or 50 to 200 adenosine nucleotides. In some embodiments, poly(A) sequence comprises at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or 500 adenosine nucleotides. In some embodiments, the RNA e.g., mRNA) comprises a poly(A) sequence. The poly(A) sequence may comprise from about 10 to 500 adenosine nucleotides, 10 to 200 adenosine nucleotides, 20 to 200 adenosine nucleotides, 30 to 200 adenosine nucleotides, 40 to 200 adenosine nucleotides, or 50 to 200 adenosine nucleotides, wherein the 3' terminal nucleotide of said nucleic acid molecule is an adenosine. In some embodiments, poly(A) sequence comprises at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or 500 adenosine nucleotides, wherein the 3’ terminal nucleotide of said nucleic acid molecule is an adenosine.

[0367] In some embodiments, the RNA (e.g., mRNA, circular RNA) comprises a poly(A)(U) sequence. In some embodiments, the RNA (e.g., mRNA, circular RNA) comprises a poly(A)(C) sequence.

[0368] In some embodiments, the RNA (e.g., mRNA) comprises a 5’-cap structure. In some embodiments, the 5'-cap structure stabilizes the RNA (e.g., mRNA), enhances expression of the encoded immunogen, and / or reduces the stimulation of the innate immune system (e.g., after administration to a subject).

[0369] Exemplary 5 ’-cap structures include, but are not limited to, capO (methylation of the first nucleobase, e.g., m7GpppN), capl (additional methylation of the ribose of the adjacent nucleotide of m7GpppN), cap2 (additional methylation of the ribose of the 2nd nucleotide downstream of the m7GpppN), cap3 (additional methylation of the ribose of the 3rd nucleotide downstream of the m7GpppN), cap4 (additional methylation of the ribose of the 4th nucleotide downstream of the m7GpppN), ARCA (anti-reverse cap analogue), modified ARCA (e.g.,phosphorothioate modified ARCA), inosine, Nl-methyi -guanosine, 2'-fluoro-guanosine, 7-deaza- guanosinc, 8-oxo-guanosinc, 2-amino-guanosinc, LNA-guanosinc, and 2-azido-guanosinc. In some embodiments, the 5' cap structure comprises m7G, capO, capl, cap2, a modified capO, or a modified capl structure.

[0370] In some embodiments, the RNA (e.g., mRNA) comprises nucleotide analogues / modifications, e.g., backbone modifications, sugar modifications, and / or base modifications. A backbone modification in the context of the present disclosure is a modification, in which phosphates of the backbone of the nucleotides of the RNA (e.g., mRNA) are chemically modified. A sugar modification in the context of the present disclosure is a chemical modification of the sugar of the nucleotides of the RNA (e.g., mRNA). A base modification in the context of the present disclosure is a chemical modification of the base moiety of the nucleotides of the RNA (e.g., mRNA).

[0371] In some embodiments, the RNA (e.g., mRNA) comprises at least one modified nucleotide. Exemplary nucleotide analogues / modifications include, but are not limited to, 2- amino-6-chloropurineriboside-5'-triphosphate, 2-Aminopurine-riboside-5'-triphosphate; 2- aminoadenosine-5'-triphosphate, 2'-Amino-2'-deoxycytidine-triphosphate, 2-thiocytidine-5'- triphosphate, 2-thiouridine-5 '-triphosphate...

Claims

CLAIMSWhat is claimed is:

1. An isolated protein comprising an amino acid sequence at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any protein set forth in Table 2 or set forth in any one of SEQ ID NOS: 129-240 that specifically binds human CD30L (hCD30L).

2. The isolated protein of claim 1, comprising an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any protein set forth in Table 2 or set forth in any one of SEQ ID NOS: 129-240.

3. The isolated protein of claim 1 or 2, comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any protein set forth in Table 2 or set forth in any one of SEQ ID NOS: 129-240.

4. The isolated protein of any one of claims 1-3, comprising an amino acid sequence about 100% identical to the amino acid sequence of any protein set forth in Table 2 or set forth in any one of SEQ ID NOS: 129-240.

5. An isolated protein comprising an amino acid sequence at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any protein set forth in Table 2 or set forth in any one of SEQ ID NOS: 129-186 that specifically binds human CD30L (hCD30L).

6. The isolated protein of claim 5, comprising an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any protein set forth in Table 2 or set forth in any one of SEQ ID NOS: 129-186.

7. The isolated protein of claim 5 or 6, comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any protein set forth in Table 2 or set forth in any one of SEQ ID NOS: 129-186.

8. The isolated protein of any one of claims 5-7, comprising an amino acid sequence about 100% identical to the amino acid sequence of any protein set forth in Table 2 or set forth in any one of SEQ ID NOS: 129-186.

9. The isolated protein of any one of the preceding claims, wherein the protein exhibits antiinflammatory properties (e.g., upon administration to a subject).

10. The isolated protein of any one of the preceding claims, wherein the protein inhibits or reduces (e.g., prevents) binding of hCD30L to human CD30 (hCD30).

11. The isolated protein of any one of the preceding claims, wherein the protein inhibits or reduces e.g., prevents) activation of hCD30 signaling.

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

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

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

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

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

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

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

19. The fusion protein of claim 18, wherein the heterologous protein comprises a half-life extension protein.

20. The fusion protein of any one of claims 18-19, wherein the heterologous protein comprises an immunoglobulin (Ig) (e.g., a human Ig (hlg)) Fc region.

21. The fusion protein of claim 20, wherein the Ig (e.g., hlg) Fc region comprises at least a portion of a hinge region, a CH2 region, and a CH3 region.

22. The fusion protein of claim 20 or 21, wherein the Ig (e.g., hlg) Fc region comprises a hinge region, a CH2 region, and a CH3 region.

23. The fusion protein of any one of claims 20-22, wherein the Ig is a hlg.

24. The fusion protein of claim 23, wherein the hlg is a human IgG (hlgG).

25. The fusion protein of any one of claims 23-24, wherein the hlgG is hlgGl or hIgG4.

26. The fusion protein of any one of claims 20-25, wherein the Ig (e.g., hlg) Fc region does not substantially mediate ADCC, docs not substantially mediate CDC, and / or docs not bind to one or more human Fc receptor (e.g., an Fey receptor (e.g., FcyRI, FcyRIIa, FcyRIIc, FcyRIIIa, and / or FcyRIIIb (e.g., FcyRI, FcyRIIa, and / or FcyRIIIa))).

27. The fusion protein of any one of claims 20-25, wherein the Ig (e.g., hlg) Fc region comprises one or more amino acid variation relative to a reference Ig (e.g., hlg) Fc region that reduces or abolishes one or more of the following effector functions relative to the reference Ig (e.g., hlg) Fc region: antibody dependent cell mediated cytotoxicity (ADCC), complement dependent cytotoxicity (CDC), and / or affinity to one or more human Fc receptor (e.g., an Fey receptor (e.g., FcyRI, FcyRIIa, FcyRIIc, FcyRIIIa, and / or FcyRIIIb (e.g., FcyRI, FcyRIIa, and / or FcyRIIIa))).

28. The fusion protein of any one of claims 20-25, wherein the Ig (e.g., hlg) Fc region is capable of mediating ADCC, capable of mediating CDC, and / or binds to one or more human Fc receptor (e.g., an Fey receptor (e.g., FcyRI, FcyRIIa, FcyRIIc, FcyRIIIa, and / or FcyRIIIb (e.g., FcyRI, FcyRIIa, and / or FcyRIIIa))).

29. The fusion protein of any one of claims 20-25 or 28, wherein the Ig (e.g., hlg) Fc region comprises one or more amino acid variation relative to a reference Ig (e.g., hlg) Fc region that enhances one or more of the following effector functions relative to the reference Ig (e.g., hlg) Fc region: ADCC, CDC, and / or affinity to one or more human Fc receptor (e.g., an Fey receptor (e.g., FcyRI, FcyRIIa, FcyRIIc, FcyRIIIa, and / or FcyRIIIb (e.g., FcyRI, FcyRIIa, and / or FcyRIIIa))).

30. The fusion protein of any one of claims 18-29, wherein the protein of any one of claims 1-15 is directly operably connected to the heterologous protein through a peptide bond.

31. The fusion protein of any one of claims 18-30, wherein the protein of any one of claims 1-15 is indirectly operably connected to the heterologous protein through a peptide linker.

32. The fusion protein of claim 31, wherein the amino acid sequence of the peptide linker comprises or consists of glycine or glycine and serine amino acid residues.

33. The fusion protein of any one of claims 18-32, wherein the fusion protein comprises from N- to C-terminus: the protein of any one of claims 1-15 and the heterologous protein.

34. The fusion protein of any one of claims 18-33, wherein the fusion protein comprises from N- to C-tcrminus: the protein of any one of claims 1-15, a peptide linker, and the heterologous protein.

35. The fusion protein of any one of claims 18-34, wherein the fusion protein comprises from N- to C-terminus: a signal peptide, the protein of any one of claims 1-15, a peptide linker, and the heterologous protein.

36. The fusion protein of any one of claims 18-35, wherein the fusion protein comprises from N- to C-terminus: the heterologous protein and the protein of any one of claims 1-15.

37. The fusion protein of any one of claims 18-36, wherein the fusion protein comprises from N- to C-terminus: the heterologous protein, a peptide linker, and the protein of any one of claims 1-15.

38. The fusion protein of any one of claims 18-37, wherein the fusion protein comprises from N- to C-terminus: a signal peptide, the heterologous protein, a peptide linker, and the protein of any one of claims 1-15.

39. The fusion protein of any one of claims 18-38, wherein the amino acid sequence of the fusion protein is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 93-128 or set forth in Table 10.

40. The fusion protein of any one of claims 18-39, wherein the amino acid sequence of the fusion protein is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 94, 97, 100, 103, 106, 109, 112, 115, 118, 121, 124, or 127.

41. A fusion protein comprising a first protein and a second protein, wherein the first protein comprises a first Ig (e.g., hlg) Fc region operably connected to a first protein of any one of claims 1-15; and wherein the second protein comprises a second Ig (e.g., hlg) Fc region operably connected to a second protein of any one of claims 1-15.

42. The fusion protein of claim 41, wherein the first Fc region and the second Fc region associate to form a dimer.

43. The fusion protein of any one of claims 41-42, wherein the first protein comprises an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of the second protein.

44. The fusion protein of claim 41-43, wherein the first Ig (e.g., hlg, mlg) Fc region comprises at least a portion of a hinge region, a CH2 region, and a CH3 region; and the second Ig (e.g., hlg) Fc region comprises at least a portion of a hinge region, a CH2 region, and a CH3 region.

45. The fusion protein of any one of claims 41-44, wherein the first Ig (e.g., hlg) Fc region comprises a hinge region, a CH2 region, and a CH3 region; and the second Ig (e.g., hlg) Fc region comprises a hinge region, a CH2 region, and a CH3 region.

46. The fusion protein of any one of claims 41-45, wherein the Ig of the first Ig Fc region is a hlg and the Ig of the second Ig Fc region is a hlg.

47. The fusion protein of claim 46, wherein the hlg of the first hlg Fc region is a hlgG and the hlg of the second hlg Fc region is a hlgG.

48. The fusion protein of claim 47, wherein the hlgG of the first hlg Fc region is h!gG4 and the hlgG of the first hlg Fc region is hIgG4.

49. The fusion protein of claim 47, wherein the hlgG of the first hlg Fc region is hlgGl and the hlgG of the first hlg Fc region is hlgGl.

50. The fusion protein of any one of claims 41-49, wherein the fusion protein does not substantially mediate ADCC, does not substantially mediate CDC, and / or does not bind to one or more Fc receptor (e.g., an Fey receptor (e.g., FcyRI, FcyRIIa, FcyRIIc, FcyRIIIa, and / or FcyRIIIb (e.g., FcyRI, FcyRIIa, and / or FcyRIIIa))).

51. The fusion protein of any one of claims 41-50, wherein the first Ig (e.g., hlg) Fc region and the second Ig (e.g., hlg) Fc region each comprises one or more amino acid variation relative to a reference Ig (e.g., hlg) Fc region that reduces or abolishes one or more of the following effector functions relative to the reference Ig (e.g., hlg) Fc region: ADCC, CDC, and / or binding affinity to one or more Fc receptor (e.g., an Fey receptor (e.g., FcyRI, FcyRIIa, FcyRIIc, FcyRIIIa, and / or FcyRIIIb (e.g., FcyRI, FcyRIIa, and / or FcyRIIIa))).

52. The fusion protein of any one of claims 41-49, wherein the fusion protein is capable of mediating ADCC, capable of mediating CDC, and / or binds to one or more Fc receptor (e.g., anFey receptor (e.g., FcyRI, FcyRIIa, FcyRIIc, FcyRIIIa, and / or FcyRIIIb (e.g., FcyRI, FcyRIIa, and / or FcyRIIIa))).

53. The fusion protein of any one of claims 41-49 or 52, wherein the first Ig (e.g., hlg) Fc region and the second Ig (e.g., hlg) Fc region each comprises one or more amino acid variation relative to a reference Ig (e.g., hlg) Fc region that reduces or abolishes one or more of the following effector functions relative to the reference Ig (e.g., hlg) Fc region: ADCC, CDC, and / or binding affinity to one or more Fc receptor (e.g., an Fey receptor (e.g., FcyRI, FcyRIIa, FcyRIIc, FcyRIIIa, and / or FcyRIIIb (e.g., FcyRI, FcyRIIa, and / or FcyRIIIa))).

54. The fusion protein of any one of claims 41-53, wherein the first Ig (e.g., hlg) Fc region comprises an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of the second Ig (e.g., hlg) Fc region.

55. The fusion protein of any one of claims 41-54, wherein the first protein comprises from N- to C-terminus: the first Ig (e.g., hlg) Fc region and the first protein of any one of claims 1-15; and the second protein comprises from N- to C-terminus: the second Ig (e.g., hlg) Fc region and the second protein of any one of claims 1-15.

56. The fusion protein of any one of claims 41-55, wherein the first protein comprises from N- to C-terminus: the first Ig (e.g., hlg) Fc region, a first peptide linker, and the first protein of any one of claims 1-15; and the second protein comprises from N- to C-terminus: the second Ig (e.g., hlg) Fc region, a second peptide linker, and the second protein of any one of claims 1-15.

57. The fusion protein of any one of claims 41-56, wherein the first protein comprises from N- to C-terminus: the first protein of any one of claims 1-15 and the first Ig (e.g., hlg) Fc region; and the second protein comprises from N- to C-terminus: the second protein of any one of claims 1-15 and the second Ig (e.g., hlg) Fc region.

58. The fusion protein of any one of claims 41-57, wherein the first protein comprises from N- to C-terminus: the first protein of any one of claims 1-15, a first peptide linker, and the first Ig (e.g., hlg) Fc region; and the second protein comprises from N- to C-terminus: the second protein of any one of claims 1-15, a second peptide linker, and the second Ig (e.g., hlg) Fc region.

59. The fusion protein of claim 56 or 58, wherein the amino acid sequence of the first peptide linker and the second peptide linker each comprises or consists of glycine or glycine and serine amino acid residues.

60. The fusion protein of any one of claims 41-59, wherein the amino acid sequence of the first protein is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 93-128 or set forth in Table 10; and the amino acid sequence of the first protein is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 93-128 or set forth in Table 10.

61. The fusion protein of any one of claims 41-60, wherein the amino acid sequence of the first protein is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 94, 97, 100, 103, 106, 109, 112, 115, 118, 121, 124, or 127; and the amino acid sequence of the first protein is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 94, 97, 100, 103, 106, 109, 112, 115, 118, 121, 124, or 127.

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

63. The immunogenic peptide or protein of claim 62, wherein the immunogenic peptide or protein does not specifically bind hCD30L or binds one or more of hCD30L with lower affinity relative to a reference protein of any one of claims 1-15.

64. The immunogenic peptide or protein of claim 62 or 63, wherein the immunogenic peptide or protein comprises a full-length protein of any one of claims 1-15.

65. The immunogenic peptide or protein of any one of claims 62-64, wherein the immunogenic peptide or protein comprises an immunogenic fragment of a protein of any one of claims 1-15.

66. The immunogenic peptide or protein of any one of claims 62-65, wherein the immunogenic peptide or protein comprises at least about 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, or 130 amino acids.

67. The immunogenic peptide or protein of any one of claims 62-66, wherein the immunogenic peptide or protein comprises from about 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, or 130 amino acids.

68. The immunogenic peptide or protein of any one of claims 62-67, wherein the amino acid sequence of the immunogenic peptide or protein comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acid variations (e.g., substitutions, additions, deletions) relative to a reference protein of any one of claims 1-15.

69. The immunogenic peptide or protein of any one of claims 62-68, wherein the immunogenic peptide or protein comprises an amino acid sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a contiguous stretch of at least about 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, or 130 amino acids set forth in any one of SEQ ID NOS: 129-240.

70. The immunogenic peptide or protein of any one of claims 62-69, wherein the immunogenic peptide or protein comprises an amino acid sequence that, other than the one or more amino acid variation (e.g., substitution, addition, deletion), is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 129-240.

71. The immunogenic peptide or protein of any one of claims 62-70, wherein the immunogenic peptide or protein is formulated with an adjuvant.

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

73. A nucleic acid molecule encoding the protein of any one of claims 1-15, the conjugate of claim 16, the radioligand of claim 17, the fusion protein of any one of claims 18-61, the immunogenic peptide or protein of any one of claims 62-71, or the antibody of claim 72.

74. The nucleic acid molecule of claim 73, wherein the nucleic acid molecule is an RNA (e.g., mRNA, circular RNA) molecule or a DNA molecule.

75. An mRNA molecule encoding the protein of any one of claims 1-15, the conjugate of claim 16, the radioligand of claim 17, the fusion protein of any one of claims 18-61, the immunogenic peptide or protein of any one of claims 62-71, or the antibody of claim 72.

76. The nucleic acid molecule of any one of claims 73-74 or the mRNA molecule of claim75, comprising a heterologous 5'-untranslated region (UTR), 3'-UTR, or both a 5'-UTR and 3'- UTR.

77. The nucleic acid molecule of any one of claims 73-74 or 76 or the mRNA molecule of any one of claims 75-76, comprising a poly(A) sequence.

78. The nucleic acid molecule of any one of claims 73-74 or 76-77 or the mRNA molecule of any one of claims 75-77, comprising a 5' cap structure.

79. The nucleic acid molecule of any one of claims 73-74 or 76-78 or the mRNA molecule of any one of claims 75-78, comprising at least one variant nucleotide.

80. The nucleic acid molecule of any one of claims 73-74 or 76-79 or the mRNA molecule of any one of claims 75-79, wherein the sequence of the nucleic acid molecule or mRNA molecule, respectively, is codon optimized.

81. A vector (e.g., expression vector) comprising the nucleic acid molecule of any one of claims 73-74 or 76-80 or the mRNA molecule of any one of claims 75-80.

82. The vector of claim 81, wherein the vector is a viral vector or a non-viral vector (e.g., a plasmid).

83. A viral particle conjugated to the protein of any one of claims 1-15, the conjugate of claim 16, the radioligand of claim 17, or the fusion protein of any one of claims 18-61.

84. A cell (e.g., host cell) comprising the protein of any one of claims 1-15, the conjugate of claim 16, the radioligand of claim 17, the fusion protein of any one of claims 18-61, the immunogenic peptide or protein of any one of claims 62-71, the antibody of claim 72, the nucleic acid molecule of any one of claims 73-74 or 76-80, the mRNA molecule of any one of claims 75-80, the vector of any one of claims 81-82, the viral particle of claim 83, the carrier of any one of claims 86-88, the lipid nanoparticle of any one of claims 89-90, the vaccine composition of claim 91, or the pharmaceutical composition of claim 92.

85. A cell (e.g., a therapeutic cell) expressing the protein of any one of claims 1-15, the conjugate of claim 16, the radioligand of claim 17, or the fusion protein of any one of claims 18- 61, on the surface of the cell.

86. A carrier comprising the protein of any one of claims 1-15, the conjugate of claim 16, the radioligand of claim 17, the fusion protein of any one of claims 18-61, the immunogenic peptide or protein of any one of claims 62-71, the antibody of claim 72, the nucleic acid molecule of any one of claims 73-74 or 76-80, the mRNA molecule of any one of claims 75-80, the vector of any one of claims 81-82, the viral particle of claim 83, the cell of any one of claims 84-85, the vaccine composition of claim 91, or the pharmaceutical composition of claim 92.

87. A carrier conjugated to the protein of any one of claims 1-15, the conjugate of claim 16, the radioligand of claim 17, or the fusion protein of any one of claims 18-61.

88. The carrier of claim 87, wherein the carrier is a lipid nanoparticle, liposome, lipoplex, or nanoliposomc.

89. A lipid nanoparticle comprising the protein of any one of claims 1-15, the conjugate of claim 16, the radioligand of claim 17, the fusion protein of any one of claims 18-61, the immunogenic peptide or protein of any one of claims 62-71, the antibody of claim 72, the nucleic acid molecule of any one of claims 73-74 or 76-80, the mRNA molecule of any one of claims 75-80, the vector of any one of claims 81-82, the viral particle of claim 83, the cell of any one of claims 84-85, the vaccine composition of claim 91, or the pharmaceutical composition of claim 92.

90. A lipid nanoparticle conjugated to the protein of any one of claims 1-15, the conjugate of claim 16, the radionuclide of claim 17, or the fusion protein of any one of claims 18-61.

91. A vaccine composition comprising the immunogenic peptide or protein of any one of claims 62-71 (or a nucleic acid molecule encoding the same (or a vector encoding the nucleic acid molecule) or a carrier comprising any of the foregoing).

92. A pharmaceutical composition comprising the protein of any one of claims 1-15, the conjugate of claim 16, the radioligand of claim 17, the fusion protein of any one of claims 18-61, the immunogenic peptide or protein of any one of claims 62-71, the antibody of claim 72, the nucleic acid molecule of any one of claims 73-74 or 76-80, the mRNA molecule of any one of claims 75-80, the vector of any one of claims 81-82, the viral particle of claim 83, the carrier of any one of claims 86-88, the lipid nanoparticle of any one of claims 89-90, the cell of any one of claims 84-85, the vaccine composition of claim 91, or the pharmaceutical composition of claim 92; and a pharmaceutically acceptable excipient.

93. A kit comprising the protein of any one of claims 1-15, the conjugate of claim 16, the radioligand of claim 17, the fusion protein of any one of claims 18-61, the immunogenic peptide or protein of any one of claims 62-71, the antibody of claim 72, the nucleic acid molecule of any one of claims 73-74 or 76-80, the mRNA molecule of any one of claims 75-80, the vector of any one of claims 81-82, the viral particle of claim 83, the carrier of any one of claims 86-88, the lipid nanoparticle of any one of claims 89-90, the cell of any one of claims 84-85, the vaccine composition of claim 91, or the pharmaceutical composition of claim 92; and optionally instructions for using any one or more of the foregoing.

94. A method of delivering a protein, fusion protein, conjugate, radioligand, nucleic acid molecule, mRNA molecule, expression vector, viral particle, cell, carrier, lipid nanoparticlc, immunogenic peptide or protein, an antibody, a vaccine composition, or pharmaceutical composition to a subject, the method comprising administering to the protein of any one of claims 1-15, the conjugate of claim 16, the radioligand of claim 17, the fusion protein of any one of claims 18-61, the immunogenic peptide or protein of any one of claims 62-71, the antibody of claim 72, the nucleic acid molecule of any one of claims 73-74 or 76-80, the mRNA molecule of any one of claims 75-80, the vector of any one of claims 81-82, the viral particle of claim 83, the carrier of any one of claims 86-88, the lipid nanoparticle of any one of claims 89-90, the cell of any one of claims 84-85, the vaccine composition of claim 91, or the pharmaceutical composition of claim 92, to thereby deliver the protein, fusion protein, conjugate, radioligand, immunogenic peptide or protein, antibody, mRNA molecule, vector, viral particle, cell, carrier, lipid nanoparticle, vaccine composition, or pharmaceutical composition to a subject.

95. A method of inhibiting or reducing (e.g., preventing) binding of CD30L to CD30 in a subject in need thereof, the method comprising administering to the subject the protein of any one of claims 1-15, the conjugate of claim 16, the radioligand of claim 17, the fusion protein of any one of claims 18-61, the immunogenic peptide or protein of any one of claims 62-71, the antibody of claim 72, the nucleic acid molecule of any one of claims 73-74 or 76-80, the mRNA molecule of any one of claims 75-80, the vector of any one of claims 81-82, the viral particle of claim 83, the carrier of any one of claims 86-88, the lipid nanoparticle of any one of claims 89- 90, the cell of any one of claims 84-85, the vaccine composition of claim 91, or the pharmaceutical composition of claim 92, to thereby inhibit or reduce (e.g., prevent) binding of CD30L to CD30 in the subject.

96. A method of inhibiting or reducing (e.g., preventing) signaling mediated by the binding of CD30L to CD30 in a subject in need thereof, the method comprising administering to the subject the protein of any one of claims 1-15, the conjugate of claim 16, the radioligand of claim 17, the fusion protein of any one of claims 18-61, the immunogenic peptide or protein of any one of claims 62-71, the antibody of claim 72, the nucleic acid molecule of any one of claims 73-74 or 76-80, the mRNA molecule of any one of claims 75-80, the vector of any one of claims 81-82, the viral particle of claim 83, the carrier of any one of claims 86-88, the lipid nanoparticle of any one of claims 89-90, the cell of any one of claims 84-85, the vaccine composition of claim 91, orthe pharmaceutical composition of claim 92, to thereby inhibit or reduce (e.g., prevent) signaling mediated by the binding of CD30L to CD30 in the subject.

97. A method of suppressing or reducing (e.g., preventing) a pro-inflammatory immune response in a subject in need thereof, the method comprising administering to the subject the protein of any one of claims 1-15, the conjugate of claim 16, the radioligand of claim 17, the fusion protein of any one of claims 18-61, the immunogenic peptide or protein of any one of claims 62-71, the antibody of claim 72, the nucleic acid molecule of any one of claims 73-74 or 76-80, the mRNA molecule of any one of claims 75-80, the vector of any one of claims 81-82, the viral particle of claim 83, the carrier of any one of claims 86-88, the lipid nanoparticle of any one of claims 89-90, the cell of any one of claims 84-85, the vaccine composition of claim 91, or the pharmaceutical composition of claim 92, to thereby suppress or reduce (e.g., prevent) a pro- inflammatory immune response in the subject.

98. A method of depleting a population of immune cells in a subject in need thereof, the method comprising administering to the subject the protein of any one of claims 1-15, the conjugate of claim 16, the radioligand of claim 17, the fusion protein of any one of claims 18-61, the immunogenic peptide or protein of any one of claims 62-71, the antibody of claim 72, the nucleic acid molecule of any one of claims 73-74 or 76-80, the mRNA molecule of any one of claims 75-80, the vector of any one of claims 81-82, the viral particle of claim 83, the carrier of any one of claims 86-88, the lipid nanoparticle of any one of claims 89-90, the cell of any one of claims 84-85, the vaccine composition of claim 91, or the pharmaceutical composition of claim 92, to thereby deplete a population of immune cells in the subject.

99. The method of claim 98, wherein the population of immune cells express CD30L on the surface.

100. The method of any one of claims 98-99, wherein the population of immune cells express one or more marker of activation.

101. The method of any one of claims 98-100, wherein the population of immune cells are activated.

102. The method of any one of claims 98-101, wherein the population of immune cells comprises T cells and / or B -cells.

103. The method of any one of claims 98-102, wherein the subject has (e.g., has been diagnosed with) a pro-inflammatory (e.g., autoimmune) disease.

104. The method of any one of claims 98- 103, wherein the pro-inflammatory disease is an autoimmune disease.

105. The method of any one of claims 98-104, wherein the pro-inflammatory (e.g., autoimmune) disease comprises flares (e.g., relapsing-remitting forms of pro-inflammatory (e.g., autoimmune) diseases).

106. The method of any one of claims 98-105, wherein the protein, fusion protein, conjugate, radioligand, immunogenic peptide or protein, antibody, mRNA molecule, vector, viral particle, cell, carrier, lipid nanoparticle, vaccine composition, or pharmaceutical composition is administered to the subject prior to, at the start of, at initial signs of, or during a flare.

107. The method of any one of claims 98-106, wherein the pro-inflammatory (e.g., autoimmune) disease is multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease, systemic lupus erythematosus, myasthenia gravis, or psoriasis.

108. The method of any one of claims 98-107, wherein the pro-inflammatory (e.g., autoimmune) disease is relapsing-remitting multiple sclerosis, relapsing-remitting rheumatoid arthritis, relapsing-remitting inflammatory bowel disease, relapsing-remitting systemic lupus erythematosus, relapsing-remitting myasthenia gravis, or relapsing-remitting psoriasis.

109. A method of treating, ameliorating, or preventing a pro-inflammatory disease in a subject in need thereof, the method comprising administering to the subject the protein of any one of claims 1-15, the conjugate of claim 16, the radioligand of claim 17, the fusion protein of any one of claims 18-61, the immunogenic peptide or protein of any one of claims 62-71, the antibody of claim 72, the nucleic acid molecule of any one of claims 73-74 or 76-80, the mRNA molecule of any one of claims 75-80, the vector of any one of claims 81-82, the viral particle of claim 83, the carrier of any one of claims 86-88, the lipid nanoparticle of any one of claims 89-90, the cell of any one of claims 84-85, the vaccine composition of claim 91, or the pharmaceutical composition of claim 92, to thereby treat, ameliorate, or prevent the pro-inflammatory immune response in the subject.

110. The method of claim 109, wherein the pro-inflammatory disease is an autoimmune disease.

111. The method of any one of claims 109- 110, wherein the pro-inflammatory (e.g., autoimmune) disease comprises flares (e.g., relapsing-remitting forms of pro-inflammatory (e.g., autoimmune) diseases).

112. The method of any one of claims 109- 111 , wherein the protein, fusion protein, conjugate, radioligand, immunogenic peptide or protein, antibody, mRNA molecule, vector, viral particle, cell, carrier, lipid nanoparticle, vaccine composition, or pharmaceutical composition is administered to the subject prior to, at the start of, at initial signs of, or during a flare.

113. The method of any one of claims 109-112, wherein the pro-inflammatory (e.g., autoimmune) disease is multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease, systemic lupus erythematosus, myasthenia gravis, or psoriasis.

114. The method of any one of claims 109-113, wherein the pro-inflammatory (e.g., autoimmune) disease is relapsing-remitting multiple sclerosis, relapsing-remitting rheumatoid arthritis, relapsing-remitting inflammatory bowel disease, relapsing-remitting systemic lupus erythematosus, relapsing-remitting myasthenia gravis, or relapsing-remitting psoriasis.

115. A method of inducing or enhancing an immune response in a subject in need thereof, the method comprising administering to the subject (i) the immunogenic peptide or protein of any one of claims 62-71 (or a conjugate or a fusion protein thereof); (ii) a nucleic acid molecule encoding (i); (iii) a vector comprising (ii); (iv) a carrier comprising (i), (ii), or (iii); a vaccine composition comprising (i), (ii), (iii), or (iv); or a pharmaceutical composition comprising (i), (ii), (iii), (iv), or (v), to thereby induce or enhance an immune response in the subject.

116. A method of vaccinating a subject in need thereof (e.g., against a viral infection), the method comprising administering to the subject (i) the immunogenic peptide or protein of any one of claims 62-71 (or a conjugate or a fusion protein thereof); (ii) a nucleic acid molecule encoding (i); (iii) a vector comprising (ii); (iv) a carrier comprising (i), (ii), or (iii); a vaccine composition comprising (i), (ii), (iii), or (iv); or a pharmaceutical composition comprising (i), (ii), (iii), (iv), or (v), to thereby vaccinate the subject in need thereof (e.g., against a virus).

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

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

119. The method of claim 117 or 118, wherein the method is an in vitro method.

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

121. The method or use of any one of claims 117-120, wherein the sample is a blood, cell, tissue, or saliva, or nasal swab.

122. The method or use of any one of claims 117-121, wherein the antibody of claim is utilized to determine the presence or absence of the protein of any one of claims 1-15 (or the fragment or variant thereof).

123. The method or use of any one of claims 94-122, wherein the subject is a human.

Images