Heterodimer fc molecules and uses thereof

Abstract

The present disclosure provides heteromultimeric complexes comprising a first polypeptide and a second polypeptide wherein the first polypeptide and the second polypeptide are different and comprise a heteromultimeric modification that reduces homomultimeric formation and promotes heteromultimeric formation. The disclosure also provides fusion proteins, multispecific ligand binding proteins, conjugates and pharmaceutical compositions comprising such heteromultimeric complexes, including methods of preparation and use for the treatment of a variety of diseases or conditions.

Description

[0001] Cross-references to related applications This PCT application claims priority to U.S. Provisional Application No. 63 / 511,726, filed July 3, 2023, which is incorporated herein by reference in its entirety.

[0002] References to sequence lists submitted electronically The contents of the sequence list submitted electronically in the .XML file (4241_042PC01_SequenceListing_ST26.xml; 85,846 bytes; July 2, 2024) submitted with this application are incorporated herein by reference in their entirety. Technical Field

[0003] This disclosure provides heteropolymeric complexes (e.g., heterodimeric proteins) comprising modified Fc and the use of such complexes. Background Technology

[0004] Antibody-based therapies have been successfully used to treat a variety of diseases, including cancer and autoimmune / inflammatory conditions. However, improvements are still needed, particularly in enhancing their clinical efficacy. The development of multispecific antibodies (e.g., bispecific antibodies) holds immense clinical potential (e.g., the ability to recognize two distinct antigens, recruit different types of effector cells, and / or modulate multiple signaling pathways). However, the production of multispecific antibodies can be challenging. The widespread use of multispecific antibodies has been hampered by the difficulty in developing platforms for producing such antibodies that exhibit favorable half-lives, high stability, lack of immunogenicity, and feasibility for large-scale production and purification. Therefore, a new and improved platform remains needed to develop heteropolymeric complexes (e.g., multispecific antibodies) for the treatment of a wide range of diseases and disorders. Summary of the Invention

[0005] This article provides a heterodimeric protein comprising a first polypeptide and a second polypeptide, wherein (a) the first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 16, and (b) the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 20.

[0006] Also provided herein is a heterodimeric protein comprising a first polypeptide and a second polypeptide, wherein (a) the first polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 27, and (b) the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 29 or SEQ ID NO: 32. In some aspects, the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO. 29. In some aspects, the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO. 32.

[0007] Some aspects of the disclosure relate to a heterodimeric protein comprising a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide are different, wherein the first polypeptide comprises a first CH3 domain and the second polypeptide comprises a second CH3 domain, wherein: (1) amino acid residue N50, S60, or both of the first CH3 domain are unmodified or modified with a positive charge modification and amino acid residue N50, S60, or both of the second CH3 domain are unmodified or modified with a negative charge modification, such that the first CH3 domain and the second CH3 domain have opposite charges; (2) amino acid residue N50, S60, or both of the first CH3 domain are unmodified or modified with a negative charge modification and amino acid residue N50, S60, or both of the second CH3 domain are unmodified or modified with a positive charge modification, such that the first CH3 domain and the second CH3 domain have opposite charges; (3) amino acid residue D59, R69, or both of the first CH3 domain are unmodified or modified with a positive charge modification and amino acid residue D59, R69, or both of the second CH3 domain are unmodified or modified with a negative charge modification, such that the first CH3 domain and the second CH3 domain have opposite charges; (4) amino acid residue D59, R69, or both of the first CH3 domain are unmodified or modified with a negative charge modification and amino acid residue D59, R69, or both of the second CH3 domain are unmodified or modified with a positive charge modification, such that the first CH3 domain and the second CH3 domain have opposite charges; (5) amino acid residue K52 of the first CH3 domain is unmodified or modified with a positive charge modification and amino acid residue F65 of the second CH3 domain is unmodified or modified with a negative charge modification, such that the first CH3 domain and the second CH3 domain have opposite charges; and / or (6) amino acid residue K52 of the first CH3 domain is unmodified or modified with a negative charge modification and amino acid residue F65 of the second CH3 domain is unmodified or modified with a positive charge modification, such that the first CH3 domain and the second CH3 domain have opposite charges.

[0008] In some aspects, the modification in the first CH3 domain comprises (a) N50K or N50R, (b) K52R, (c) D59K or D59R, (d) S60K or S60R, (e) R69K, or (f) any combination thereof; and wherein the modification in the second CH3 domain comprises (a) N50D or N50E, (b) D59E, (c) S60D or S60E, (d) R69D or R69E, (e) F65D or F65E, or (f) any combination thereof. In some aspects, the modification in the first CH3 domain comprises (a) N50D or N50E, (b) K52D or K52E, (c) D59E, (d) S60D or S60E, (e) R69D or R69E, or (f) any combination thereof; and wherein the modification in the second CH3 domain comprises (a) N50K or N50R, (b) D59K or D59R, (c) S60K or S60R, (d) R69K, (e) F65K or F65R, or (f) any combination thereof.

[0009] In some aspects, (a) amino acid residue N50 of the first CH3 domain is modified to N50K or N50R and amino acid residue S60 of the second CH3 domain is modified to S60D or S60E; (b) amino acid residue S60 of the first CH3 domain is modified to S60D or S60E and amino acid residue N50 of the second CH3 domain is modified to N50K or N50R; (c) amino acid residue K52 of the first CH3 domain is unmodified or modified to K52R and amino acid residue F65 of the second CH3 domain is modified to F65D or F65E; (d) amino acid residue D59 of the first CH3 domain is modified to D59K or D59R and amino acid residue R69 of the second CH3 domain is modified to R69D or R69E; (e) amino acid residue R69 of the first CH3 domain is modified to R69D or R69E and amino acid residue D59 of the second CH3 domain is modified to D59K or D59R, or (f) any combination of (a) to (e). In some aspects, the first CH3 domain or the second CH3 domain comprises the amino acid sequence set forth in SEQ ID NO: 28, SEQ ID NO: 31, or SEQ ID NO: 33. In some aspects, the first CH3 domain comprises the amino acid sequence set forth in SEQ ID NO: 28 and the second CH3 domain comprises the amino acid sequence set forth in SEQ ID NO: 31 or SEQ ID NO: 33.

[0010] For any of the heterodimeric proteins described herein, in some aspects, (a) the first polypeptide further comprises a first hinge domain, a first CH2 domain, or both; (b) the second polypeptide further comprises a second hinge domain, a second CH2 domain, or both; or (c) both (a) and (b).

[0011] In some aspects, the first hinge domain and the second hinge domain are each selected from an IgGl hinge, an IgG2 hinge, an IgG3 hinge, an IgG4 hinge, an IgD hinge, or a synthetic linker. In some aspects, the synthetic linker comprises a glycine-serine linker, a glycine-alanine linker, an alanine-serine linker, or a combination thereof. In some aspects, the first hinge domain and the second hinge domain are the same or different. In some aspects, (a) the first hinge domain comprises the amino acid sequence set forth in SEQ ID NO: 22, (b) the second hinge domain comprises the amino acid sequence set forth in SEQ ID NO: 22, or (c) both (a) and (b).

[0012] In some aspects, the first CH2 domain and the second CH2 domain are the same or different. In some aspects, (a) the first CH2 domain comprises the amino acid sequence set forth in SEQ ID NO: 25, (b) the second CH2 domain comprises the amino acid sequence set forth in SEQ ID NO: 25, or (c) both (a) and (b).

[0013] For any of the heterodimeric proteins described herein, in some aspects, the heterodimeric protein further comprises a first bioactive molecule. In some aspects, the first bioactive molecule is attached to the N-terminus of the first polypeptide, the C-terminus of the first polypeptide, the N-terminus of the second polypeptide, the C-terminus of the second polypeptide, or any combination thereof. In some aspects, the first bioactive molecule is attached directly or via a linker. In some aspects, the linker is selected from an IgGl hinge, an IgG2 hinge, an IgG3 hinge, an IgG4 hinge, an IgD hinge, or a synthetic linker.

[0014] In some aspects, the heterodimeric protein comprising the first bioactive molecule further comprises a second bioactive molecule. In some aspects, the second bioactive molecule is attached to the N-terminus of the first polypeptide, the C-terminus of the first polypeptide, the N-terminus of the second polypeptide, the C-terminus of the second polypeptide, the first bioactive molecule, or any combination thereof. In some aspects, the second bioactive molecule is attached directly or via a linker. In some aspects, the linker is selected from an IgGl hinge, an IgG2 hinge, an IgG3 hinge, an IgG4 hinge, an IgD hinge, or a synthetic linker.

[0015] In some aspects, the heterodimeric protein further comprises one or more additional biologically active molecules. In some aspects, the one or more additional biologically active molecules is attached to the N-terminus of the first polypeptide, the C-terminus of the first polypeptide, the N-terminus of the second polypeptide, the C-terminus of the second polypeptide, the first biologically active molecule, the second biologically active molecule, or any combination thereof.

[0016] In some aspects, the first biologically active molecule, the second biologically active molecule, the one or more additional biologically active molecules, or a combination thereof comprises a ligand binding protein, a cytokine, or both.

[0017] In some aspects, the ligand binding protein comprises a T cell receptor, an antibody, or both. In some aspects, the antibody comprises an IgNAR, a Fab fragment, a Fab' fragment, a F(ab)'2 fragment, a F(ab)'3 fragment, an Fv, a single-chain variable fragment (scFv), a bis-scFv, a (scFv)2, a minibody, a diabody, a triabody, a tetrabody, an intrabody, a disulfide stabilized Fv protein (dsFv), a unibody, a nanobody, an aptamer, a single domain antibody (sdAB), or a combination thereof. In some aspects, the antibody comprises a T cell engager (e.g., a bispecific T cell engager (BiTE) antibody), a dual-affinity retargeting molecule (DART), a CrossMAb antibody, a DutaMab™ antibody, a DuoBody antibody, a Triomab, a TandAb, a bispecific nanobody, a tandem scFv, a diabody, a single-chain diabody, a HSA antibody, a (scFv)2HSA antibody, a scFv-IgG antibody, a Dock and Lock bispecific antibody, a DVD-IgG antibody, a TBTI DVD-IgG, an IgG-fynomer, a tetravalent bispecific tandem IgG antibody, a dual targeting domain antibody, a chemically linked bispecific (Fab')2 molecule, a cross-linked mAb, a dual acting Fab IgG (DAF-IgG), a vicinal Fab-IgG, a bispecific CovX-Body, a bispecific hexavalent trimer, 2 scFv linked to diphtheria toxin, an ART-Ig, an IgM T cell engager, a Humabody™ human heavy chain only antibody (HCAb), a UniAb™ HCAb, a shark heavy chain only antibody (VNAR), or a combination thereof.

[0018] In some aspects, the ligand binding protein binds to a tumor antigen. In some aspects, the ligand binding protein binds to an antigen expressed on an immune cell. In some aspects, the tumor antigen comprises guanylate cyclase C (GC-C), epidermal growth factor receptor (EGFR or erbB-1), human epidermal growth factor receptor 2 (HER2 or erbB2), erbB-3, erbB-4, MUC-1, melanoma-associated chondroitin sulfate proteoglycan (MCSP), mesothelin (MSLN), folate receptor 1 (FOLR1), CD4, CD19, CD20, CD22, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD123, CD138, CD171, CEA, CSPG4, CXCR5, c-Met, HERV-Envelope protein, eriostin, Bigh3, SPARC, BCR, CD79, CD37, EGFRvin, EGP2, EGP40, IGFr, LICAM, AXL, tissue factor (TF), CD74, EpCAM, EphA2, MRP3 cadherin 19 (CDH19), epidermal growth factor 2 (FIER2), 5T4, 8H9, integrin, BCMA, B7-H3, B7-H6, CAIX, CA9, FAP, FBP, fetal AchR, FRcc, GD2, GD3, Glypican-1 (GPC1), Glypican-2 (GPC2), Glypican-3 (GPC3), HLA-A1+MAGE1, HLA-A1+NY-ESO-1, IL-13Rcc2, Lewis-Y, KDR, MCSP, mesothelin, Mud, Muc16, NCAM, NKG2D ligand, NY-ESO-1, PRAME, PSC1, PSCA, PSMA, ROR1, ROR2, SP17, survivin, TAG72, TEM, carcinoembryonic antigen, HMW-MAA, VEGF, CLDN18.2, Programmed Death Ligand 1 (PDL-1), or combinations thereof. In some aspects, the antigen expressed on an immune cell comprises CD3, CD27, B7H3, CD2, CD4, CD5, CD8, CD11b, CD14, CD16, CD19, CD28, CD32, CD45, CD56, CD64, KLRG-1, NKG2D, NKp30, DNAM-1, TIM-3, LAG-3, TIGIT, PD-1, CTLA-4, ILT2, ILT3, ILT4, LAIR1, VISTA, 4-1BB, OX40, CD40L, ICOS, LIGHT, or combinations thereof.

[0019] In some aspects, the first biologically active molecule, the second biologically active molecule, the one or more additional biologically active molecules, or a combination thereof comprises a cytokine. In some aspects, the cytokine comprises IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21, IL-23, IFNa, IFP, IFNy, TNF, a combination thereof, IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21, IL-23, IFNa, IFP, IFNy, TNF, a combination thereof.

[0020] Also provided herein is a fusion protein comprising any of the heterodimeric proteins described herein.

[0021] Also provided herein is a bispecific antibody comprising (a) a first antigen binding domain, (b) a second antigen binding domain, and (c) any of the heterodimeric proteins described herein. In some aspects, the first antigen binding domain, the second antigen binding domain, or both are conjugated to the heterodimeric protein. In some aspects, (a) the first antigen binding domain is directly conjugated, (b) the first antigen binding domain is conjugated via a first linker, (c) the second antigen binding domain is directly conjugated, (d) the second antigen binding domain is conjugated via a second linker, or (e) any combination of (a) to (d). In some aspects, the first linker, the second linker, or both are selected from an IgGl hinge, an IgG2 hinge, an IgG3 hinge, an IgG4 hinge, an IgD hinge, or a synthetic linker.

[0022] In some aspects, the first antigen binding domain, the second antigen binding domain, or both bind to a tumor antigen. In some aspects, the first antigen binding domain, the second antigen binding domain, or both bind to an antigen expressed on an immune cell. In some aspects, the first antigen binding domain binds to a tumor antigen and the second antigen binding domain binds to an antigen expressed on an immune cell.

[0023] In some aspects, the tumor antigen comprises guanylyl cyclase C (GC-C), epidermal growth factor receptor (EGFR or erbB-1), human epidermal growth factor receptor 2 (HER2 or erbB2), erbB-3, erbB-4, MUC-1, melanoma-associated chondroitin sulfate proteoglycan (MCSP), mesothelin (MSLN), folate receptor 1 (FOLR1), CD4, CD19, CD20, CD22, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD123, CD138, CD171, CEA, CSPG4, CXCR5, c-Met, HERV- Envelope protein, eriostin, Bigh3, SPARC, BCR, CD79, CD37, EGFRvin, EGP2, EGP40, IGFr, LICAM, AXL, tissue factor (TF), CD74, EpCAM, EphA2, MRP3, CA19 (CDH19), epidermal growth factor 2 (FIER2), 5T4, 8H9, integrin, BCMA, B7-H3, B7-H6, CAIX, CA9, FAP, FBP, fetal AchR, FRcc, GD2, GD3, Glypican-1 (GPC1), Glypican-2 (GPC2), Glypican-3 (GPC3), HLA-A1+MAGE1, HLA-A1+NY-ESO-1, IL-13Rcc2, Lewis-Y, KDR, MCSP, mesothelin, Mud, Muc16, NCAM, NKG2D ligand, NY-ESO-1, PRAME, PSC1, PSCA, PSMA, ROR1, ROR2, SP17, survivin, TAG72, TEM, carcinoembryonic antigen, HMW-MAA, VEGF, CLDN18.2, Programmed Death Ligand 1 (PDL-1), or a combination thereof. In some aspects, the antigen expressed on the immune cell comprises CD3, CD27, B7H3, CD2, CD4, CD5, CD8, CD11b, CD14, CD16, CD19, CD28, CD32, CD45, CD56, CD64, KLRG-1, NKG2D, NKp30, DNAM-1, TIM-3, LAG-3, TIGIT, PD-1, CTLA-4, ILT2, ILT3, ILT4, LAIR1, VISTA, 4-1BB, OX40, CD40L, ICOS, LIGHT, or a combination thereof.

[0024] In some aspects, the bispecific antibodies described herein comprise an Ig NAR, a Fab fragment, a Fab' fragment, a F(ab)'2 fragment, a F(ab)'3 fragment, a Fv, a single chain variable fragment (scFv), a bis-scFv, a (scFv)2, a minibody, a diabody, a triabody, a tetrabody, an intrabody, a disulfide stabilized Fv protein (dsFv), a unibody, a nanobody, an aptamer, a Humabody™ human heavy chain only antibody (HCAb), a UniAb™ HCAb, a shark heavy chain only antibody (VNAR), or a combination thereof.

[0025] Also provided herein is a multispecific antibody comprising (a) a first antigen binding domain, (b) a second antigen binding domain, (c) a third antigen binding domain, and (d) any of the heterodimeric proteins described herein.

[0026] In some aspects, the first antigen binding domain, the second antigen binding domain, the third antigen binding domain, or a combination thereof is conjugated to the heterodimeric protein. In some aspects, (a) the first antigen binding domain is directly conjugated, (b) the first antigen binding domain is conjugated via a first linker, (c) the second antigen binding domain is directly conjugated, (d) the second antigen binding domain is conjugated via a second linker, (e) the third antigen binding domain is directly conjugated, (f) the third antigen binding domain is conjugated via a third linker, or (g) any combination of (a) to (f). In some aspects, the first linker, the second linker, the third linker, or a combination thereof is selected from an IgGl hinge, an IgG2 hinge, an IgG3 hinge, an IgG4 hinge, an IgD hinge, or a synthetic linker.

[0027] In some aspects, the first antigen binding domain is attached to the N-terminus of a first polypeptide via a first linker, the second antigen binding domain is attached to the N-terminus of a second polypeptide via a second linker, and the third antigen binding domain is attached to the N-terminus of the second polypeptide via a third linker, and wherein the first linker, the second linker, and the third linker are selected from an IgGl hinge, an IgG2 hinge, an IgG3 hinge, an IgG4 hinge, an IgD hinge, or a synthetic linker. In some aspects, the first antigen binding domain is attached to the N-terminus of a first polypeptide via a first linker, the second antigen binding domain is attached to the C-terminus of the first polypeptide via a second linker, and the third antigen binding domain is attached to the C-terminus of a second polypeptide via a third linker, and wherein the first linker, the second linker, and the third linker are selected from an IgGl hinge, an IgG2 hinge, an IgG3 hinge, an IgG4 hinge, an IgD hinge, or a synthetic linker.

[0028] In some aspects, the first, second, third, or combination thereof, antigen binding domain binds to a tumor antigen. In some aspects, the first, second, third, or combination thereof, antigen binding domain binds to an antigen expressed on an immune cell.

[0029] In some aspects, the multispecific antibody comprises an additional antigen binding domain. In some aspects, the additional antigen binding domain binds to a tumor antigen or an antigen expressed on an immune cell.

[0030] In some aspects, the tumor antigen comprises guanylate cyclase C (GC-C), epidermal growth factor receptor (EGFR or erbB-1), human epidermal growth factor receptor 2 (HER2 or erbB2), erbB-3, erbB-4, MUC-1, melanoma-associated chondroitin sulfate proteoglycan (MCSP), mesothelin (MSLN), folate receptor 1 (FOLR1), CD4, CD19, CD20, CD22, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD123, CD138, CD171, CEA, CSPG4, CXCR5, c-Met, HERV- Envelope protein, eriostin, Bigh3, SPARC, BCR, CD79, CD37, EGFRvin, EGP2, EGP40, IGFr, LICAM, AXL, tissue factor (TF), CD74, EpCAM, EphA2, MRP3 cadherin 19 (CDH19), epidermal growth factor 2 (FIER2), 5T4, 8H9, integrin, BCMA, B7-H3, B7-H6, CAIX, CA9, FAP, FBP, fetal AchR, FRcc, GD2, GD3, Glypican-1 (GPC1), Glypican-2 (GPC2), Glypican-3 (GPC3), HLA-A1+MAGE1, HLA-A1+NY-ESO-1, IL-13Rcc2, Lewis-Y, KDR, MCSP, mesothelin, Mud, Muc16, NCAM, NKG2D ligand, NY-ESO-1, PRAME, PSC1, PSCA, PSMA, ROR1, ROR2, SP17, survivin, TAG72, TEM, carcinoembryonic antigen, HMW-MAA, VEGF, CLDN18.2, Programmed Death Ligand 1 (PDL-1), or a combination thereof. In some aspects, the antigen expressed on the immune cell comprises CD3, CD27, B7H3, CD2, CD4, CD5, CD8, CD11b, CD14, CD16, CD19, CD28, CD32, CD45, CD56, CD64, KLRG-1, NKG2D, NKp30, DNAM-1, TIM-3, LAG-3, TIGIT, PD-1, CTLA-4, ILT2, ILT3, ILT4, LAIR1, VISTA, 4-1BB, OX40, CD40L, ICOS, LIGHT, or a combination thereof.

[0031] Some aspects of the present disclosure relate to a conjugate comprising any of the heterodimeric proteins, fusion proteins, bispecific antibodies, or multispecific antibodies provided herein linked to one or more conjugate moieties. In some aspects, the one or more conjugate moieties comprise a clearance modulator, a chemotherapeutic agent, a toxin, a radioisotope, a lanthanide element, a luminescent label, a fluorescent label, an enzyme substrate label, a DNA alkylating agent, a topoisomerase inhibitor, a tubulin binding agent, an anticancer drug, or a combination thereof.

[0032] Provided herein is an isolated nucleic acid encoding a first polypeptide and / or a second polypeptide of any of the heterodimeric proteins described herein. Some aspects of the present disclosure relate to a vector comprising such an isolated nucleic acid.

[0033] Also provided herein is an isolated nucleic acid encoding any of the fusion proteins, bispecific antibodies, multispecific antibodies, or conjugates provided herein. Also provided herein is a vector comprising such an isolated nucleic acid.

[0034] The present disclosure also provides a pharmaceutical composition comprising any of the heterodimeric proteins, fusion proteins, bispecific antibodies, multispecific antibodies, conjugates, isolated nucleic acids, or vectors described herein, and a pharmaceutically acceptable carrier. Also provided herein is a kit comprising any of the heterodimeric proteins, fusion proteins, bispecific antibodies, multispecific antibodies, conjugates, isolated nucleic acids, vectors, pharmaceutical compositions, and instructions for use provided herein.

[0035] Some aspects of the present disclosure relate to a method of making a heterodimeric protein described herein, wherein the method comprises culturing a cell comprising any of the isolated nucleic acids or vectors provided herein, and optionally recovering the produced heterodimeric protein.

[0036] Also provided herein is a method of producing a fusion protein comprising conjugating one or more biologically active molecules to any of the heterodimeric proteins described herein. In some aspects, the one or more biologically active molecules are conjugated directly or via a linker. In some aspects, the linker is selected from an IgGl hinge, an IgG2 hinge, an IgG3 hinge, an IgG4 hinge, an IgD hinge, or a synthetic linker.

[0037] Provided herein is a method of treating a disease or condition in a subject in need thereof comprising administering to the subject any of the heterodimeric proteins, fusion proteins, bispecific antibodies, multispecific antibodies, conjugates, isolated nucleic acids, vectors, or pharmaceutical compositions provided herein.

[0038] In some aspects, the disease or condition comprises a cancer, an infectious disease, or both. In some aspects, the cancer comprises a breast cancer, a head and neck cancer, a uterine cancer, a brain cancer, a skin cancer, a kidney cancer, a lung cancer, a colorectal cancer, a prostate cancer, a liver cancer, a bladder cancer, a kidney cancer, a pancreatic cancer, a thyroid cancer, an esophageal cancer, an eye cancer, a stomach cancer / gastric cancer, a gastrointestinal cancer, a carcinoma, a sarcoma, a leukemia, a lymphoma, a myeloma, or a combination thereof. In some aspects, the infectious disease comprises progressive multifocal leukoencephalopathy (PML; caused by the polyomavirus JC), sepsis, HIV, cytomegalovirus (CMV) infection, Epstein-Barr virus (EBV) infection, a respiratory infectious disease, or a combination thereof.

[0039] In some aspects, the heterodimeric protein, fusion protein, bispecific antibody, multispecific antibody, conjugate, isolated nucleic acid, vector, or pharmaceutical composition is administered intramuscularly, parenterally, subcutaneously, ocularly, intravenously, intraperitoneally, intradermally, intraorbitally, intracerebrally, intracranially, intraspinally, intraventricularly, intrathecally, intracisternally, intracapsularly, or intratumorally to the subject.

[0040] In some aspects, the method comprises administering an additional therapeutic agent to the subject. In some aspects, the additional therapeutic agent is administered intramuscularly, parenterally, subcutaneously, ocularly, intravenously, intraperitoneally, intradermally, intraorbitally, intracerebrally, intracranially, intraspinally, intraventricularly, intrathecally, intracisternally, intracapsularly, or intratumorally to the subject.

[0041] In some aspects, the additional therapeutic agent comprises an immune checkpoint inhibitor, an immune checkpoint activator, a standard of care treatment, a cytokine, or a combination thereof. In some aspects, the immune checkpoint inhibitor comprises a CTLA-4 antagonist (e.g., an anti-CTLA-4 antibody), a PD-1 antagonist (e.g., an anti-PD-1 antibody, an anti-PD-L1 antibody), a TIM3 antagonist (e.g., an anti-TIM3 antibody), or a combination thereof. In some aspects, the immune checkpoint activator comprises an OX40 agonist (e.g., an anti-OX40 antibody), a LAG-3 agonist (e.g., an anti-LAG3 antibody), a 4-1BB (CD137) (e.g., an anti-CD137 antibody), a GITR agonist (e.g., an anti-GITR antibody), or a combination thereof. In some aspects, the standard of care treatment comprises chemotherapy, radiation therapy, or both. In some aspects, the cytokine comprises IL-7. BRIEF DESCRIPTION OF DRAWINGS

[0042] Figure 1 Provided are predicted structural models of exemplary trispecific antibodies comprising the heterodimeric proteins described herein (also referred to herein as “trispecific heterodimeric antibodies”). In a first polypeptide (CH3 A ; left chain; “Chain A”) and a second polypeptide (CH3 BIn the context of the heterodimeric Fc portion of the trispecific heterodimeric antibody of the disclosure, the first polypeptide chain comprises (from N-terminus to C-terminus): (i) an IgD hinge, (ii) an IgD / IgG4 chimeric CH2 domain, and (iii) an IgG4 CH3 domain. The second polypeptide chain further comprises an anti-CD27 sdAB and an anti-CD3 sdAb, wherein the anti-CD27 sdAb is attached to the N-terminus of the IgD hinge and the anti-CD3 sdAb is attached to the anti-CD27 sdAb. The CH3 domain of Chain A comprises the amino acid sequence set forth in SEQ ID NO: 40 with the following amino acid modifications: T10V, L11Y, F65A, and Y67V (i.e., T148V, L149Y, F203A, and Y205V in SEQ ID NO: 4). The CH3 domain of Chain B comprises the amino acid sequence set forth in SEQ ID NO: 40 with the following amino acid modifications: T10V, T26L, K52L, and T54W (i.e., T148V, T164L, K190L, and T192W in SEQ ID NO: 4).

[0043] Figure 2 Exemplary methods for producing multispecific ligand binding proteins conjugated to the heterodimeric proteins described herein (e.g., trispecific heterodimeric antibodies as set forth in Figure 1 An overview of exemplary methods for producing multispecific ligand binding proteins conjugated to the heterodimeric proteins described herein (e.g., trispecific heterodimeric antibodies as set forth in

[0044] Figure 3 Schematic diagrams of different constructs for producing trispecific heterodimeric antibodies (e.g., as set forth in Figure 1 The constructs shown include: (a) Construct #1, which comprises (from N-terminus to C-terminus): (i) an anti-B7H3 sdAb, (ii) a first polypeptide CH3 A(i.e., comprising IgD hinge, IgD / IgG4 CH2, and IgG4 CH3), (iv) first cleavage site, (v) mCherry tag, (vi) second cleavage site, and (vii) ALFA tag; (c) Construct #3, comprising (from N- to C-terminus): (i) anti-CD3 sdAb, (ii) anti-CD27 sdAb, (iii) second polypeptide CH3 B (i.e., comprising IgD hinge, IgD / IgG4 CH2, and IgG4 CH3), (iv) first cleavage site, (v) mCherry tag, (vi) second cleavage site, and (vii) ALFA tag; (c) Construct #3, comprising (from N- to C-terminus): (i) anti-CD3 sdAb, (ii) anti-CD27 sdAb, (iii) second polypeptide CH3 B (i.e., comprising IgD hinge, IgD / IgG4 CH2, and IgG4 CH3), (iv) first cleavage site, (v) mCherry tag, (vi) second cleavage site, and (vii) ALFA tag; (c) Construct #3, comprising (from N- to C-terminus): (i) anti-CD3 sdAb, (ii) anti-CD27 sdAb, (iii) second polypeptide CH3 A .

[0045] Figure 4A and Figure 4B A purification analysis of the trispecific heterodimeric antibody (comprising Chain A encoded in Construct #1 and Chain B encoded in Construct #2; see Figure 3 ) using ALFA nanobody resin under non-reducing conditions is provided. Figure 4A An SDS-PAGE analysis of anti-ALFA column fractions using fluorescent imaging (left) and Coomassie staining (right) is provided. The lanes of these gels are labeled as follows: (i) “M”: molecular weight marker, (ii) “R”: protein bound to ALFA nanobody resin, and (iii) “E1-4”: protein eluted by thrombin. CD3a-CD27-CH B -mCherry polypeptide chain (Chain B + mCherry) and B7H3-CH A The expected molecular weights of the CD3a-CD27-CH Figure 4B An SDS-PAGE analysis of the pre- and post-cleavage of the preScission site is provided. The lanes of these gels are labeled as follows: (i) “M”: molecular weight marker, (ii) “pre-cleavage”: pre-cleavage, and (iii) “post-cleavage”: post-cleavage.

[0046] Figure 5 An SDS-PAGE analysis is provided demonstrating Figure 4A and Figure 4BThe trispecific heterodimeric antibody described herein (i.e., containing chains A and B encoded in constructs #1 and #2, respectively) was successfully formed. The lane labels of these gels are as follows: (i) “R”: containing β-mercaptoethanol (reduced), (ii) “M”: molecular weight marker, and (iii) “NR”: not containing β-mercaptoethanol (non-reduced).

[0047] Figure 6A and Figure 6B Analysis was provided, showing that after purification under non-reducing conditions, Figure 4A and Figure 4B The purity of the trispecific heterodimer antibody described in [the text] (i.e., containing chain A and chain B encoded in construct #1 and construct #2, respectively). Figure 6A An SDS-PAGE gel was provided, which confirmed the molecular weight of the purified trispecific heterodimer antibody (see the band at 116.7 kDa). Figure 6B ChemiDoc readings are provided, showing the estimated purity of the trispecific heterodimer antibody (see band 2; purity approximately 75%).

[0048] Figure 7 SDS-PAGE analysis was provided, showing the presence of chain A (B7H3-CH) after purification using anti-ALFA and anti-GFP columns, respectively. A (Encoded in constructor #1) and chain B (CD3a-CD27-CH) B The relative amounts of (encoded in construct #2) were shown. The gel on the left was developed using fluorescence, and the gel on the right was developed using Coomassie staining. The lane labels for both gels were as follows: (i) “sup”: supernatant, (ii) “FT”: flow-through, (iii) “R”: protein bound to ALFA nanobody or GFP nanobody resin, and (iv) “M”: molecular weight marker. The expected molecular weights of chain B+mCherry and chain A+GFP were 103.2 kDa and 72 kDa, respectively.

[0049] Figure 8 SDS-PAGE analysis was provided, showing the purified chain A (B7H3-CH) using baculovirus:human cell infection ratios (4:1, 8:1, and 10:1). A (Encoded in constructor #1) and chain B (CD3a-CD27-CH) B The relative value of (encoded in constructor #2). Figure 7 As shown, chain A (B7H3-CH A ) and chain B (CD3a-CD27-CH BPurification was performed using anti-ALFA and anti-GFP columns, respectively. All gel lanes were labeled as follows: (i) “M”: molecular weight marker, (ii) “sup”: supernatant, (iii) “FT”: flow-through, (iv) “R”: protein bound to ALFA or GFP nanobody resin, and (v) “E”: protein eluted with thrombin. The gel on the left was fluorescently visualized, and the gel on the right was visualized using Coomassie staining.

[0050] Figure 9A SDS-PAGE analysis was provided, showing the presence of chains A and B (i.e., encoded in constructs #1 and #2, respectively) under non-reducing conditions and at three different infection ratios: (i) 4:1, (ii) 8:1, and (iii) 10⁻¹. Chain B (i.e., CD3a-CD27-CH B ) and chain A (i.e., B7H3-CH) A The expected molecular weights are 72 kDa and 43.6 kDa, respectively. Figure 9B SDS-PAGE analysis was provided, which confirmed the successful formation of samples containing different infection rates. Figure 9A The three-specific heterodimer antibodies of chain A and chain B are shown in the figure.

[0051] Figure 10 Provided Figure 9B The mass spectrophotometric analysis of the heterodimer trispecific antibody described in the paper showed a peak at approximately 115.5 kDa.

[0052] Figure 11 SDS-PAGE analysis of the eluted fractions after purification of the trispecific heterodimer antibody (containing chains A and B encoded in constructs #4 and #3, respectively) using an anti-His column is provided. The lane labels of the gel are as follows: (i) “M”: molecular weight marker, (ii) “Sup”: supernatant, (iii) “FT”: flow-through, (iv) “W”: wash, and (v) “E1-3”: eluted proteins.

[0053] Figure 12 Provides further purification of protein A column. Figure 11 SDS-PAGE analysis of the eluted fractions was performed on the eluted proteins. The lane labels on the gel were as follows: (i) "input": imidazole eluted proteins, (ii) "FT": flow through, (iii) "W": wash, (iv) "E1-5": eluted proteins, and (v) "M": marker.

[0054] Figure 13SDS-PAGE analysis was provided, which confirmed that heterodimer formation of chain A (encoded in construct #4) and chain B (encoded in construct #3) under both reducing and non-reducing conditions yielded trispecific heterodimer antibodies (such as...). Figure 1 (As shown in the image). The lane markings for the gel are as follows: (i) "NR": non-reducing conditions, (ii) "R": reducing conditions, (iii) "NR (1 / 10)": non-reducing conditions (1 / 10 protein dilution), (iv) "R (1 / 10)": reducing conditions (1 / 10 protein dilution). Chain B (i.e., CD3a-CD27-CH B Chain A (i.e., B7H3-CH) A The expected molecular weights of the α and β antibodies are 72 kDa, 43.6 kDa, and 115.5 kDa, respectively.

[0055] Figure 14 ChemiDoc readings are provided, which show... Figure 13 The estimated purity of the purified trispecific heterodimeric antibody described is shown in [the diagram]. Band 1 corresponds to chain A (encoded in construct #4). Band 4 corresponds to chain B (encoded in construct #3). The final purified protein has a purity of 83%.

[0056] Figure 15 Provided under restored (top, blue) and non-restored (bottom, black) conditions Figure 9B CE-SDS analysis of the purified trispecific heterodimer antibody described in the paper.

[0057] Figure 16 Western blotting analysis was provided, which confirmed the formation of heterodimers of chain A (encoded in construct #4) and chain B (encoded in construct #3) to produce trispecific heterodimer antibodies, such as... Figure 1 As shown in the diagram. For comparison, homodimers of chain B and monomers of chains A and B are also shown. The lane markings of the gel are as follows: (i) "NR": non-reducing, (ii) "R": reducing conditions, and (iii) "M": molecular weight gradient. Chain B (CD3a-CD27-CH B Chain A (B7H3-CH) A ), trispecific heterodimer protein and chain B (CD3a-CD27-CH) B The expected molecular weights of the homodimers are 72 kDa, 43.6 kDa, 115.5 kDa and 144 kDa, respectively.

[0058] Figures 17A-17CExemplary positive charge amino acid modifications and negative charge amino acid modifications of the CH3 domains of the first and second polypeptides (Chains A and B, respectively) described herein are provided. For positive charge modifications of Chain A, amino acid residues N188, K190, D197, S198, and / or R207 of Chain A (e.g., comprising the amino acid sequence set forth in SEQ ID NO: 16) are modified to arginine (R) or lysine (K). As described herein, these amino acid residues correspond to amino acid residues N50, K52, D59, S60, and R69 (e.g., comprising the amino acid sequence set forth in SEQ ID NO: 24) of the CH3 domains described herein, respectively. For negative charge modifications of Chain B, amino acid residues N188, D197, S198, R207, and / or F203 of Chain B (e.g., comprising the amino acid sequence set forth in SEQ ID NO: 20) are modified to aspartic acid (D) or glutamic acid (E). Also as described herein, these amino acid residues correspond to amino acid residues N50, D59, S60, R69, and F65 (e.g., comprising the amino acid sequence set forth in SEQ ID NO: 26) of the CH3 domains described herein, respectively. Figure 17A Exemplary positive charge combinations #1-#8 and exemplary negative charge combinations #1-#12 are shown. It will be apparent from the present disclosure that, based on the positive charge and negative charge combinations provided in Figure 17A Figure 17B Exemplary positive charge combinations #9-#28 and exemplary negative charge combinations #13-#35 are shown. Figure 17C Exemplary positive charge combinations #29-#38 and exemplary negative charge combinations #36-#60 are shown. Based on the positive charge and negative charge combinations provided in Figure 17B and Figure 17C Exemplary positive charge combinations #29-#38 and exemplary negative charge combinations #36-#60 are shown. Based on the positive charge and negative charge combinations provided in

[0059] Figures 18A-18D ​Exemplary lists of negative charge amino acid modifications and positive charge amino acid modifications of the CH3 domains of the first and second polypeptides (Chains A and B, respectively) described herein are provided. For negative charge modifications of Chain A, amino acid residues N188, K190, D197, S198, and / or R207 of the amino acid sequence set forth in SEQ ID NO: 16 (or N50, K52, D59, S60, and / or R69 of the amino acid sequence set forth in SEQ ID NO: 24) are modified to aspartic acid (D) or glutamic acid (E). For positive charge modifications of Chain B, amino acid residues N188, D197, S198, R207, and / or F203 of the amino acid sequence set forth in SEQ ID NO: 20 (or N50, D59, S60, R69, and / or F65 of the amino acid sequence set forth in SEQ ID NO: 26) are modified to arginine (R) or lysine (K). Figure 18A Exemplary negative charge combinations #1-#12 and exemplary positive charge combinations #1-#12 are shown. Based on this positive and negative charge combinations, 144 different combinations of Chain A (with a negative charge) and Chain B (with a positive charge) heterodimers can be produced. Figure 18B Exemplary negative charge combinations #13-#33 and exemplary positive charge combinations #13-#33 are shown. Figure 18C Exemplary negative charge combinations #34-#54 and exemplary positive charge combinations #34-#54 are shown. Figure 18D Exemplary negative charge combinations #55-#60 and exemplary positive charge combinations #55-#60 are shown. Based on Figures 18B-18D Based on the negative and positive charge combinations provided in Table 2, 2,304 different combinations of Chain A (with a negative charge) and Chain B (with a positive charge) heterodimers can be produced.

[0060] Figure 19A schematic diagram of amino acid residues of an exemplary polypeptide chain A (comprising the amino acid sequence set forth in SEQ ID NO: 4 with the following amino acid modifications: T148V, L149Y, F203A, and Y205V, i.e., SEQ ID NO: 16) and an exemplary polypeptide chain B (comprising the amino acid sequence set forth in SEQ ID NO: 4 with the following amino acid modifications: T148V, T164L, K190L, and T192W, i.e., SEQ ID NO: 20) that are capable of interacting with one another to form a heterodimeric protein (such as a heterodimeric protein described herein) is provided. As shown, amino acid residue N188 on the first polypeptide is capable of interacting with amino acid residue S198 on the second polypeptide. Similarly, amino acid residue S198 on the first polypeptide is capable of interacting with amino acid residue N188 on the second polypeptide. Amino acid residue K190 on the first polypeptide is capable of interacting with amino acid residue F203 on the second polypeptide. Amino acid residue D197 on the first polypeptide is capable of interacting with amino acid residue R207 on the second polypeptide. Amino acid residue R207 on the first polypeptide is capable of interacting with amino acid residue D197 on the second polypeptide. As described further herein, amino acid residues N188, K190, D197, S198, and R207 correspond to amino acid residues N50, N52, D59, S60, and R69 of a CH3 domain described herein (e.g., comprising the amino acid sequence set forth in SEQ ID NO: 40, SEQ ID NO: 24, or SEQ ID NO: 26).

[0061] Figure 20 A schematic diagram is provided showing exemplary amino acid modifications that can be made to polypeptide chain A (e.g., comprising the amino acid sequence set forth in SEQ ID NO: 4 with the following amino acid modifications: T148V, L149Y, F203A, and Y205V; i.e., SEQ ID NO: 16) and polypeptide chain B (e.g., comprising the amino acid sequence set forth in SEQ ID NO: 4 with the following amino acid modifications: T148V, T164L, K190L, and T192W; i.e., SEQ ID NO: 20) to prevent interaction (“repulsion between same chains”) or to promote interaction (“attraction between different chains”).

[0062] Figure 21 A nano-differential scanning fluorimetry assay is provided for determining the melting temperature (Tm) of a heterodimeric protein (e.g., a heterodimeric protein described herein) to a heterodimeric protein (e.g., a heterodimeric protein described herein) comprising a polypeptide chain A (e.g., comprising the amino acid sequence set forth in SEQ ID NO: 4 with the following amino acid modifications: T148V, L149Y, F203A, and Y205V; i.e., SEQ ID NO: 16) and a polypeptide chain B (e.g., comprising the amino acid sequence set forth in SEQ ID NO: 4 with the following amino acid modifications: T148V, T164L, K190L, and T192W; i.e., SEQ ID NO: 20). m) curves: (1) NIT-Fc-01, (2) NIT-Fc-02, (3) NIT-Fc-03, (4) NIT-Fc-04, and (5) NIT-Fc-05. Tm was determined by detecting the maximum of the first derivative of the fluorescence ratio (F350 / 330).

[0063] Figure 22 Provided is a comparison of the thermal stability of the following heterodimers as measured at 37 °C: (1) NIT-Fc-01, (2) NIT-Fc-02, (3) NIT-Fc-03, (4) NIT-Fc-04, and (5) NIT-Fc-05. Figure 21 Provided is a comparison of the thermal stability of the following heterodimers as measured at 37 °C: (1) NIT-Fc-01, (2) NIT-Fc-02, (3) NIT-Fc-03, (4) NIT-Fc-04, and (5) NIT-Fc-05.

[0064] Figure 23 Provided is a comparison of the aggregation temperature (T agg ) of the following heterodimers as determined using DLS experiments: (1) NIT-Fc-01, (2) NIT-Fc-02, (3) NIT-Fc-03, (4) NIT-Fc-04, and (5) NIT-Fc-05. DLS-T agg was determined as the point at which the sample radius increased to three times its initial value, indicating the onset of denaturation and aggregation.

[0065] Figure 24 Provided is a comparison of the thermal stability of the following heterodimers as measured at 37 °C: (1) NIT-Fc-01, (2) NIT-Fc-02, (3) NIT-Fc-03, (4) NIT-Fc-04, and (5) NIT-Fc-05. DETAILED DESCRIPTION

[0066] Disclosed herein are heteromultimeric proteins comprising at least a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide are different, wherein: (a) the first polypeptide comprises a first CH3 domain comprising a heterodimerization modification (e.g., a positive charge or a negative charge modification described herein), and (b) the second polypeptide comprises a second CH3 domain comprising a heterodimerization modification (e.g., a negative charge or a positive charge modification described herein), and wherein the heterodimerization modification of the first CH3 domain and the heterodimerization modification of the second CH3 domain promote interaction of the first CH3 domain and the second CH3 domain to produce the heteromultimeric protein. As described further herein, the heteromultimeric proteins described herein are capable of increasing the productivity and / or stability of a protein. Additional aspects of the disclosure are provided throughout the application.

[0067] To facilitate an understanding of the disclosure disclosed herein, a number of terms and phrases are defined. Additional definitions are set forth throughout the detailed description.

[0068] I. Definitions Throughout this disclosure, the term "a" or "an" entity refers to one or more than one of that entity; for example, "a polypeptide" should be understood as meaning one or more polypeptides. As such "a" or "an" and "one or more" and "at least one" are used interchangeably herein.

[0069] Also, as used herein, "and / or" is to be taken as specific disclosure of each of the various components that the phrases "and / or" connects. For example, a list of items joined by "and / or" means that any of the items can be present or none of the items are present. Likewise, a list of items joined by "at least one of" means any of the items can be present or none of the items are present. Similarly, "comprises" (and all of the variations of that term such as "comprising" or a statement of comprising) means that an item can be present, but not exclusive, and that the item does not have to be present. Also, as used herein, "coupled" means that two or more items are in some way present with one another, such as being bound by adhesive, covalent bond, chemical bond, electromagnetic force, heat bonding, pressure bonding, use of an intermediate, etc.

[0070] It should be understood that wherever aspects are described herein with the language "comprising" either "a" or "an" entity, "comprising" is intended to not exclude the possibility that the other entity or entities are also present in the system, whether or not expressly identified or not.

[0071] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure is related. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; the Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised Edition, 2000, Oxford University Press, give one of ordinary skill in the art access to a glossary of terms used in the art.

[0072] Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, amino acid sequences are written left to right in amino to carboxy orientation. The headings provided herein are not limitations of the various aspects of the disclosure, which can be had by reference to the specification as a whole. Accordingly, the terms defined herein have meanings ascribed to them by reference to the specification as a whole and to each and every specification and art- referenced publication and / or document herein.

[0073] The term“about” is used herein to mean approximately, roughly, around, or in the region of. When the term“about” is used in conjunction with a numerical value, it modifies that value by extending it by the margin of error of the devices, methods, and materials involved with a bit of latitude on both ends of the range. Generally, the term“about” can modify a value to be higher and lower than stated values by variations of, e.g., 10%.

[0074] As used herein, the terms“heteromultimer,”“heteromultimeric complex,” and“heteromultimeric protein” can be used interchangeably and refer to a molecule comprising at least a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide are different. For example, in some aspects, the first polypeptide and the second polypeptide differ by at least one amino acid residue in their amino acid sequence. Thus, in some aspects, the amino acid sequence of the first polypeptide and the amino acid sequence of the second polypeptide share less than about 99%, less than about 98%, less than about 97%, less than about 96%, less than about 95%, less than about 94%, less than about 93%, less than about 92%, less than about 91%, less than about 90%, less than about 85%, less than about 80%, or less than about 75% sequence identity. In some aspects, the amino acid sequence of the first polypeptide and the amino acid sequence of the second polypeptide differ by about one amino acid residue, about two amino acid residues, about three amino acid residues, about four amino acid residues, about five amino acid residues, about six amino acid residues, about seven amino acid residues, about eight amino acid residues, about nine amino acid residues, or about 10 amino acid residues or more. In some aspects, the heteromultimer can comprise a“heterodimer” formed by the first polypeptide and the second polypeptide. In some aspects, the heteromultimer can comprise a higher order tertiary structure in which there are polypeptides in addition to the first polypeptide and the second polypeptide. The polypeptides of the heteromultimer can interact with one another by various means known in the art, such as non-peptide bonds, covalent bonds (e.g., disulfide bonds), and / or non-covalent interactions (e.g., hydrogen bonds, ionic bonds, van der Waals forces, and / or hydrophobic interactions). Unless otherwise specified, heteromultimers and heterodimers (and variants thereof) can be used interchangeably.

[0075] As used herein, the term “heteromultimerization modification” (or grammatical variants thereof) refers to an alteration that promotes heteromultimer formation and reduces / inhibits homomultimer formation. Examples of such modifications are known in the art, e.g., WO 2013 / 063702, which is incorporated by reference herein in its entirety. Additional non-limiting examples of such modifications are provided throughout the present disclosure. In some aspects, the heteromultimerization modification comprises an amino acid modification. For example, in some aspects, the first polypeptide comprises a first amino acid modification and the second polypeptide comprises a second amino acid modification, wherein the first amino acid modification and the second amino acid modification are such that they promote the interaction of the first polypeptide and the second polypeptide. In some aspects, the first amino acid modification and the second amino acid modification result in opposite charges (e.g., a positive charge modification and a negative charge modification described herein).

[0076] As used herein, the term “amino acid modification” refers to any alteration of an amino acid at a particular residue of a polypeptide. Non-limiting examples of amino acid modifications include amino acid insertions, deletions, substitutions, and rearrangements.

[0077] As used herein, “first polypeptide” refers to any polypeptide that will associate with a second polypeptide. As used herein, “second polypeptide” refers to any polypeptide that will associate with a first polypeptide. Unless otherwise specified, the first polypeptide and the second polypeptide are different (e.g., their sequences differ by at least one or more amino acids) such that the interaction of the first polypeptide and the second polypeptide does not form a homodimer. It will be apparent to one of skill in the art that any heteromultimerization modification (e.g., an amino acid modification described herein) can be made on either the first polypeptide or the second polypeptide so long as the heteromultimerization modification promotes the interaction of the first polypeptide and the second polypeptide. In some aspects, the first polypeptide is referred to herein as “chain A” and the second polypeptide is referred to herein as “chain B.”

[0078] The term “Fc region” (fragment crystallizable region) or “Fc domain” or “Fc” refers to a C-terminal region of an antibody heavy chain that mediates the binding of an immunoglobulin to host tissues or factors including binding to Fc receptors located on various cells of the immune system (e.g., effector cells) or to the first component (Clq) of the classical complement system. Thus, the Fc region comprises the constant region of an antibody excluding the first constant region immunoglobulin domain (e.g., CHI or CL). For example, in some aspects, an Fc useful for the present disclosure comprises a CH3 domain. In some aspects, an Fc further comprises: (a) a hinge domain, (b) a CH2 domain, or (c) both (a) and (b). For example, in some aspects, an Fc comprises a hinge domain and a CH3 domain. In some aspects, an Fc comprises a CH2 domain and a CH3 domain. In some aspects, an Fc comprises a hinge domain, a CH2 domain, and a CH3 domain. In some aspects, an Fc is a human IgG Fc. In some aspects, a human IgG Fc is a human IgGl, IgG2, IgG3, or IgG4 Fc. In some aspects, an Fc is from an immunoglobulin selected from IgG, IgA, IgD, IgE, or IgM.

[0079] In IgG, IgA, and IgD antibody isotypes, the Fc region comprises two identical protein fragments derived from the second (CH2) and third (CH3) constant domains of the two heavy chains of the antibody; IgM and IgE Fc regions comprise three heavy chain constant domains (CH domains 2-4) in each polypeptide chain. For IgG, the Fc region comprises immunoglobulin domains CH2 and CH3 and a hinge between the CHI and CH2 domains. While the definition of the boundaries of the Fc region of an immunoglobulin heavy chain can vary slightly, as defined herein, a human IgG heavy chain Fc region is defined as stretching from an amino acid residue D221 of IgGl, V222 of IgG2, L221 of IgG3, and P224 of IgG4, to the carboxy-terminal end of the heavy chain, with numbering according to the EU index as in Kabat. The CH2 domain of a human IgG Fc region extends from amino acid 237 to amino acid 340, and the CH3 domain is located C-terminal to the CH2 domain in the Fc region, i.e., it extends from amino acid 341 to amino acid 447 or 446 (if the C-terminal lysine residue is not present) or 445 (if the C-terminal glycine and lysine residues are not present) of IgG. As used herein, an Fc region can be a native sequence Fc, including any allotypic variant, or a variant Fc (e.g., a non-naturally occurring Fc). Fc can also refer to this region in isolation or in the context of a protein polypeptide comprising an Fc, such as a “binding protein comprising an Fc region,” also referred to as an “Fc fusion protein” (e.g., an antibody or immunoadhesin). Exemplary Fc sequences are provided elsewhere in the disclosure (see, e.g., Table 3).

[0080] As described herein, in some aspects, the Fc comprises domains (e.g., hinge domains, CH2 domains, and / or CH3 domains) from different types of antibodies. Such Fc molecules are also referred to herein as “hybrid Fc.” In some aspects, the hybrid Fc comprises: (a) a human IgD hinge region, (b) a portion of a human IgD CH2 domain and a portion of a human IgG4 CH2 domain, and (c) a human IgG4 CH3 domain. Additional disclosure related to such hybrid Fc is provided in WO 2008 / 147143 Al, which is incorporated by reference herein in its entirety.

[0081] As used herein, the term “hinge domain” refers to any moiety capable of linking two or more molecules. In some aspects, the hinge domain comprises a hinge region of an antibody (e.g., IgG, IgA, IgD, IgE, or IgM). The hinge region of an antibody is a short heavy chain sequence that connects the antigen-binding fragment (Fab) and the Fc region. As further described elsewhere in the disclosure, in some aspects, the hinge domain comprises a linker (e.g., a synthetic linker). Examples of linkers useful as hinge domains are known in the art. See, e.g., WO 2014 / 087248, which is incorporated by reference herein in its entirety. Thus, unless otherwise specified, the terms “hinge domain” and “linker” can be used interchangeably in the disclosure.

[0082] As used herein, the term “CH2 domain” refers to a heavy chain immunoglobulin constant chain located between the hinge and CH3 domains. The CH2 domain can be a naturally occurring CH2 domain, or a naturally occurring CH2 domain in which one or more amino acids have been modified (e.g., substituted, inserted, deleted, and / or rearranged), provided that the CH2 domain has a desired biological property. The desired biological activity can be a native biological activity, an enhanced biological activity relative to the naturally occurring domain, or a decreased biological activity.

[0083] As used herein, the term “CH3 domain” refers to a heavy chain immunoglobulin constant chain located C-terminal to the CH2 domain and spanning about 110 residues, e.g., about positions 341-446b (EU numbering system), from the N-terminus of the CH2 domain. The CH3 domain can be a naturally occurring CH3 domain, or a naturally occurring CH3 domain in which one or more amino acids have been modified (e.g., substituted, inserted, deleted, and / or rearranged), provided that the CH3 domain has a desired biological property. The desired biological activity can be a native biological activity, an enhanced biological activity relative to the naturally occurring domain, or a decreased biological activity. In some aspects, the CH3 domains described herein comprise a C-terminal lysine. In some aspects, the CH3 domains described herein do not comprise a C-terminal lysine.

[0084] “EU” indicates that the amino acid position (e.g., in a heavy chain constant region, such as within the hinge domain, CH2 domain, and / or CH3 domain) is numbered herein according to the EU index numbering system (see Kabat et al., in “Sequences of Proteins of Immunological Interest,” U.S. Dept. Health and Human Services, 5th Ed. (1991).

[0085] The term “fusion protein” refers to a protein produced by joining two or more genes that originally coded for separate proteins. Translation of such a fusion gene results in a single polypeptide or multiple polypeptides with functional properties derived from each of the original proteins. In some aspects, two or more genes can include substitutions, deletions, and / or additions in their nucleotide sequences.

[0086] As used herein, the term “conjugate” refers to two or more molecules (e.g., a heterodimeric protein described herein and a conjugate moiety) linked into a larger construct.

[0087] As used herein, “attached,” “conjugated,” and “linked” can be used interchangeably and refer to the linkage of a first moiety to a second moiety (e.g., the linkage of a heterodimeric protein to a biologically active molecule described herein). In some aspects, the first moiety can be directly attached to the second moiety (e.g., the first moiety and the second moiety are fused together). In some aspects, the first moiety can be attached to the second moiety via a linker. Non-limiting examples of useful linkers are described elsewhere in the disclosure.

[0088] The term “antibody(ies)” is a term of art and is used interchangeably herein and refers to a molecule having an antigen binding site that specifically binds an antigen. The term as used herein includes intact antibodies and any antigen binding fragment (i.e., “antigen binding moiety” or “antigen binding fragment”) or single chain thereof. In some aspects, “antibody” refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen binding portion thereof. In some aspects, “antibody” refers to a single chain antibody comprising a single variable domain (e.g., a VHH domain). Each heavy chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. In some naturally occurring antibodies, the heavy chain constant region comprises three domains, CH1, CH2, and CH3. In some naturally occurring antibodies, each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region comprises one domain, CL.

[0089] Unless otherwise indicated, an antibody can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, or IgY), any class (e.g., IgD, IgG2, IgG3, IgG4, IgAl, or IgA2), or any subclass (e.g., IgGl, IgG2, IgG3, and IgG4 in humans, and IgGl, IgG2a, IgG2b, and IgG3 in mice) of immunoglobulin molecule. Immunoglobulins (e.g., IgGl) exist in several allotypes that differ from one another by up to several amino acids. The antibodies disclosed herein can be from any of the well-known isotypes, classes, subclasses, or allotypes. In some aspects, the antibodies described herein belong to the IgGl, IgG2, IgG3, or IgG4 subclass, or any hybrid thereof. In some aspects, the antibodies belong to the human IgGl subclass, the human IgG2 subclass, or the human IgG4 subclass.

[0090] As used herein, the term “naturally-occurring” or “naturally-existing” refers to the fact that an object (e.g., a protein) can be found in nature. For example, a polypeptide or polynucleotide sequence that exists in an organism (including a virus) is naturally-occurring, which can be isolated from a natural source, and cannot be intentionally modified by humans in a laboratory. As further described elsewhere in the disclosure, the polypeptides useful for the disclosure are not naturally-occurring.

[0091] “Polypeptide” refers to a chain comprising at least two contiguous linked amino acid residues, with no upper limit on the length of the chain. One or more amino acid residues in a protein can contain modifications, such as but not limited to glycosylation, phosphorylation, or disulfide bond formation. A “protein” can comprise one or more polypeptides. Unless otherwise specified, the terms “protein” and “polypeptide” are used interchangeably.

[0092] As used herein, the term "nucleic acid," "nucleic acid molecule," or "polynucleotide" (or variants thereof) refers to polymers of nucleotides of any length (including ribonucleotides, deoxyribonucleotides, analogs thereof, or mixtures thereof). The term refers to the primary structure of the molecule. Thus, the term includes triple-stranded, double-stranded, and single- stranded deoxyribonucleic acids ("DNA") as well as triple-stranded, double-stranded, and single-stranded ribonucleic acids ("RNA"). It also includes modified and unmodified polynucleotide forms, for example, by alkylation and / or by capping. In particular, the term "polynucleotide" includes polydeoxyribonucleotides (containing 2-deoxy-D-ribose), and polyribonucleotides (containing D-ribose), including mRNA, whether spliced or unspliced, any other type of polynucleotide (which is an N- or C-glycoside of a purine or pyrimidine base), and other polymers containing a positive nucleotide backbone, for example, polyamides (e.g., peptide nucleic acids "PNAs") and polyphosphoramide polymers, as well as other synthetic sequence-specific nucleic acid polymers (provided that the polymer contains nucleobases whose configuration allows for base pairing and base stacking, such as found in DNA and RNA).

[0093] For nucleic acids, the term "substantial homology" indicates that two nucleic acids or specified sequences thereof, when optimally aligned and compared, are at least about 80% identical, at least about 90% to 95% identical, or at least about 98% to 99.5% identical, with appropriate insertions, deletions, or substitutions. Alternatively, substantial homology exists when a fragment of a sequence hybridizes to the complement of a sequence under selective hybridization conditions. For polypeptides, the term "substantial homology" indicates that two polypeptides or specified sequences thereof, when optimally aligned and compared, are at least about 80% identical, at least about 90% to 95% identical, or at least about 98% to 99.5% identical, with appropriate insertions, deletions, or substitutions.

[0094] The percent identity between two sequences is the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap (i.e., homology %= number of identical positions / total number of positions x 100). Comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, for example, as described in the following non-limiting examples.

[0095] The percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package (available at worldwideweb.gcg.com), using a NWSgapdna.CMP matrix and a 40, 50, 60, 70, or 80 gap weight, and a 1, 2, 3, 4, 5, or 6 length weight. The percent identity between two nucleotide or amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller CABIOS , 4:11-17 (1989)), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch J. Mol. Biol . (48):444-453 (1970)), using a Blossum 62 matrix or a PAM250 matrix, and a gap penalty of 16, 14, 12, 10, 8, 6, or 4, and a gap length penalty of 1, 2, 3, 4, 5, or 6.

[0096] The nucleic acid and protein sequences described herein can further be used as a "query sequence" to perform a search against public databases to, for example, identify related sequences. Such searches can be performed using the Altschul et al. (1990) J. Mol. Biol .215:403-10. BLAST nucleotide searches can be performed with the NBLAST program (score = 100, word length = 12) to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. BLAST protein searches can be performed with the XBLAST program (score = 50, word length = 3) to obtain amino acid sequences homologous to the protein molecules described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids Res .25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See worldwideweb.ncbi.nlm.nih.gov.

[0097] As used herein, the term "vector" is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors"). In general, expression vectors of utility in recombinant DNA technologies are often in the form of plasmids. In this specification, "plasmid" and "vector" can be used interchangeably as the plasmid is the most commonly used form of vector. However, other forms of expression vectors which serve equivalent functions are also included, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses).

[0098] As used herein, the term "recombinant host cell" (or simply "host cell") is intended to refer to a cell that contains a nucleic acid that is not naturally present in the cell and can be a cell into which a recombinant expression vector has been introduced. It should be understood that such terms are intended to also include progeny of the original cell which progeny carry the heterologous nucleic acid(s) and which progeny, for example, express the heterologous nucleic acid(s) under conditions compatible with the prese

[0099] As used herein, “administering” refers to the physical introduction of an agent (e.g., a polypeptide or molecule described herein) or a composition comprising the agent into a subject using any of a variety of methods and delivery systems known to those of skill in the art. Non-limiting examples of routes of administration that can be used include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal or other parenteral routes of administration, e.g., by injection or infusion. The phrase “parenteral administration” as used herein means modes of administration other than enteral or topical administration, usually by injection, and includes, without limitation, intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intra-lymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intracerebroventricular, intravitreal, intraspinal, and intrasternal injection as well as other parenteral injections and infusion, and in vivo electroporation. Alternatively, a polypeptide or molecule described herein can be administered via a non-parenteral route, such as a topical, epidermal, or mucosal route of administration, e.g., intranasal, oral, vaginal, rectal, sublingual, or topical administration. Administration can also be carried out, e.g., once, multiple times, and / or over one or more extended periods.

[0100] As used herein, the term “subject” includes any human or non-human animal. The term “non-human animal” includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cows, chickens, amphibians, reptiles, etc.

[0101] As used herein, the term “therapeutically effective amount” refers to the amount of a substance, alone or in combination with another therapeutic agent, that is effective in “treating” a disease or condition in a subject or reducing the risk, potential, likelihood, or incidence of a disease or condition (e.g., a cancer and / or infectious disease described herein). A “therapeutically effective amount” includes the amount of a substance or therapeutic agent that provides some improvement or benefit to a subject having or likely to have a disease or condition. Thus, a “therapeutically effective” amount is an amount that reduces the risk, potential, likelihood, or incidence of a disease or provides some alleviation, mitigation, and / or reduction of at least one indicator of a disease or condition and / or at least one clinical symptom.

[0102] As used herein, the terms “treat,” “treating,” and “treatment” refer to any type of intervention or process carried out by or on a subject with an active agent with the objective of reversing, alleviating, ameliorating, inhibiting, or slowing down or preventing the progression, development, severity, or recurrence of symptoms, complications, conditions, or biochemical indicia associated with a disease. Treatment can be of a subject having a disease or of a subject not having a disease (e.g., for prophylaxis).

[0103] As used herein, the terms “ug” and “uM” are used interchangeably with “pg” and “pM,” respectively.

[0104] Various aspects described herein are described in greater detail in the following subsections.

[0105] II. Polypeptides Provided herein are polypeptides comprising one or more amino acid modifications that: (a) promote heteromultimer formation, (b) reduce homomultimer formation, or (c) both (a) and (b). Such amino acid modifications are also referred to herein as “heteromultimerization modifications.” In some aspects, a polypeptide comprises a single heteromultimerization modification (e.g., a single amino acid modification that promotes heteromultimer formation). In some aspects, a polypeptide comprises multiple heteromultimerization modifications (e.g., two or more amino acid modifications that promote heteromultimer formation). In cases involving multiple heteromultimerization modifications, in some aspects, each heteromultimerization modification is the same type of modification (e.g., all amino acid modifications). In some aspects, one or more of the multiple heteromultimerization modifications is a different type of modification.

[0106] It is evident from the present disclosure that a polypeptide comprising a heteromultimerization modification described herein is less likely to form a homomultimer (e.g., an interaction between two polypeptides having the same amino acid sequence) compared to a corresponding polypeptide lacking the heteromultimerization modification (also referred to herein as a “reference polypeptide”). In some aspects, a polypeptide described herein (i.e., comprising one or more heteromultimerization modifications) has at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% less ability to form a homomultimer compared to a reference polypeptide. It is also evident from the present disclosure that a polypeptide comprising a heteromultimerization modification described herein is more likely to form a heteromultimer (e.g., an interaction between two polypeptides having an amino acid sequence that differs by at least one amino acid residue) compared to a reference polypeptide. In some aspects, a polypeptide described herein (i.e., comprising one or more heteromultimerization modifications) has at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% greater ability to form a heteromultimer compared to a reference polypeptide. In some aspects, a polypeptide described herein (i.e., comprising one or more heteromultimerization modifications) has at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, or at least about 50-fold greater ability to form a heteromultimer compared to a reference polypeptide.

[0107] Heterodimeric Fc proteins As demonstrated herein, the heteromultimerization modifications described herein are particularly useful in producing heteromultimeric proteins. Accordingly, in some aspects, provided herein are heterodimeric proteins comprising a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide are different, and wherein the first polypeptide, the second polypeptide, or both the first polypeptide and the second polypeptide comprise a heteromultimerization modification (e.g., an amino acid modification described herein). In some aspects, the first polypeptide comprises a heteromultimerization modification. In some aspects, the second polypeptide comprises a heteromultimerization modification. In some aspects, both the first polypeptide and the second polypeptide comprise a heteromultimerization modification.

[0108] CH3 domain In some aspects, the heterodimeric protein comprises an Fc protein or fragment thereof. For example, in some aspects, provided herein are heterodimeric proteins comprising a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide are different, wherein the first polypeptide comprises a first CH3 domain and the second polypeptide comprises a second CH3 domain, and wherein: (a) the first CH3 domain comprises a heteromultimerization modification, (b) the second CH3 domain comprises a heteromultimerization modification, or (c) both (a) and (b). In cases where both the first CH3 domain and the second CH3 domain comprise a heteromultimerization modification, in some aspects, the heteromultimerization modification of the first CH3 domain and the heteromultimerization modification of the second CH3 domain enable the first CH3 domain and the second CH3 domain to interact with one another to form a heterodimer.

[0109] For example, in some aspects, the heteromultimerization modification of the first CH3 domain comprises a substitution of one or more amino acid residues of the first CH3 domain with an amino acid having a positive charge (also referred to herein as a “positive charge modification”), and the heteromultimerization modification of the second CH3 domain comprises a substitution of one or more amino acid residues of the second CH3 domain with an amino acid having a negative charge (also referred to herein as a “negative charge modification”), wherein the positive charge modification and the negative charge modification promote interaction of the first CH3 domain and the second CH3 domain. Unless otherwise specified, the positive charge modifications and the negative charge modifications useful for the present disclosure include any amino acid modification (e.g., substitution) that results in a positive charge and a negative charge, respectively, thereby promoting interaction between a polypeptide comprising a positive charge modification and a polypeptide comprising a negative charge modification.

[0110] In some aspects, a positive charge modification comprises substitution of an amino acid with lysine (K) or arginine (R). In some aspects, a negative charge modification comprises substitution of an amino acid with aspartic acid (D) or glutamic acid (E). It will be apparent to one of skill in the art that, in cases where a CH3 domain is modified to comprise a positive charge and the amino acid residue within the CH3 domain already has a positive charge (e.g., amino acid residue K190 in SEQ ID NO: 4 or SEQ ID NO: 16), in some aspects, the amino acid residue with a positive charge is not further modified. Similarly, in cases where a CH3 domain is modified to comprise a negative charge and the amino acid residue within the CH3 domain already has a negative charge (e.g., amino acid residue D197 in SEQ ID NO: 4, SEQ ID NO: 16, or SEQ ID NO: 20), in some aspects, the amino acid residue with a negative charge is not further modified. Non-limiting examples of positive charge and negative charge modifications that can be made within the CH3 domain of the polypeptides provided herein (e.g., comprising the amino acid sequence set forth in SEQ ID NO: 4) are provided in Table 1 (below). Figures 17A-17C and 18A to Figure 18D Exemplary combinations of positive charge modifications and negative charge modifications that can be introduced into the CH3 domains of the polypeptides described herein are provided. Based on the Figures 17A-17C and 18A to Figure 18D Combinations of the combinations of positive charge and negative charge combinations provided in Tables 1-3 are possible. Each of the different combinations of a first polypeptide (chain A; comprising a first CH3 domain) and a second polypeptide (chain B; comprising a second CH3 domain) are within the scope of the present disclosure. Thus, in some aspects, a heterodimeric protein provided herein comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises a first CH3 domain, and the second polypeptide comprises a second CH3 domain, and wherein the first CH3 domain and the second CH3 domain comprise Figures 17A-17C and 18A to Figure 18D any one of Tables 1-3, wherein the combination of positive charge modifications and negative charge modifications facilitates interaction of the first CH3 domain and the second CH3 domain.

[0111] Table 1. Exemplary positive charge modifications and negative charge modifications In some aspects, the heterodimeric protein described herein comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises a first CH3 domain comprising a positive charge modification, and the second polypeptide comprises a second CH3 domain comprising a negative charge modification. As will be apparent to those of skill in the art, in some aspects, the first CH3 domain can comprise a negative charge modification, and the second CH3 domain can comprise a positive charge modification. For example, in some aspects, the heterodimeric protein described herein comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises a first CH3 domain comprising a negative charge modification, and the second polypeptide comprises a second CH3 domain comprising a positive charge modification. Thus, any of the heteromultimerization modifications (e.g., a positive charge modification or a negative charge modification) of the first polypeptides described herein can be present on the second polypeptide, and any of the heteromultimerization modifications (e.g., a negative charge modification or a positive charge modification) of the second polypeptides described herein can be present on the first polypeptide, so long as the heteromultimerization modifications of the first polypeptide and the second polypeptide promote the interaction of the first polypeptide and the second polypeptide to form a heterodimer.

[0112] As described elsewhere in the disclosure, the CH3 domains useful in the disclosure can be derived from the heavy chain of any suitable antibody. In some aspects, the CH3 domain is a domain of an IgG antibody, an IgA antibody, an IgD antibody, an IgE antibody, or an IgM antibody. In some aspects, the CH3 domain is a domain of an IgG antibody (e.g., IgG1, IgG2, IgG3, or IgG4). For example, in some aspects, the CH3 domain is a domain of an IgG4 antibody (referred to herein as an “IgG4 CH3 domain”). An exemplary amino acid sequence of an IgG4 CH3 domain is set forth in SEQ ID NO: 40 (see, e.g., Table 3).

[0113] Accordingly, in some aspects, the heterodimeric protein provided herein comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises a first IgG4 CH3 domain comprising a heteromultimerization domain (e.g., an amino acid modification described herein), and the second polypeptide comprises a second IgG4 CH3 domain comprising a heteromultimerization domain (e.g., an amino acid modification described herein), wherein the heteromultimerization domain of the first IgG4 CH3 domain and the heteromultimerization domain of the second IgG4 CH3 domain are different. For example, in some aspects, the first IgG4 CH3 domain comprises a positive charge modification, and the second IgG4 CH3 domain comprises a negative charge modification, wherein the positive charge modification and the negative charge modification promote interaction of the first IgG4 CH3 domain with the second IgG4 CH3 domain, thereby forming the heterodimeric protein. As described herein, in some aspects, the first IgG4 CH3 domain comprises a negative charge modification, and the second IgG4 CH3 domain comprises a positive charge modification, wherein the positive charge modification and the negative charge modification promote interaction of the first IgG4 CH3 domain with the second IgG4 CH3 domain, thereby forming the heterodimeric protein.

[0114] In some aspects, the CH3 domain provided herein comprises the amino acid sequence set forth in SEQ ID NO: 40, but with one or more amino acid modifications. In some aspects, a CH3 domain useful for the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 40, but with about one, about two, about three, about four, about five, about six, about seven, about eight, about nine, or about 10 amino acid modifications. Accordingly, in some aspects, provided herein is a heterodimeric protein comprising a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide are different, wherein the first polypeptide comprises a first CH3 domain comprising the amino acid sequence set forth in SEQ ID NO: 40, but with one or more amino acid modifications (e.g., about one, about two, about three, about four, about five, about six, about seven, about eight, about nine, or about 10), wherein the second polypeptide comprises a second CH3 domain comprising the amino acid sequence set forth in SEQ ID NO: 40, but with one or more amino acid modifications (e.g., about one, about two, about three, about four, about five, about six, about seven, about eight, about nine, or about 10), and wherein the one or more amino acid modifications of the first CH3 domain and the one or more amino acid modifications of the second CH3 domain promote interaction of the first CH3 domain and the second CH3 domain.

[0115] In some aspects, the first CH3 domain or the second CH3 domain comprises the amino acid sequence set forth in SEQ ID NO: 40, wherein amino acid residue T10, L11, F65, Y67, or a combination thereof has been modified. For example, in some aspects, the threonine (T) at amino acid residue 10 of SEQ ID NO: 40 has been modified to a valine (V). In some aspects, the leucine (L) at amino acid residue 11 of SEQ ID NO: 40 has been modified to a tyrosine (Y). In some aspects, the phenylalanine (F) at residue 65 of SEQ ID NO: 40 has been modified to an alanine (A). In some aspects, the tyrosine (Y) at amino acid residue Y67 of SEQ ID NO: 40 has been modified to a valine (V). Thus, in some aspects, the first CH3 domain or the second CH3 domain comprises the amino acid sequence set forth in SEQ ID NO: 40 with one or more amino acid modifications selected from the group consisting of T10V, L11Y, F65A, Y67V, or a combination thereof. In some aspects, the first CH3 domain or the second CH3 domain comprises the amino acid sequence set forth in SEQ ID NO: 40 with the following amino acid modifications: T10V, L11Y, F65A, and Y67V. It is evident from the disclosure that amino acid residues T10, L11, F65, and Y67 of SEQ ID NO: 40 correspond to amino acid residues T148, L149, F203, and Y205, respectively, of SEQ ID NO: 4. Thus, in some aspects, the polypeptide of the heterodimeric protein (e.g., the first polypeptide and / or the second polypeptide described herein) comprises the amino acid sequence set forth in SEQ ID NO: 4 with one or more amino acid modifications selected from the group consisting of T148V, L149Y, F203A, Y205V, or a combination thereof. In some aspects, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 4 with the following amino acid modifications: T148V, L149Y, F203A, and Y205V.

[0116] In some aspects, the first CH3 domain or the second CH3 domain comprises the amino acid sequence set forth in SEQ ID NO: 40, wherein amino acid residue T10, T26, K52, T54, or a combination thereof has been modified. In some aspects, the threonine (T) at amino acid residue 10 has been modified to a valine (V). In some aspects, the threonine (T) at amino acid residue 26 has been modified to a leucine (L). In some aspects, the lysine (K) at amino acid residue 52 has been modified to a leucine (L). In some aspects, the threonine at amino acid residue 54 has been modified to a tryptophan (W). Thus, in some aspects, the first CH3 domain or the second CH3 domain comprises the amino acid sequence set forth in SEQ ID NO: 40 with one or more amino acid modifications selected from the group consisting of T10V, T26L, K52L, T54W, or a combination thereof. In some aspects, the first CH3 domain or the second CH3 domain comprises the amino acid sequence set forth in SEQ ID NO: 40 with the following amino acid modifications: T10V, T26L, K52L, and T54W. It will be apparent from the disclosure that amino acid residues T10, T26, K52, and T54 correspond to amino acid residues T148, T164, K190, and T192, respectively, of SEQ ID NO: 4. Thus, in some aspects, the polypeptide of the heterodimeric protein (e.g., the first polypeptide and / or the second polypeptide described herein) comprises the amino acid sequence set forth in SEQ ID NO: 4 with one or more amino acid modifications selected from the group consisting of T148V, T164L, K190L, T192W, or a combination thereof. In some aspects, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 4 with the following amino acid modifications: T148V, T164L, K190L, and T192W.

[0117] In some aspects, provided herein are heterodimeric proteins comprising a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide are different, wherein the first polypeptide comprises a first CH3 domain and the second polypeptide comprises a second CH3 domain, wherein: (a) the first CH3 domain comprises the amino acid sequence set forth in SEQ ID NO: 40 with one or more amino acid modifications selected from T10V, L11Y, F65A, Y67V, or combinations thereof, and (b) the second CH3 domain comprises the amino acid sequence set forth in SEQ ID NO: 40 with one or more amino acid modifications selected from T10V, T26L, K52L, T54W, or combinations thereof, and wherein the one or more amino acid modifications of the first CH3 domain and the one or more amino acid modifications of the second CH3 domain promote interaction of the first CH3 domain and the second CH3 domain. In some aspects, the heterodimeric proteins comprise a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide are different, wherein the first polypeptide comprises a first CH3 domain and the second polypeptide comprises a second CH3 domain, wherein: (a) the first CH3 domain comprises the amino acid sequence set forth in SEQ ID NO: 40 with the following amino acid modifications: T10V, L11Y, F65A, and Y67V, and (b) the second CH3 domain comprises the amino acid sequence set forth in SEQ ID NO: 40 with the following amino acid modifications: T10V, T26L, K52L, and T54W. In some aspects, provided herein are heterodimeric proteins comprising a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide are different, wherein: (a) the first polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 4 with one or more amino acid modifications selected from T148V, L149Y, F203A, Y205V, or combinations thereof, and (b) the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 4 with one or more amino acid modifications selected from T148V, T164L, K190L, T192W, or combinations thereof. In some aspects, (a) the first polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 4 with the following amino acid modifications: T148V, L149Y, F203A, and Y205V, and (b) the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 4 with the following amino acid modifications: T148V, T164L, K190L, and T192W.

[0118] In some aspects, the first CH3 domain or the second CH3 domain comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 24 or SEQ ID NO: 26. In some aspects, the heterodimeric protein provided herein comprises a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide are different, and wherein: (a) the first polypeptide comprises a first CH3 domain comprising the amino acid sequence set forth in SEQ ID NO: 24, and (b) the second polypeptide comprises a second CH3 domain comprising the amino acid sequence set forth in SEQ ID NO: 26.

[0119] As demonstrated herein, in some aspects, either of the first CH3 domain and the second CH3 domain described above can comprise one or more additional modifications that help to further promote heteromultimer formation. For example, in some aspects, the first CH3 domain or the second CH3 domain comprises the amino acid sequence set forth in SEQ ID NO: 40, SEQ ID NO: 24, or SEQ ID NO: 26, wherein amino acid residue N50, K52, D59, S60, R69, or a combination thereof has been modified.

[0120] In some aspects, the amino acid residue N50, K52, D59, S60, R69, or a combination thereof has been modified to comprise a positive charge (referred to herein as a “positive charge modification”). For example, in some aspects, the asparagine (N) at amino acid residue 50 has been modified to lysine (K) or arginine (R). In some aspects, the lysine (K) at amino acid residue 52 is unmodified or has been modified to arginine (R). In some aspects, the aspartic acid (D) at amino acid residue 59 has been modified to lysine (K) or arginine (R). In some aspects, the serine (S) at amino acid residue 60 has been modified to lysine (K) or arginine (R). In some aspects, the arginine (R) at amino acid residue 69 is unmodified or has been modified to lysine (K).

[0121] Accordingly, in some aspects, the first CH3 domain or the second CH3 domain comprises the amino acid sequence set forth in SEQ ID NO: 40 with one or more amino acid modifications selected from (a) N50K or N50R, (b) K52R, (c) D59K or D59R, (d) S60K or S60R, (e) R69K, or (f) any combination of (a)-(e). In some aspects, the first CH3 domain or the second CH3 domain comprises the amino acid sequence set forth in SEQ ID NO: 40 with the following amino acid modifications: (a) N50K or N50R, (b) K52R, (c) D59K or D59R, (d) S60K or S60R, and (e) R69K. In some aspects, the first CH3 domain or the second CH3 domain comprises the amino acid sequence set forth in SEQ ID NO: 24 with one or more amino acid modifications selected from (a) N50K or N50R, (b) K52R, (c) D59K or D59R, (d) S60K or S60R, (e) R69K, or (f) any combination of (a)-(e). In some aspects, the first CH3 domain or the second CH3 domain comprises the amino acid sequence set forth in SEQ ID NO: 24 with the following amino acid modifications: (a) N50K or N50R, (b) K52R, (c) D59K or D59R, (d) S60K or S60R, and (e) R69K. In some aspects, the first CH3 domain or the second CH3 domain comprises the amino acid sequence set forth in SEQ ID NO: 26 with one or more amino acid modifications selected from (a) N50K or N50R, (b) K52R, (c) D59K or D59R, (d) S60K or S60R, (e) R69K, or (f) any combination of (a)-(e). In some aspects, the first CH3 domain or the second CH3 domain comprises the amino acid sequence set forth in SEQ ID NO: 26 with the following amino acid modifications: (a) N50K or N50R, (b) K52R, (c) D59K or D59R, (d) S60K or S60R, and (e) R69K.

[0122] As evident from the disclosure, amino acid residues N50, K52, D59, S60, and R69 correspond to amino acid residues N188, K190, D197, S198, and R207 of SEQ ID NO: 4, 16, or 20, respectively. Thus, in some aspects, the polypeptide of the heterodimeric protein comprises the amino acid sequence set forth in SEQ ID NO: 4 having one or more amino acid modifications selected from the group consisting of: (a) N188K or N188R, (b) K190R, (c) D197K or D197R, (d) S198K or S198R, (e) R207K, or (f) any combination of (a) to (e). In some aspects, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 4 having the following amino acid modifications: (a) N188K or N188R, (b) K190R, (c) D197K or D197R, (d) S198K or S198R, and (e) R207K. In some aspects, the polypeptide of the heterodimeric protein comprises the amino acid sequence set forth in SEQ ID NO: 16 having one or more amino acid modifications selected from the group consisting of: (a) N188K or N188R, (b) K190R, (c) D197K or D197R, (d) S198K or S198R, (e) R207K, or (f) any combination of (a) to (e). In some aspects, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 16 having the following amino acid modifications: (a) N188K or N188R, (b) K190R, (c) D197K or D197R, (d) S198K or S198R, and (e) R207K. In some aspects, the polypeptide of the heterodimeric protein comprises the amino acid sequence set forth in SEQ ID NO: 20 having one or more amino acid modifications selected from the group consisting of: (a) N188K or N188R, (b) K190R, (c) D197K or D197R, (d) S198K or S198R, (e) R207K, or (f) any combination of (a) to (e). In some aspects, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 16 having the following amino acid modifications: (a) N188K or N188R, (b) K190R, (c) D197K or D197R, (d) S198K or S198R, and (e) R207K.

[0123] In some aspects, the first CH3 domain or the second CH3 domain comprises the amino acid sequence set forth in SEQ ID NO: 40, SEQ ID NO: 24, or SEQ ID NO: 26, wherein amino acid residue N50, D59, S60, F65, R69, or a combination thereof has been modified to comprise a negative charge (referred to herein as a“negative charge modification”). For example, in some aspects, the asparagine (N) at amino acid residue 50 has been modified to an aspartic acid (D) or a glutamic acid (E). In some aspects, the aspartic acid (D) at amino acid residue 59 is unmodified or has been modified to a glutamic acid (E). In some aspects, the serine (S) at amino acid residue 60 has been modified to an aspartic acid (D) or a glutamic acid (E). In some aspects, the phenylalanine (F) at amino acid residue 65 has been modified to an aspartic acid (D) or a glutamic acid (E). In some aspects, the arginine (R) at amino acid residue 69 has been modified to an aspartic acid (D) or a glutamic acid (E).

[0124] Accordingly, in some aspects, the first CH3 domain or the second CH3 domain comprises the amino acid sequence set forth in SEQ ID NO: 40 with one or more amino acid modifications selected from (a) N50D or N50E, (b) D59E, (c) S60D or S60E, (d) F65D or F65E, (e) R69D or R69E, or (f) any combination of (a)-(e). In some aspects, the first CH3 domain or the second CH3 domain comprises the amino acid sequence set forth in SEQ ID NO: 40 with the following amino acid modifications: (a) N50D or N50E, (b) D59E, (c) S60D or S60E, (d) F65D or F65E, and (e) R69D or R69E. In some aspects, the first CH3 domain or the second CH3 domain comprises the amino acid sequence set forth in SEQ ID NO: 24 with one or more amino acid modifications selected from (a) N50D or N50E, (b) D59E, (c) S60D or S60E, (d) F65D or F65E, (e) R69D or R69E, or (f) any combination of (a)-(e). In some aspects, the first CH3 domain or the second CH3 domain comprises the amino acid sequence set forth in SEQ ID NO: 24 with the following amino acid modifications: (a) N50D or N50E, (b) D59E, (c) S60D or S60E, (d) F65D or F65E, and (e) R69D or R69E. In some aspects, the first CH3 domain or the second CH3 domain comprises the amino acid sequence set forth in SEQ ID NO: 26 with one or more amino acid modifications selected from (a) N50D or N50E, (b) D59E, (c) S60D or S60E, (d) F65D or F65E, (e) R69D or R69E, or (f) any combination of (a)-(e). In some aspects, the first CH3 domain or the second CH3 domain comprises the amino acid sequence set forth in SEQ ID NO: 26 with the following amino acid modifications: (a) N50D or N50E, (b) D59E, (c) S60D or S60E, (d) F65D or F65E, and (e) R69D or R69E.

[0125] In some aspects, the polypeptides of the heterodimeric proteins described herein comprise the amino acid sequence set forth in SEQ ID NO: 4 having one or more amino acid modifications selected from (a) N188D or N188E, (b) D197E, (c) S198D or S198E, (d) F203D or F203E, (e) R207D or R207E, or (f) any combination of (a) to (e). In some aspects, the polypeptides comprise the amino acid sequence set forth in SEQ ID NO: 4 having the following amino acid modifications: (a) N188D or N188E, (b) D197E, (c) S198D or S198E, (d) F203D or F203E, and (e) R207D or R207E. In some aspects, the polypeptides of the heterodimeric proteins described herein comprise the amino acid sequence set forth in SEQ ID NO: 16 having one or more amino acid modifications selected from (a) N188D or N188E, (b) D197E, (c) S198D or S198E, (d) F203D or F203E, (e) R207D or R207E, or (f) any combination of (a) to (e). In some aspects, the polypeptides comprise the amino acid sequence set forth in SEQ ID NO: 16 having the following amino acid modifications: (a) N188D or N188E, (b) D197E, (c) S198D or S198E, (d) F203D or F203E, and (e) R207D or R207E. In some aspects, the polypeptides of the heterodimeric proteins described herein comprise the amino acid sequence set forth in SEQ ID NO: 20 having one or more amino acid modifications selected from (a) N188D or N188E, (b) D197E, (c) S198D or S198E, (d) F203D or F203E, (e) R207D or R207E, or (f) any combination of (a) to (e). In some aspects, the polypeptides comprise the amino acid sequence set forth in SEQ ID NO: 20 having the following amino acid modifications: (a) N188D or N188E, (b) D197E, (c) S198D or S198E, (d) F203D or F203E, and (e) R207D or R207E.

[0126] In some aspects, provided herein are heterodimeric proteins comprising a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide are different, wherein the first polypeptide comprises a first CH3 domain and the second polypeptide comprises a second CH3 domain, wherein: (1) the first CH3 domain comprises an amino acid sequence set forth in SEQ ID NO: 40 with one or more amino acid modifications selected from the group consisting of: (a) N50K or N50R, (b) K52R, (c) D59K or D59R, (d) S60K or S60R, (e) R69K, (f) any combination of (a) to (e), and (2) the second CH3 domain comprises an amino acid sequence set forth in SEQ ID NO: 40 with one or more amino acid modifications selected from the group consisting of: (a) N50D or N50E, (b) D59E, (c) S60D or S60E, (d) F65D or F65E, (e) R69D or R69E, or (f) any combination of (a) to (e), and wherein the one or more amino acid modifications of the first CH3 domain and the one or more amino acid modifications of the second CH3 domain promote interaction of the first CH3 domain and the second CH3 domain. In some aspects, (1) the first CH3 domain comprises an amino acid sequence set forth in SEQ ID NO: 40 with the following amino acid modifications: (a) N50K or N50R, (b) K52R, (c) D59K or D59R, (d) S60K or S60R, and (e) R69K, and (2) the second CH3 domain comprises an amino acid sequence set forth in SEQ ID NO: 40 with the following amino acid modifications: (a) N50D or N50E, (b) D59E, (c) S60D or S60E, (d) F65D or F65E, (e) R69D or R69E.

[0127] In some aspects, provided herein are heterodimeric proteins comprising a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide are different, wherein the first polypeptide comprises a first CH3 domain and the second polypeptide comprises a second CH3 domain, wherein: (1) the first CH3 domain comprises an amino acid sequence set forth in SEQ ID NO: 24 with one or more amino acid modifications selected from the group consisting of: (a) N50K or N50R, (b) K52R, (c) D59K or D59R, (d) S60K or S60R, (e) R69K, or (f) any combination of (a) to (e), and (2) the second CH3 domain comprises an amino acid sequence set forth in SEQ ID NO: 26 with one or more amino acid modifications selected from the group consisting of: (a) N50D or N50E, (b) D59E, (c) S60D or S60E, (d) F65D or F65E, (e) R69D or R69E, or (f) any combination of (a) to (e), and wherein the one or more amino acid modifications of the first CH3 domain and the one or more amino acid modifications of the second CH3 domain promote interaction of the first CH3 domain and the second CH3 domain. In some aspects, (1) the first CH3 domain comprises an amino acid sequence set forth in SEQ ID NO: 24 with the following amino acid modifications: (a) N50K or N50R, (b) K52R, (c) D59K or D59R, (d) S60K or S60R, and (e) R69K, and (2) the second CH3 domain comprises an amino acid sequence set forth in SEQ ID NO: 26 with the following amino acid modifications: (a) N50D or N50E, (b) D59E, (c) S60D or S60E, (d) F65D or F65E, (e) R69D or R69E.

[0128] In some aspects, provided herein are heterodimeric proteins comprising a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide are different, wherein the first polypeptide comprises a first CH3 domain and the second polypeptide comprises a second CH3 domain, wherein: (1) the first CH3 domain comprises an amino acid sequence set forth in SEQ ID NO: 26 with one or more amino acid modifications selected from the group consisting of: (a) N50K or N50R, (b) K52R, (c) D59K or D59R, (d) S60K or S60R, (e) R69K, (f) any combination of (a) to (e), and (2) the second CH3 domain comprises an amino acid sequence set forth in SEQ ID NO: 24 with one or more amino acid modifications selected from the group consisting of: (a) N50D or N50E, (b) D59E, (c) S60D or S60E, (d) F65D or F65E, (e) R69D or R69E, or (f) any combination of (a) to (e), and wherein the one or more amino acid modifications of the first CH3 domain and the one or more amino acid modifications of the second CH3 domain promote interaction of the first CH3 domain and the second CH3 domain. In some aspects, (1) the first CH3 domain comprises an amino acid sequence set forth in SEQ ID NO: 26 with the following amino acid modifications: (a) N50K or N50R, (b) K52R, (c) D59K or D59R, (d) S60K or S60R, and (e) R69K, and (2) the second CH3 domain comprises an amino acid sequence set forth in SEQ ID NO: 24 with the following amino acid modifications: (a) N50D or N50E, (b) D59E, (c) S60D or S60E, (d) F65D or F65E, (e) R69D or R69E.

[0129] In some aspects, the first CH3 domain or the second CH3 domain useful for the present disclosure comprises an amino acid sequence set forth in SEQ ID NO: 40, wherein amino acid residues T10, L11, F65, Y67, T26, K52, T54, D59, R69, or a combination thereof have been modified. In some aspects, the first CH3 domain or the second CH3 domain comprises an amino acid sequence set forth in SEQ ID NO: 40 with a D59R amino acid modification and one or more of the following amino acid modifications: T10V, L11Y, F65A, Y67V, or a combination thereof. In some aspects, the first CH3 domain or the second CH3 domain comprises an amino acid sequence set forth in SEQ ID NO: 40 with the following amino acid modifications: T10V, L11Y, F65A, Y67V, and D59R.

[0130] In some aspects, the first CH3 domain or the second CH3 domain comprises the amino acid sequence set forth in SEQ ID NO: 40 with a R69D amino acid modification and one or more of the following amino acid modifications: T10V, T26L, K52L, T54W, or a combination thereof. In some aspects, the first CH3 domain or the second CH3 domain comprises the amino acid sequence set forth in SEQ ID NO: 40 with the following amino acid modifications: T10V, T26L, K52L, T54W, and R69D.

[0131] Accordingly, in some aspects, the heterodimeric protein described herein comprises a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide are different, wherein the first polypeptide comprises a first CH3 domain and the second polypeptide comprises a second CH3 domain, wherein: (a) the first CH3 domain comprises the amino acid sequence set forth in SEQ ID NO: 40 with a D59R amino acid modification and one or more of the following amino acid modifications: T10V, L11Y, F65A, Y67V, or a combination thereof, and (b) the second CH3 domain comprises the amino acid sequence set forth in SEQ ID NO: 40 with a R69D amino acid modification and one or more of the following amino acid modifications: T10V, T26L, K52L, T54W, or a combination thereof, wherein the one or more amino acid modifications of the first CH3 domain and the one or more amino acid modifications of the second CH3 domain promote interaction of the first CH3 domain and the second CH3 domain. In some aspects, the heterodimeric protein comprises a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide are different, wherein the first polypeptide comprises a first CH3 domain and the second polypeptide comprises a second CH3 domain, wherein: (a) the first CH3 domain comprises the amino acid sequence set forth in SEQ ID NO: 40 with the following amino acid modifications: D59R, T10V, L11Y, F65A, and Y67V, and (b) the second CH3 domain comprises the amino acid sequence set forth in SEQ ID NO: 40 with the following amino acid modifications: R69D, T10V, T26L, K52L, and T54W.

[0132] In some aspects, the first CH3 domain or the second CH3 domain comprises the amino acid sequence set forth in SEQ ID NO: 40 with a R69E amino acid modification and one or more of the following amino acid modifications: T10V, T11L, K52L, T54W, or a combination thereof. In some aspects, the first CH3 domain or the second CH3 domain comprises the amino acid sequence set forth in SEQ ID NO: 40 with the following amino acid modifications: T10V, T26L, K52L, T54W, and R69E.

[0133] Accordingly, in some aspects, the heterodimeric protein described herein comprises a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide are different, wherein the first polypeptide comprises a first CH3 domain and the second polypeptide comprises a second CH3 domain, wherein: (a) the first CH3 domain comprises the amino acid sequence set forth in SEQ ID NO: 40 with a D59R amino acid modification and one or more of the following amino acid modifications: T10V, L11Y, F65A, Y67V, or a combination thereof, and (b) the second CH3 domain comprises the amino acid sequence set forth in SEQ ID NO: 40 with a R69E amino acid modification and one or more of the following amino acid modifications: T10V, T26L, K52L, T54W, or a combination thereof, wherein the one or more amino acid modifications of the first CH3 domain and the one or more amino acid modifications of the second CH3 domain promote interaction of the first CH3 domain and the second CH3 domain. In some aspects, (a) the first CH3 domain comprises the amino acid sequence set forth in SEQ ID NO: 40 with the following amino acid modifications: D59R, T10V, L11Y, F65A, and Y67V, and (b) the second CH3 domain comprises the amino acid sequence set forth in SEQ ID NO: 40 with the following amino acid modifications: R69E, T10V, T26L, K52L, and T54W.

[0134] In some aspects, (a) amino acid residue N50 of the first CH3 domain is modified to N50K or N50R and amino acid residue S60 of the second CH3 domain is modified to S60D or S60E; (b) amino acid residue S60 of the first CH3 domain is modified to S60D or S60E and amino acid residue N50 of the second CH3 domain is modified to N50K or N50R; (c) amino acid residue K52 of the first CH3 domain is unmodified or modified to K52R and amino acid residue F65 of the second CH3 domain is modified to F65D or F65E; (d) amino acid residue D59 of the first CH3 domain is modified to D59K or D59R and amino acid residue R69 of the second CH3 domain is modified to R69D or R69E; (e) amino acid residue R69 of the first CH3 domain is modified to R69D or R69E and amino acid residue D59 of the second CH3 domain is modified to D59K or D59R; or (f) any combination of (a) to (e). In some aspects, amino acid residue N50 of the first CH3 domain is modified to N50K or N50R and amino acid residue S60 of the second CH3 domain is modified to S60D or S60E. In some aspects, amino acid residue S60 of the first CH3 domain is modified to S60D or S60E and amino acid residue N50 of the second CH3 domain is modified to N50K or N50R. In some aspects, amino acid residue K52 of the first CH3 domain is unmodified or modified to K52R and amino acid residue F65 of the second CH3 domain is modified to F65D or F65E. In some aspects, amino acid residue D59 of the first CH3 domain is modified to D59K or D59R and amino acid residue R69 of the second CH3 domain is modified to R69D or R69E. In some aspects, amino acid residue R69 of the first CH3 domain is modified to R69D or R69E and amino acid residue D59 of the second CH3 domain is modified to D59K or D59R.

[0135] In some aspects, the first CH3 domain or the second CH3 domain comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 28, SEQ ID NO: 31, or SEQ ID NO: 33. In some aspects, a heterodimeric protein provided herein comprises a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide are different, and wherein: (a) the first polypeptide comprises a first CH3 domain comprising the amino acid sequence set forth in SEQ ID NO: 28, and (b) the second polypeptide comprises a second CH3 domain comprising the amino acid sequence set forth in SEQ ID NO: 31. In some aspects, a heterodimeric protein provided herein comprises a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide are different, and wherein: (a) the first polypeptide comprises a first CH3 domain comprising the amino acid sequence set forth in SEQ ID NO: 28, and (b) the second polypeptide comprises a second CH3 domain comprising the amino acid sequence set forth in SEQ ID NO: 33.

[0136] Hinge domain As further described elsewhere in the present disclosure, in some aspects, the first polypeptide and / or the second polypeptide further comprises a hinge domain. Thus, in some aspects, a heterodimeric protein provided herein comprises a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide are different, and wherein: (a) the first polypeptide comprises (i) a first hinge domain and (ii) a first CH3 domain comprising a heteromultimerization modification (e.g., an amino acid modification described herein), and (b) the second polypeptide comprises (i) a second hinge domain and (ii) a second CH3 domain comprising a heteromultimerization modification (e.g., an amino acid modification described herein).

[0137] In some aspects, a hinge domain useful for the present disclosure (e.g., a first hinge domain and / or a second hinge domain) can be derived from the heavy chain of any suitable antibody. In some aspects, the hinge domain is a hinge domain of an IgG antibody, an IgA antibody, an IgD antibody, an IgE antibody, or an IgM antibody. In some aspects, the hinge domain is a hinge domain of an IgG antibody (referred to herein as an “IgG hinge domain”). For example, in some aspects, the hinge domain is a hinge domain of an IgG1 antibody (“IgG1 hinge domain”). In some aspects, the hinge domain is a hinge domain of an IgG2 antibody (“IgG2 hinge domain”). In some aspects, the hinge domain is a hinge domain of an IgG3 antibody (“IgG3 hinge domain”). In some aspects, the hinge domain is a hinge domain of an IgG4 antibody (“IgG4 hinge domain”). In some aspects, the hinge domain is a hinge domain of an IgD antibody (referred to herein as an “IgD hinge domain”). Exemplary amino acid sequences of such hinge domains are provided elsewhere in the present disclosure (see, e.g., Table 3).

[0138] In some aspects, a hinge domain useful for the present disclosure (e.g., a first hinge domain and / or a second hinge domain) comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 22. In some aspects, the hinge domain comprises the amino acid sequence set forth in SEQ ID NO: 22.

[0139] Linker In some aspects, a hinge domain useful for the present disclosure comprises a linker, e.g., a synthetic linker. Non-limiting examples of linkers include: m-maleimidobenzoyl-N- hydroxysuccinimide ester (MBS) (see, e.g., Ramakrishnan et al., Bioconjugate Chem. 1999, 10(6): 971-7), and the like. Cancer Research44(1): 201-208 (1984), incorporated herein in its entirety by reference); (i) EDC (1-ethyl-3-(3-dimethylamino-propyl) carbodiimide hydrochloride; (ii) SMPT (4-succinimidoxycarbonyl-alpha-methyl-alpha-(2-pyridyl-disulfide)-toluene (Pierce Chem. Co. Catalog No. 21558G); (iii) SPDP (succinimidyl-6 [3-(2-pyridyldithio) propionamido] hexanoate (Pierce Chem. Co., Catalog No. 21651g); (iv) Sulfo-LC-SPDP (6 [3-(2-pyridyldithio)-propionamido] hexanoic acid sulfosuccinimidyl ester (Pierce Chem. Co. Catalog No. 2165-G); and (v) sulfo-NHS (N-hydroxysulfosuccinimide: Pierce Chem. Co., Catalog No. 24510) conjugated to EDC.

[0140] In some aspects, the linker comprises glycine, serine, alanine, or a combination thereof. Thus, in some aspects, a linker useful in the present disclosure comprises a glycine polymer (G)n, where n is an integer of at least 1 (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, etc.). In some aspects, the linker comprises a glycine-serine polymer (GS)n, where n is an integer of at least 1 (SEQ ID NO: 41) (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, etc.). In some aspects, the linker comprises a glycine-alanine polymer (GA)n, where n is an integer of at least 1 (SEQ ID NO: 42) (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, etc.). In some aspects, the linker comprises an alanine-serine polymer (AS)n, where n is an integer of at least 1 (SEQ ID NO: 43) (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, etc.). Additional disclosures related to useful linkers are provided in WO2023072177A1, which is incorporated by reference herein in its entirety.

[0141] In some aspects, the hinge domain comprises a glycine / serine linker according to the formula [(Gly)n-Ser]m (SEQ ID NO: 44), wherein n is any integer from 1 to 100, and m is any integer from 1 to 100. In other aspects, the glycine / serine linker is according to the formula [(Gly)x-Sery]z (SEQ ID NO: 45), wherein x is an integer from 1 to 4, y is 0 or 1, and z is an integer from 1 to 50. In some aspects, the optional linker comprises the sequence (G)n (SEQ ID NO: 46), wherein n can be an integer from 1 to 100. In some aspects, the linker can comprise the sequence (GlyAla)n (SEQ ID NO: 47), wherein n is an integer between 1 and 100. Non-limiting examples of glycine / serine linkers useful in the present disclosure are described, for example, in US 2021 / 0206806 and US 2017 / 0114151, each of which is incorporated by reference herein in its entirety.

[0142] In some aspects, the linker is GGGG (SEQ ID NO: 48). In some aspects, the linker is GGGSG (SEQ ID NO: 49). In some aspects, the linker comprises the sequence (GGGSG)n (SEQ ID NO: 50). In some aspects, the linker comprises the sequence (GGGGS)n (SEQ ID NO: 51). In some aspects, the linker can comprise the sequence (GGGS)n (SEQ ID NO: 52). In some aspects, the linker can comprise the sequence (GGS)n (SEQ ID NO: 53). In these aspects, n can be an integer from 1 to 100. In other instances, n can be an integer from one to 20, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some aspects, n is an integer from 1 to 100.

[0143] Non-limiting examples of linkers that can be used with the present disclosure include: GGGGSGGGGS (SEQ ID NO: 11), GSGSGS (SEQ ID NO: 54), GGSGG (SEQ ID NO: 55), SGGSGGS (SEQ ID NO: 56), GGSGGSGGSGGSGGG (SEQ ID NO: 57), GGSGGSGGGGSGGGGS (SEQ ID NO: 58), GGSGGSGGSGGSGGSGGS (SEQ ID NO: 59), or GGGGSGGGGSGGGGS (SEQ ID NO: 60).

[0144] In some aspects, the linker comprises the sequence PGG. In some aspects, the linker comprises additional amino acids in addition to glycine and serine. In some aspects, the linker comprises at least about 60%, at least about 65%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or at least 95% glycine or serine amino acids.

[0145] In some particular aspects, the length of the hinge domain (e.g., linker) is between 1 and 10 amino acids. In some aspects, the length of the hinge domain (e.g., linker) is between about 5 and about 10 amino acids, between about 10 and about 20 amino acids, between about 20 and about 30 amino acids, between about 30 and about 40 amino acids, between about 40 and about 50 amino acids, between about 50 and about 60 amino acids, between about 60 and about 70 amino acids, between about 70 and about 80 amino acids, between about 80 and about 90 amino acids, or between about 90 and about 100 amino acids.

[0146] In some aspects, the linker is a non-cleavable linker. In some aspects, the linker is a cleavable linker. As used herein, the term “cleavable linker” refers to a linker that comprises a cleavage site such that when expressed can be selectively cleaved to produce two or more products. In some aspects, the linker is selected from a P2A linker, a T2A linker, a F2A linker, an E2A linker, a furin cleavage site, or any combination thereof (see Table 2 below). In some aspects, the linker further comprises a GSG linker sequence. In some aspects, linkers useful with the present disclosure comprise an internal ribosome entry site (IRES) such that during translation separate polypeptides encoded by the first and second genes are produced. Additional descriptions of linkers useful with the present disclosure are provided in WO 2020 / 223625 Al and US 2019 / 0276801 Al, each of which is incorporated by reference herein in its entirety.

[0147] Table 2: Linker Sequences Accordingly, in some aspects, the present disclosure provides a heterodimeric protein comprising a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide are different, wherein: (a) the first polypeptide comprises (i) a first hinge domain selected from an IgGl hinge domain, an IgG2 hinge domain, an IgG3 hinge domain, an IgG4 hinge domain, an IgD hinge domain, a synthetic linker, or a combination thereof, and (ii) a first CH3 domain comprising a heteromultimerization modification (e.g., an amino acid modification described herein); and (b) the second polypeptide comprises (i) a second hinge domain selected from an IgGl hinge domain, an IgG2 hinge domain, an IgG3 hinge domain, an IgG4 hinge domain, an IgD hinge domain, a synthetic linker, or a combination thereof, and (ii) a second CH3 domain comprising a heteromultimerization modification (e.g., an amino acid modification described herein). In some aspects, the first hinge domain and the second hinge domain are the same. In some aspects, the first hinge domain and the second hinge domain are different.

[0148] In some aspects, the heterodimeric protein provided herein comprises a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide are different, wherein: (a) the first polypeptide comprises (i) a first hinge domain comprising the amino acid sequence set forth in SEQ ID NO: 22, and (ii) a first CH3 domain comprising the amino acid sequence set forth in SEQ ID NO: 40 with one or more amino acid modifications selected from: T10V, L11Y, F65A, Y67V, or a combination thereof; and (b) the second polypeptide comprises (i) a second hinge domain comprising the amino acid sequence set forth in SEQ ID NO: 22, and (ii) a second CH3 domain comprising the amino acid sequence set forth in SEQ ID NO: 40 with one or more amino acid modifications selected from: T10V, T26L, K52L, T54W, or a combination thereof. In some aspects, the heterodimeric protein comprises a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide are different, wherein: (a) the first polypeptide comprises (i) a first hinge domain comprising the amino acid sequence set forth in SEQ ID NO: 22, and (ii) a first CH3 domain comprising the amino acid sequence set forth in SEQ ID NO: 40 with the following amino acid modifications: T10V, L11Y, F65A, and Y67V; and (b) the second polypeptide comprises (i) a second hinge domain comprising the amino acid sequence set forth in SEQ ID NO: 22, and (ii) a second CH3 domain comprising the amino acid sequence set forth in SEQ ID NO: 40 with the following amino acid modifications: T10V, T26L, K52L, and T54W.

[0149] In some aspects, provided herein are heterodimeric proteins comprising a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide are different, wherein: (1) the first polypeptide comprises (i) a first hinge domain comprising the amino acid sequence set forth in SEQ ID NO: 22, and (ii) a first CH3 domain comprising the amino acid sequence set forth in SEQ ID NO: 40 with one or more amino acid modifications selected from: (a) N50K or N50R, (b) K52R, (c) D59K or D59R, (d) S60K or S60R, (e) R69K, or (f) any combination of (a)-(e), and (2) the second polypeptide comprises (i) a second hinge domain comprising the amino acid sequence set forth in SEQ ID NO: 22, and (ii) a second CH3 domain comprising the amino acid sequence set forth in SEQ ID NO: 40 with one or more amino acid modifications selected from: (a) N50D or N50E, (b) D59E, (c) S60D or S60E, (d) F65D or F65E, (e) R69D or R69E, or (f) any combination of (a)-(e), and wherein the one or more amino acid modifications of the first CH3 domain and the one or more amino acid modifications of the second CH3 domain promote interaction of the first CH3 domain and the second CH3 domain. In some aspects, the heterodimeric proteins described herein comprise a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide are different, wherein: (1) the first polypeptide comprises (i) a first hinge domain comprising the amino acid sequence set forth in SEQ ID NO: 22, and (ii) a first CH3 domain comprising the amino acid sequence set forth in SEQ ID NO: 40 with the following amino acid modifications: (a) N50K or N50R, (b) K52R, (c) D59K or D59R, (d) S60K or S60R, and (e) R69K, and (2) the second polypeptide comprises (i) a second hinge domain comprising the amino acid sequence set forth in SEQ ID NO: 22, and (ii) a second CH3 domain comprising the amino acid sequence set forth in SEQ ID NO: 40 with the following amino acid modifications: (a) N50D or N50E, (b) D59E, (c) S60D or S60E, (d) F65D or F65E, and (e) R59D or R59E.

[0150] In some aspects, provided herein are heterodimeric proteins comprising a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide are different, wherein: (1) the first polypeptide comprises (i) a first hinge domain comprising the amino acid sequence set forth in SEQ ID NO: 22, and (ii) a first CH3 domain comprising the amino acid sequence set forth in SEQ ID NO: 24 having one or more amino acid modifications selected from the group consisting of: (a) N50K or N50R, (b) K52R, (c) D59K or D59R, (d) S60K or S60R, (e) R69K, or (f) any combination of (a) to (e), and (2) the second polypeptide comprises (i) a second hinge domain comprising the amino acid sequence set forth in SEQ ID NO: 22, and (ii) a second CH3 domain comprising the amino acid sequence set forth in SEQ ID NO: 26 having one or more amino acid modifications selected from the group consisting of: (a) N50D or N50E, (b) D59E, (c) S60D or S60E, (d) F65D or F65E, (e) R69D or R69E, or (f) any combination of (a) to (e), and wherein the one or more amino acid modifications of the first CH3 domain and the one or more amino acid modifications of the second CH3 domain promote interaction of the first CH3 domain and the second CH3 domain. In some aspects, the heterodimeric proteins described herein comprise a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide are different, wherein: (1) the first polypeptide comprises (i) a first hinge domain comprising the amino acid sequence set forth in SEQ ID NO: 22, and (ii) a first CH3 domain comprising the amino acid sequence set forth in SEQ ID NO: 24 having the following amino acid modifications: (a) N50K or N50R, (b) K52R, (c) D59K or D59R, (d) S60K or S60R, and (e) R69K, and (2) the second polypeptide comprises (i) a second hinge domain comprising the amino acid sequence set forth in SEQ ID NO: 22, and (ii) a second CH3 domain comprising the amino acid sequence set forth in SEQ ID NO: 26 having the following amino acid modifications: (a) N50D or N50E, (b) D59E, (c) S60D or S60E, (d) F65D or F65E, and (e) R69D or R69E.

[0151] In some aspects, provided herein are heterodimeric proteins comprising a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide are different, wherein: (1) the first polypeptide comprises (i) a first hinge domain comprising the amino acid sequence set forth in SEQ ID NO: 22, and (ii) a first CH3 domain comprising the amino acid sequence set forth in SEQ ID NO: 26 having one or more amino acid modifications selected from the group consisting of: (a) N50K or N50R, (b) K52R, (c) D59K or D59R, (d) S60K or S60R, (e) R69K, or (f) any combination of (a) to (e), and (2) the second polypeptide comprises (i) a second hinge domain comprising the amino acid sequence set forth in SEQ ID NO: 22, and (ii) a second CH3 domain comprising the amino acid sequence set forth in SEQ ID NO: 24 having one or more amino acid modifications selected from the group consisting of: (a) N50D or N50E, (b) D59E, (c) S60D or S60E, (d) F65D or F65E, (e) R69D or R69E, or (f) any combination of (a) to (e), and wherein the one or more amino acid modifications of the first CH3 domain and the one or more amino acid modifications of the second CH3 domain promote interaction of the first CH3 domain and the second CH3 domain. In some aspects, the heterodimeric proteins described herein comprise a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide are different, wherein: (1) the first polypeptide comprises (i) a first hinge domain comprising the amino acid sequence set forth in SEQ ID NO: 22, and (ii) a first CH3 domain comprising the amino acid sequence set forth in SEQ ID NO: 26 having the following amino acid modifications: (a) N50K or N50R, (b) K52R, (c) D59K or D59R, (d) S60K or S60R, and (e) R69K, and (2) the second polypeptide comprises (i) a second hinge domain comprising the amino acid sequence set forth in SEQ ID NO: 22, and (ii) a second CH3 domain comprising the amino acid sequence set forth in SEQ ID NO: 24 having the following amino acid modifications: (a) N50D or N50E, (b) D59E, (c) S60D or S60E, (d) F65D or F65E, and (e) R69D or R69E.

[0152] In some aspects, the hetero-dimeric protein provided herein comprises a first polypeptide and a second polypeptide, wherein: (a) the first polypeptide comprises (i) a first hinge domain comprising the amino acid sequence set forth in SEQ ID NO: 22, and (ii) a first CH3 domain comprising the amino acid sequence set forth in SEQ ID NO: 40 with a D59R amino acid modification and one or more of the following amino acid modifications: T10V, L11Y, F65A, Y67V, or a combination thereof; and (b) the second polypeptide comprises (i) a second hinge domain comprising the amino acid sequence set forth in SEQ ID NO: 22, and (ii) a second CH3 domain comprising the amino acid sequence set forth in SEQ ID NO: 40 with a R69D amino acid modification and one or more of the following amino acid modifications: T10V, T26L, K52L, T54W, or a combination thereof. In some aspects, the hetero-dimeric protein comprises a first polypeptide and a second polypeptide, wherein: (a) the first polypeptide comprises (i) a first hinge domain comprising the amino acid sequence set forth in SEQ ID NO: 22, and (ii) a first CH3 domain comprising the amino acid sequence set forth in SEQ ID NO: 40 with the following amino acid modifications: D59R, T10V, L11Y, F65A, and Y67V; and (b) the second polypeptide comprises (i) a second hinge domain comprising the amino acid sequence set forth in SEQ ID NO: 22, and (ii) a second CH3 domain comprising the amino acid sequence set forth in SEQ ID NO: 40 with the following amino acid modifications: R69D, T10V, T26L, K52L, and T54W.

[0153] In some aspects, the heterodimeric protein provided herein comprises a first polypeptide and a second polypeptide, wherein: (a) the first polypeptide comprises (i) a first hinge domain comprising the amino acid sequence set forth in SEQ ID NO: 22, and (ii) a first CH3 domain comprising the amino acid sequence set forth in SEQ ID NO: 40 having the D59R amino acid modification and one or more of the following amino acid modifications: T10V, L11Y, F65A, Y67V, or a combination thereof; and (b) the second polypeptide comprises (i) a second hinge domain comprising the amino acid sequence set forth in SEQ ID NO: 22, and (ii) a second CH3 domain comprising the amino acid sequence set forth in SEQ ID NO: 40 having the R69E amino acid modification and one or more of the following amino acid modifications: T10V, T26L, K52L, T54W, or a combination thereof. In some aspects, the heterodimeric protein comprises a first polypeptide and a second polypeptide, wherein: (a) the first polypeptide comprises (i) a first hinge domain comprising the amino acid sequence set forth in SEQ ID NO: 22, and (ii) a first CH3 domain comprising the amino acid sequence set forth in SEQ ID NO: 40 having the following amino acid modifications: D59R, T10V, L11Y, F65A, and Y67V; and (b) the second polypeptide comprises (i) a second hinge domain comprising the amino acid sequence set forth in SEQ ID NO: 22, and (ii) a second CH3 domain comprising the amino acid sequence set forth in SEQ ID NO: 40 having the following amino acid modifications: R69E, T10V, T26L, K52L, and T54W.

[0154] CH2 domain In some aspects, the first and / or second polypeptides provided herein (e.g., comprising a CH3 domain having one or more heteromultimerization modifications) further comprise a CH2 domain. CH2 domains useful in the present disclosure can be derived from the heavy chain of any suitable antibody. For example, in some aspects, the CH2 domain is the domain of an IgG antibody (“IgG CH2 domain”), an IgA antibody (“IgA CH2 domain”), an IgD antibody (“IgD CH2 domain”), an IgE antibody (“IgE CH2 domain”), or an IgM antibody (“IgM CH2 domain”). In some aspects, the CH2 domain comprises the CH2 domain of multiple antibodies. For example, in some aspects, the CH2 domain comprises a portion of a human IgD CH2 domain and a portion of a human IgG4 CH2 domain (referred to herein as an “IgD / IgG4 CH2 domain”). An exemplary amino acid sequence of an IgD / IgG4 CH2 domain is set forth in SEQ ID NO: 25 (see, e.g., Table 3).

[0155] In some aspects, a CH2 domain useful for the present disclosure comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 25. In some aspects, a CH2 domain comprises the amino acid sequence set forth in SEQ ID NO: 25.

[0156] It will be apparent from the present disclosure that, in some aspects, a heterodimeric protein described herein comprises a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide are different, wherein: (a) the first polypeptide comprises (i) a first CH2 domain (e.g., an IgD / IgG4 CH2 domain), and (ii) a first CH3 domain comprising a heteromultimerization modification (e.g., an amino acid modification described herein, e.g., a positive charge modification and / or a negative charge modification described herein); and (b) the second polypeptide comprises (i) a second CH2 domain (e.g., an IgD / IgG4 CH2 domain), and (ii) a second CH3 domain comprising a heteromultimerization modification (e.g., an amino acid modification described herein, e.g., a negative charge modification and / or a positive charge modification described herein). In some aspects, the first CH2 domain and the second CH2 domain are the same. In some aspects, the first CH2 domain and the second CH2 domain are different.

[0157] It will be apparent from the present disclosure that, in some aspects, a heterodimeric protein described herein comprises a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide are different, wherein: (a) the first polypeptide comprises (i) a first CH2 domain (e.g., an IgD / IgG4 CH2 domain), and (ii) a first CH3 domain comprising a heteromultimerization modification (e.g., an amino acid modification described herein, e.g., a positive charge modification and / or a negative charge modification described herein); and (b) the second polypeptide comprises (i) a second CH2 domain (e.g., an IgD / IgG4 CH2 domain), and (ii) a second CH3 domain comprising a heteromultimerization modification (e.g., an amino acid modification described herein, e.g., a negative charge modification and / or a positive charge modification described herein). In some aspects, the first CH2 domain and the second CH2 domain are the same. In some aspects, the first CH2 domain and the second CH2 domain are different.

[0158] In some aspects, the hetero-dimeric protein described herein comprises a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide are different, wherein: (a) the first polypeptide comprises (i) a first CH2 domain comprising the amino acid sequence set forth in SEQ ID NO: 25, and (ii) a first CH3 domain comprising the amino acid sequence set forth in SEQ ID NO: 40 with one or more amino acid modifications selected from T10V, L11Y, F65A, Y67V, or combinations thereof; and (b) the second polypeptide comprises (i) a second CH2 domain comprising the amino acid sequence set forth in SEQ ID NO: 25, and (ii) a second CH3 domain comprising the amino acid sequence set forth in SEQ ID NO: 40 with one or more amino acid modifications selected from T10V, T26L, K52L, T54W, or combinations thereof. In some aspects, (a) the first polypeptide comprises (i) a first CH2 domain comprising the amino acid sequence set forth in SEQ ID NO: 25, and (ii) a first CH3 domain comprising the amino acid sequence set forth in SEQ ID NO: 40 with the following amino acid modifications: T10V, L11Y, F65A, and Y67V; and (b) the second polypeptide comprises (i) a second CH2 domain comprising the amino acid sequence set forth in SEQ ID NO: 25, and (ii) a second CH3 domain comprising the amino acid sequence set forth in SEQ ID NO: 40 with the following amino acid modifications: T10V, T26L, K52L, T54W. In some aspects, the first polypeptide of such hetero-dimeric protein further comprises a first hinge domain comprising the amino acid sequence set forth in SEQ ID NO: 22. In some aspects, the second polypeptide of such hetero-dimeric protein further comprises a second hinge domain comprising the amino acid sequence set forth in SEQ ID NO: 22.

[0159] In some aspects, provided herein are heterodimeric proteins comprising a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide are different, wherein: (1) the first polypeptide comprises (i) a first CH2 domain comprising the amino acid sequence set forth in SEQ ID NO: 25, and (ii) a first CH3 domain comprising the amino acid sequence set forth in SEQ ID NO: 40 with one or more amino acid modifications selected from: (a) N50K or N50R, (b) K52R, (c) D59K or D59R, (d) S60K or S60R, (e) R69K, or (f) any combination of (a)-(e), and (2) the second polypeptide comprises (i) a second CH2 domain comprising the amino acid sequence set forth in SEQ ID NO: 25, and (ii) a second CH3 domain comprising the amino acid sequence set forth in SEQ ID NO: 40 with one or more amino acid modifications selected from: (a) N50D or N50E, (b) D59E, (c) S60D or S60E, (d) F65D or F65E, (e) R69D or R69E, or (f) any combination of (a)-(e), and wherein the one or more amino acid modifications of the first CH3 domain and the one or more amino acid modifications of the second CH3 domain promote interaction of the first CH3 domain and the second CH3 domain. In some aspects, (1) the first polypeptide comprises (i) a first CH2 domain comprising the amino acid sequence set forth in SEQ ID NO: 25, and (ii) a first CH3 domain comprising the amino acid sequence set forth in SEQ ID NO: 40 with the following amino acid modifications: (a) N50K or N50R, (b) K52R, (c) D59K or D59R, (d) S60K or S60R, and (e) R69K, and (2) the second polypeptide comprises (i) a second CH2 domain comprising the amino acid sequence set forth in SEQ ID NO: 25, and (ii) a second CH3 domain comprising the amino acid sequence set forth in SEQ ID NO: 40 with the following amino acid modifications: (a) N50D or N50E, (b) D59E, (c) S60D or S60E, (d) F65D or F65E, and (e) R69D or R69E. In some aspects, the first polypeptide of such heterodimeric proteins further comprises a first hinge domain comprising the amino acid sequence set forth in SEQ ID NO: 22. In some aspects, the second polypeptide of such heterodimeric proteins further comprises a second hinge domain comprising the amino acid sequence set forth in SEQ ID NO: 22.

[0160] In some aspects, provided herein are heterodimeric proteins comprising a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide are different, wherein: (1) the first polypeptide comprises (i) a first CH2 domain comprising the amino acid sequence set forth in SEQ ID NO: 25, and (ii) a first CH3 domain comprising the amino acid sequence set forth in SEQ ID NO: 24 with one or more amino acid modifications selected from: (a) N50K or N50R, (b) K52R, (c) D59K or D59R, (d) S60K or S60R, (e) R69K, or (f) any combination of (a)-(e), and (2) the second polypeptide comprises (i) a second CH2 domain comprising the amino acid sequence set forth in SEQ ID NO: 25, and (ii) a second CH3 domain comprising the amino acid sequence set forth in SEQ ID NO: 26 with one or more amino acid modifications selected from: (a) N50D or N50E, (b) D59E, (c) S60D or S60E, (d) F65D or F65E, (e) R69D or R69E, or (f) any combination of (a)-(e), and wherein the one or more amino acid modifications of the first CH3 domain and the one or more amino acid modifications of the second CH3 domain promote interaction of the first CH3 domain and the second CH3 domain. In some aspects, (1) the first polypeptide comprises (i) a first CH2 domain comprising the amino acid sequence set forth in SEQ ID NO: 25, and (ii) a first CH3 domain comprising the amino acid sequence set forth in SEQ ID NO: 24 with the following amino acid modifications: (a) N50K or N50R, (b) K52R, (c) D59K or D59R, (d) S60K or S60R, and (e) R69K, and (2) the second polypeptide comprises (i) a second CH2 domain comprising the amino acid sequence set forth in SEQ ID NO: 25, and (ii) a second CH3 domain comprising the amino acid sequence set forth in SEQ ID NO: 26 with the following amino acid modifications: (a) N50D or N50E, (b) D59E, (c) S60D or S60E, (d) F65D or F65E, and (e) R69D or R69E. In some aspects, the first polypeptide of such a heterodimeric protein further comprises a first hinge domain comprising the amino acid sequence set forth in SEQ ID NO: 22. In some aspects, the second polypeptide of such a heterodimeric protein further comprises a second hinge domain comprising the amino acid sequence set forth in SEQ ID NO: 22.

[0161] In some aspects, provided herein are heterodimeric proteins comprising a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide are different, wherein: (1) the first polypeptide comprises (i) a first CH2 domain comprising the amino acid sequence set forth in SEQ ID NO: 25, and (ii) a first CH3 domain comprising the amino acid sequence set forth in SEQ ID NO: 26 with one or more amino acid modifications selected from: (a) N50K or N50R, (b) K52R, (c) D59K or D59R, (d) S60K or S60R, (e) R69K, or (f) any combination of (a)-(e), and (2) the second polypeptide comprises (i) a second CH2 domain comprising the amino acid sequence set forth in SEQ ID NO: 25, and (ii) a second CH3 domain comprising the amino acid sequence set forth in SEQ ID NO: 24 with one or more amino acid modifications selected from: (a) N50D or N50E, (b) D59E, (c) S60D or S60E, (d) F65D or F65E, (e) R69D or R69E, or (f) any combination of (a)-(e), and wherein the one or more amino acid modifications of the first CH3 domain and the one or more amino acid modifications of the second CH3 domain promote interaction of the first CH3 domain and the second CH3 domain. In some aspects, (1) the first polypeptide comprises (i) a first CH2 domain comprising the amino acid sequence set forth in SEQ ID NO: 25, and (ii) a first CH3 domain comprising the amino acid sequence set forth in SEQ ID NO: 26 with the following amino acid modifications: (a) N50K or N50R, (b) K52R, (c) D59K or D59R, (d) S60K or S60R, and (e) R69K, and (2) the second polypeptide comprises (i) a second CH2 domain comprising the amino acid sequence set forth in SEQ ID NO: 25, and (ii) a second CH3 domain comprising the amino acid sequence set forth in SEQ ID NO: 24 with the following amino acid modifications: (a) N50D or N50E, (b) D59E, (c) S60D or S60E, (d) F65D or F65E, and (e) R69D or R69E. In some aspects, the first polypeptide of such a heterodimeric protein further comprises a first hinge domain comprising the amino acid sequence set forth in SEQ ID NO: 22. In some aspects, the second polypeptide of such a heterodimeric protein further comprises a second hinge domain comprising the amino acid sequence set forth in SEQ ID NO: 22.

[0162] In some aspects, the hetero-dimeric protein provided herein comprises a first polypeptide and a second polypeptide, wherein: (a) the first polypeptide comprises (i) a first CH2 domain comprising the amino acid sequence set forth in SEQ ID NO: 25, and (ii) a first CH3 domain comprising the amino acid sequence set forth in SEQ ID NO: 40 having a D59R amino acid modification and one or more of the following amino acid modifications: T10V, L11Y, F65A, Y67V, or a combination thereof; and (b) the second polypeptide comprises (i) a second CH2 domain comprising the amino acid sequence set forth in SEQ ID NO: 25, and (ii) a second CH3 domain comprising the amino acid sequence set forth in SEQ ID NO: 40 having a R69D amino acid modification and one or more of the following amino acid modifications: T10V, T26L, K52L, T54W, or a combination thereof. In some aspects, (a) the first polypeptide comprises (i) a first CH2 domain comprising the amino acid sequence set forth in SEQ ID NO: 25, and (ii) a first CH3 domain comprising the amino acid sequence set forth in SEQ ID NO: 40 having the following amino acid modifications: D59R, T10V, L11Y, F65A, and Y67V; and (b) the second polypeptide comprises (i) a second CH2 domain comprising the amino acid sequence set forth in SEQ ID NO: 25, and (ii) a second CH3 domain comprising the amino acid sequence set forth in SEQ ID NO: 40 having the following amino acid modifications: R69D, T10V, T26L, K52L, and T54W. In some aspects, the first polypeptide of such hetero-dimeric protein further comprises a first hinge domain comprising the amino acid sequence set forth in SEQ ID NO: 22. In some aspects, the second polypeptide of such hetero-dimeric protein further comprises a second hinge domain comprising the amino acid sequence set forth in SEQ ID NO: 22.

[0163] In some aspects, the hetero-dimeric protein provided herein comprises a first polypeptide and a second polypeptide, wherein: (a) the first polypeptide comprises (i) a first CH2 domain comprising the amino acid sequence set forth in SEQ ID NO: 25, and (ii) a first CH3 domain comprising the amino acid sequence set forth in SEQ ID NO: 40 having the D59R amino acid modification and one or more of the following amino acid modifications: T10V, L11Y, F65A, Y67V, or a combination thereof; and (b) the second polypeptide comprises (i) a second CH2 domain comprising the amino acid sequence set forth in SEQ ID NO: 25, and (ii) a second CH3 domain comprising the amino acid sequence set forth in SEQ ID NO: 40 having the R69E amino acid modification and one or more of the following amino acid modifications: T10V, T26L, K52L, T54W, or a combination thereof. In some aspects, (a) the first polypeptide comprises (i) a first CH2 domain comprising the amino acid sequence set forth in SEQ ID NO: 25, and (ii) a first CH3 domain comprising the amino acid sequence set forth in SEQ ID NO: 40 having the following amino acid modifications: D59R, T10V, L11Y, F65A, and Y67V; and (b) the second polypeptide comprises (i) a second CH2 domain comprising the amino acid sequence set forth in SEQ ID NO: 25, and (ii) a second CH3 domain comprising the amino acid sequence set forth in SEQ ID NO: 40 having the following amino acid modifications: R69E, T10V, T26L, K52L, and T54W. In some aspects, the first polypeptide of such hetero-dimeric protein further comprises a first hinge domain comprising the amino acid sequence set forth in SEQ ID NO: 22. In some aspects, the second polypeptide of such hetero-dimeric protein further comprises a second hinge domain comprising the amino acid sequence set forth in SEQ ID NO: 22.

[0164] In some aspects, the heterodimeric protein provided herein comprises a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide are different, wherein the first polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 4 with an amino acid modification at one or more of the following amino acid residues: T148, L149, F203, and Y205, and wherein the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 4 with an amino acid modification at one or more of the following amino acid residues: T148, T164, K190, and T192. In some aspects, the first polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 4 with one or more amino acid modifications selected from the group consisting of: T148V, L149Y, F203A, and Y205V; and the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 4 with one or more amino acid modifications selected from the group consisting of: T148V, T164L, K190L, and T192W. In some aspects, the first polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 4 with the following amino acid modifications: T148V, L149Y, F203A, and Y205V; and the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 4 with the following amino acid modifications: T148V, T164L, K190L, and T192W.

[0165] In some aspects, the heterodimeric protein provided herein comprises a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide are different, wherein: (1) the first polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 4 having one or more amino acid modifications selected from: (a) N188K or N188R, (b) K190R, (c) D197K or D197R, (d) S198K or S198R, (e) R207K, or (f) any combination of (a) to (e); and (2) the second polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 4 having one or more amino acid modifications selected from: (a) N188D or N188E, (b) D197E, (c) S198D or S198E, (d) F203D or F203E, (e) R207D or R207E, or (f) any combination of (a) to (e). In some aspects, (1) the first polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 4 having the following amino acid modifications: (a) N188K or N188R, (b) K190R, (c) D197K or D197R, (d) S198K or S198R, and (e) R207K; and (2) the second polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 4 having the following amino acid modifications: (a) N188D or N188E, (b) D197E, (c) S198D or S198E, (d) F203D or F203E, and (e) R207D or R207E.

[0166] In some aspects, the heterodimeric protein provided herein comprises a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide are different, wherein: (1) the first polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 16 having one or more amino acid modifications selected from: (a) N188K or N188R, (b) K190R, (c) D197K or D197R, (d) S198K or S198R, (e) R207K, or (f) any combination of (a) to (e), and (2) the second polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 20 having one or more amino acid modifications selected from: (a) N188D or N188E, (b) D197E, (c) S198D or S198E, (d) F203D or F203E, (e) R207D or R207E, or (f) any combination of (a) to (e). In some aspects, (1) the first polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 16 having the following amino acid modifications: (a) N188K or N188R, (b) K190R, (c) D197K or D197R, (d) S198K or S198R, and (e) R207K; and (2) the second polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 20 having the following amino acid modifications: (a) N188D or N188E, (b) D197E, (c) S198D or S198E, (d) F203D or F203E, and (e) R207D or R207E.

[0167] In some aspects, the heterodimeric protein provided herein comprises a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide are different, wherein: (1) the first polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 20 with one or more amino acid modifications selected from: (a) N188K or N188R, (b) K190R, (c) D197K or D197R, (d) S198K or S198R, (e) R207K, or (f) any combination of (a) to (e); and (2) the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 16 with one or more amino acid modifications selected from: (a) N188D or N188E, (b) D197E, (c) S198D or S198E, (d) F203D or F203E, (e) R207D or R207E, or (f) any combination of (a) to (e). In some aspects, (1) the first polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 20 with the following amino acid modifications: (a) N188K or N188R, (b) K190R, (c) D197K or D197R, (d) S198K or S198R, and (e) R207K; and (2) the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 16 with the following amino acid modifications: (a) N188D or N188E, (b) D197E, (c) S198D or S198E, (d) F203D or F203E, and (e) R207D or R207E.

[0168] In some aspects, the heterodimeric protein provided herein comprises a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide are different, wherein: (1) the first polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 4 with a D197R amino acid modification and one or more of the following amino acid modifications: T148V, L149Y, F203A, Y205V, or a combination thereof; and (b) the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 4 with a R207D amino acid modification and one or more of the following amino acid modifications: T148V, T164L, K190L, T192W, or a combination thereof. In some aspects, (1) the first polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 4 with the following amino acid modifications: D197R, T148V, L149Y, F203A, and Y205V; and (b) the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 4 with the following amino acid modifications: R207D T148V, T164L, K190L, and T192W.

[0169] In some aspects, the heterodimeric protein provided herein comprises a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide are different, wherein: (1) the first polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 4 with a D197R amino acid modification and one or more of the following amino acid modifications: T148V, L149Y, F203A, Y205V, or a combination thereof; and (b) the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 4 with a R207E amino acid modification and one or more of the following amino acid modifications: T148V, T164L, K190L, T192W, or a combination thereof. In some aspects, (1) the first polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 4 with the following amino acid modifications: D197R, T148V, L149Y, F203A, and Y205V; and (b) the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 4 with the following amino acid modifications: R207E, T148V, T164L, K190L, and T192W.

[0170] In some aspects, the heterodimeric protein provided herein comprises a first polypeptide and a second polypeptide, wherein: (a) the first polypeptide comprises an amino acid sequence with at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 16; and (b) the second polypeptide comprises an amino acid sequence with at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 20. In some aspects, the heterodimeric protein of the present disclosure comprises a first polypeptide and a second polypeptide, wherein: (a) the first polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 16, and (b) the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 20.

[0171] In some aspects, a heterodimeric protein provided herein comprises a first polypeptide and a second polypeptide, wherein (a) the first polypeptide comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 27; and (b) the second polypeptide comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 29. In some aspects, a heterodimeric protein of the present disclosure comprises a first polypeptide and a second polypeptide, wherein: (a) the first polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 27, and (b) the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 29.

[0172] In some aspects, a heterodimeric protein provided herein comprises a first polypeptide and a second polypeptide, wherein (a) the first polypeptide comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 27; and (b) the second polypeptide comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 32. In some aspects, a heterodimeric protein of the present disclosure comprises a first polypeptide and a second polypeptide, wherein: (a) the first polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 27, and (b) the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 32.

[0173] Additional moiety Any of the heterodimeric proteins of the present disclosure (e.g., such as those described above) can be conjugated to one or more additional moieties. As used herein, the term “additional moiety” (or grammatical variants thereof) is not particularly limited, and includes any molecule that can be conjugated to a heterodimeric protein described herein. Non-limiting examples of suitable additional moieties are provided elsewhere in the present disclosure.

[0174] In some aspects, one or more additional moieties can be conjugated directly to the heterodimeric protein (e.g., fused directly to the heterodimeric protein). In some aspects, one or more additional moieties can be conjugated to the heterodimeric protein via a linker (e.g., a synthetic linker described herein). Unless otherwise specified, the expression “conjugated to the heterodimeric protein” (or variations thereof) includes both direct conjugation (e.g., fused directly to the heterodimeric protein) and conjugation via a linker.

[0175] For example, in some aspects, a heterodimeric protein of the present disclosure comprises a first polypeptide, a second polypeptide, and one or more additional moieties, wherein the first polypeptide and the second polypeptide are different, wherein: (a) the first polypeptide comprises a first CH3 domain (e.g., an IgG4 CH3 domain) comprising a heterodimerization modification (e.g., an amino acid modification described herein), and (b) the second polypeptide comprises a second CH3 domain (e.g., an IgG4 CH3 domain) comprising a heterodimerization modification (e.g., an amino acid modification described herein), and wherein the one or more additional moieties are conjugated to: (1) the N-terminus of the first polypeptide, (2) the C-terminus of the first polypeptide, (3) the N-terminus of the second polypeptide, (4) the C-terminus of the second polypeptide, or (5) any combination of (1) to (4). In some aspects, when multiple (e.g., two or more) additional moieties are attached, (a) a first additional moiety is conjugated to: (1) the N-terminus of the first polypeptide, (2) the C-terminus of the first polypeptide, (3) the N-terminus of the second polypeptide, (4) the C-terminus of the second polypeptide, or (5) any combination of (1) to (4); and (b) a second additional moiety is conjugated to the first additional moiety (either directly or via a linker). It will be apparent to those skilled in the art that, in some aspects, the second additional moiety can be conjugated to the heterodimeric protein, and then the first additional moiety can be conjugated to the second additional moiety.

[0176] In some aspects, the heterodimeric protein described herein comprises a first polypeptide, a second polypeptide, and one or more additional moieties, wherein the first polypeptide and the second polypeptide are different, wherein: (a) the first polypeptide comprises (i) a first hinge domain (e.g., selected from an IgGl hinge domain, an IgG2 hinge domain, an IgG3 hinge domain, an IgG4 hinge domain, an IgD hinge domain, a synthetic linker, or a combination thereof), and (ii) a first CH3 domain (e.g., an IgG4 CH3 domain) comprising a heterodimerization modification (e.g., an amino acid modification described herein), and (b) the second polypeptide comprises (i) a second hinge domain (e.g., selected from an IgGl hinge domain, an IgG2 hinge domain, an IgG3 hinge domain, an IgG4 hinge domain, an IgD hinge domain, a synthetic linker, or a combination thereof), and (ii) a second CH3 domain (e.g., an IgG4 CH3 domain) comprising a heterodimerization modification (e.g., an amino acid modification described herein); and wherein the one or more additional moieties are conjugated to: (1) the N-terminus of the first polypeptide, (2) the C-terminus of the first polypeptide, (3) the N-terminus of the second polypeptide, (4) the C-terminus of the second polypeptide, or (5) any combination of (1) to (4). In some aspects, a first additional moiety is conjugated to (1) the N-terminus of the first polypeptide, (2) the C-terminus of the first polypeptide, (3) the N-terminus of the second polypeptide, (4) the C-terminus of the second polypeptide, or (5) any combination of (1) to (4); and (b) a second additional moiety is conjugated to the first additional moiety (directly conjugated or conjugated via a linker).

[0177] In some aspects, the heterodimeric protein described herein comprises a first polypeptide, a second polypeptide, and one or more additional moieties, wherein the first polypeptide and the second polypeptide are different, wherein: (a) the first polypeptide comprises (i) a first CH2 domain (e.g., an IgD / IgG4 CH2 domain), and (ii) a first CH3 domain (e.g., an IgG4 CH3 domain) comprising a heterodimerization modification (e.g., an amino acid modification described herein), and (b) the second polypeptide comprises (i) a second CH2 domain (e.g., an IgD / IgG4 CH2 domain), and (ii) a second CH3 domain (e.g., an IgG4 CH3 domain) comprising a heterodimerization modification (e.g., an amino acid modification described herein); and wherein the one or more additional moieties are conjugated to: (1) the N-terminus of the first polypeptide, (2) the C-terminus of the first polypeptide, (3) the N-terminus of the second polypeptide, (4) the C-terminus of the second polypeptide, or (5) any combination of (1) to (4). In some aspects, a first additional moiety is conjugated to (1) the N-terminus of the first polypeptide, (2) the C-terminus of the first polypeptide, (3) the N-terminus of the second polypeptide, (4) the C-terminus of the second polypeptide, or (5) any combination of (1) to (4); and (b) a second additional moiety is conjugated to the first additional moiety (directly conjugated or conjugated via a linker).

[0178] In some aspects, the hetero-dimeric protein described herein comprises a first polypeptide, a second polypeptide, and one or more additional moieties, wherein the first polypeptide and the second polypeptide are different, wherein: (a) the first polypeptide comprises (i) a first hinge domain (e.g., selected from an IgGl hinge domain, an IgG2 hinge domain, an IgG3 hinge domain, an IgG4 hinge domain, an IgD hinge domain, a synthetic linker, or a combination thereof), (ii) a first CH2 domain (e.g., an IgD / IgG4 CH2 domain), and (iii) a first CH3 domain (e.g., an IgG4 CH3 domain) comprising a hetero-dimerization modification (e.g., an amino acid modification described herein), and (b) the second polypeptide comprises (i) a second hinge domain (e.g., selected from an IgGl hinge domain, an IgG2 hinge domain, an IgG3 hinge domain, an IgG4 hinge domain, an IgD hinge domain, a synthetic linker, or a combination thereof), (ii) a second CH2 domain (e.g., an IgD / IgG4 CH2 domain), and (iii) a second CH3 domain (e.g., an IgG4 CH3 domain) comprising a hetero-dimerization modification (e.g., an amino acid modification described herein); and wherein the one or more additional moieties are conjugated to: (1) the N-terminus of the first polypeptide, (2) the C-terminus of the first polypeptide, (3) the N-terminus of the second polypeptide, (4) the C-terminus of the second polypeptide, or (5) any combination of (1) to (4). In some aspects, a first additional moiety is conjugated to (1) the N-terminus of the first polypeptide, (2) the C-terminus of the first polypeptide, (3) the N-terminus of the second polypeptide, (4) the C-terminus of the second polypeptide, or (5) any combination of (1) to (4); and (b) a second additional moiety is conjugated to the first additional moiety (directly conjugated or conjugated via a linker).

[0179] For any of the above aspects involving multiple (e.g., two or more) additional moieties, in some aspects, each of the additional moieties is the same. In some aspects, one or more of the additional moieties is different. In some aspects, the heterodimeric protein described herein comprises about two additional moieties, about three additional moieties, about four additional moieties, about five additional moieties, about six additional moieties, about seven additional moieties, about eight additional moieties, about nine additional moieties, or about 10 additional moieties. In some aspects, all of the additional moieties are conjugated to the first polypeptide (e.g., at the N-terminus and / or C-terminus). In some aspects, all of the additional moieties are conjugated to the second polypeptide (e.g., at the N-terminus and / or C-terminus). In some aspects, some (e.g., at least one) of the additional moieties are conjugated to the first polypeptide (e.g., at the N-terminus and / or C-terminus), and some (e.g., at least one) of the additional moieties are conjugated to the second polypeptide (e.g., at the N-terminus and / or C-terminus). As described herein, in some aspects, a first additional moiety is conjugated to (1) the N-terminus of the first polypeptide, (2) the C-terminus of the first polypeptide, (3) the N-terminus of the second polypeptide, (4) the C-terminus of the second polypeptide, or (5) any combination of (1) to (4); and (b) a second additional moiety is conjugated to the first additional moiety (either directly conjugated or conjugated via a linker). In some aspects, the heterodimeric protein is conjugated to about eight additional moieties, wherein four of the additional moieties are attached to the first polypeptide (e.g., N-terminus and / or C-terminus), and four of the additional moieties are attached to the second polypeptide (e.g., N-terminus and / or C-terminus).

[0180] In some aspects, the additional moiety that can be conjugated to a heterodimeric protein of the present disclosure comprises a biologically active molecule. As used herein, the term “biologically active molecule” refers to an agent that is active in a biological system (e.g., a cell or a human subject), including but not limited to a protein, polypeptide, or peptide, including but not limited to a structural protein, an enzyme, a cytokine (such as an interferon and / or an interleukin), an antibiotic, a ligand binding protein (e.g., a polyclonal or monoclonal antibody, or an effective portion thereof, such as an Fv fragment, which antibody or portion thereof can be natural, synthetic, or humanized), a peptide hormone, a receptor, a signaling molecule, or other protein; a nucleic acid, including but not limited to an oligonucleotide or modified oligonucleotide, an antisense oligonucleotide or modified antisense oligonucleotide, a cDNA, a genomic DNA, an artificial or natural chromosome (e.g., a yeast artificial chromosome) or portion thereof, an RNA, including an mRNA, tRNA, rRNA, or ribozyme, or a peptide nucleic acid (PNA); a virus or virus-like particle; a nucleotide or ribonucleotide or synthetic analog thereof, which can be modified or unmodified; an amino acid or analog thereof, which can be modified or unmodified; a nonpeptide (e.g., a steroid) hormone; a proteoglycan; a lipid; or a carbohydrate. In some aspects, the biologically active molecule comprises a macromolecule (e.g., a protein, an antibody, an enzyme, a peptide, DNA, RNA, or any combination thereof). In some aspects, the biologically active molecule comprises a small molecule.

[0181] In some aspects, the biologically active molecule is a ligand binding protein. As used herein, “ligand binding protein” refers to any protein that is capable of binding to a molecule. Non-limiting examples of such ligand binding proteins include an antibody (including an antigen binding fragment thereof), a T cell receptor, or both. As further described elsewhere in the present disclosure, in some aspects, the heterodimeric proteins provided herein are conjugated to multiple ligand binding proteins. For example, in some aspects, the heterodimeric protein is conjugated to about two, about three, about four, about five, about six, about seven, about eight, about nine, or about 10 ligand binding proteins. Where the heterodimeric protein is conjugated to multiple ligand binding proteins, the multiple ligand binding proteins can bind to the same molecule (e.g., antigen), which can help to improve binding affinity. In some aspects, one or more of the multiple ligand binding proteins can bind to different molecules (e.g., different antigens), such that the heterodimeric protein exhibits multispecificity.

[0182] In some aspects, the ligand binding proteins useful for the present disclosure are antibodies. Non-limiting examples of such antibodies include: single domain antibodies (sdABs), Ig NARs, Fab fragments, Fab' fragments, F(ab)'2 fragments, F(ab)'3 fragments, Fv, single chain variable fragments (scFv), bis-scFv, (scFv)2, minibodies, diabodies, triabodies, tetrabodies, intrabodies, disulfide stabilized Fv proteins (dsFv), unibodies, nanobodies, aptamers, or combinations thereof. In some aspects, the ligand binding protein is a sdAb. Non-limiting examples of sdAbs useful for the present disclosure include: human heavy chain only antibodies, rabbit single domain antibodies, camelid VHHs, shark heavy chain only antibodies (VNAR), or combinations thereof.

[0183] In some aspects, the ligand binding protein comprises a T cell engager (e.g., a bispecific T cell engager (BiTE) antibody), a dual affinity retargeting molecule (DART), a CrossMAb antibody, a DutaMab™ antibody, a DuoBody antibody, a Triomab, a TandAb, a bispecific nanobody, a tandem scFv, a diabody, a single chain diabody, a HSA antibody, a (scFv)2 HSA antibody, a scFv-IgG antibody, a dock and lock bispecific antibody, a DVD-IgG antibody, a TBTI DVD-IgG, an IgG-fynomer, a tetravalent bispecific tandem IgG antibody, a dual targeting domain antibody, a chemically linked bispecific (Fab')2 molecule, a cross-linked mAb, a dual acting Fab IgG (DAF-IgG), a vicinal Fab-IgG, a bispecific CovX-Body, a bispecific hexavalent trimer, a 2 scFv linked to diphtheria toxin, an ART-Ig, an IgM T cell engager, a Humabody™ human heavy chain only antibody (HCAb), a UniAb™ HCAb, a shark heavy chain only antibody (VNAR), or combinations thereof.

[0184] It will be apparent from the disclosure that ligand binding proteins useful in the present disclosure can bind to a variety of molecules known in the art. In some aspects, the ligand binding protein binds to a tumor antigen. Non-limiting examples of such tumor antigens include guanylate cyclase-C (GC-C), epidermal growth factor receptor (EGFR or erbB-1), human epidermal growth factor receptor 2 (HER2 or erbB2), erbB-3, erbB-4, MUC-1, melanoma-associated chondroitin sulfate proteoglycan (MCSP), mesothelin (MSLN), folate receptor 1 (FOLR1), CD4, CD19, CD20, CD22, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD123, CD138, CD171, CEA, CSPG4, CXCR5, c-Met, HERV- Envelope protein, eriostin, Bigh3, SPARC, BCR, CD79, CD37, EGFRvin, EGP2, EGP40, IGFr, LICAM, AXL, tissue factor (TF), CD74, EpCAM, EphA2, MRP3, Cadherin 19 (CDH19), epidermal growth factor 2 (FIER2), 5T4, 8H9, integrin, BCMA, B7-H3, B7-H6, CAIX, CA9, FAP, FBP, fetal AchR, FRcc, GD2, GD3, Glypican-1 (GPC1), Glypican-2 (GPC2), Glypican-3 (GPC3), HLA-A1 + MAGE 1, HLA-A1 + NY-ESO-1, IL-13Rcc2, Lewis-Y, KDR, MCSP, mesothelin, Mud, Muc16, NCAM, NKG2D ligand, NY-ESO-1, PRAME, PSC1, PSCA, PSMA, ROR1, ROR2, SP17, surviving, TAG72, TEM, carcinoembryonic antigen, HMW-MAA, VEGF, CLDN18.2, Programmed Death Ligand 1 (PDL-1), or combinations thereof.

[0185] In some aspects, the ligand binding protein binds to an antigen expressed on an immune cell. In some aspects, the antigen expressed on an immune cell comprises a checkpoint molecule. In some aspects, the antigen expressed on an immune cell comprises a stimulatory molecule. Non-limiting examples of such antigens include CD3, CD27, B7H3, CD2, CD4, CD5, CD8, CD11b, CD14, CD16, CD19, CD28, CD32, CD45, CD56, CD64, KLRG-1, NKG2D, NKp30, DNAM-1, TIM-3, LAG-3, TIGIT, PD-1, CTLA-4, ILT2, ILT3, ILT4, LAIR1, VISTA, 4-1BB, OX40, CD40L, ICOS, LIGHT, or a combination thereof.

[0186] Accordingly, in some aspects, the heterodimeric protein provided herein comprises a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide are different, wherein: (a) the first polypeptide comprises a first CH3 domain comprising a heterodimerization modification (e.g., an amino acid modification described herein), and is conjugated to a first ligand binding protein at the N-terminus and / or C-terminus of the first polypeptide, and (b) the second polypeptide comprises a second CH3 domain comprising a heterodimerization modification (e.g., an amino acid modification described herein, e.g., a negative charge modification and / or a positive charge modification described herein), and is conjugated to a second ligand binding protein at the N-terminus and / or C-terminus of the second polypeptide; and wherein the first ligand binding protein specifically binds to a tumor antigen, and the second ligand binding protein specifically binds to an antigen expressed on an immune cell. As described further herein, in some aspects, the first ligand binding protein (i.e., that binds to a tumor antigen) can alternatively be conjugated to the second polypeptide, and the second ligand binding protein (i.e., that binds to an antigen on an immune cell) can be conjugated to the first polypeptide.

[0187] III. Bispecific / Multispecific Ligand Binding Proteins As evident at least from the foregoing, some aspects of the disclosure relate to bispecific ligand binding proteins (e.g., bispecific antibodies). In some aspects, the bispecific ligand binding protein comprises: (a) a first antigen binding domain, (b) a second antigen binding domain, and (c) a heterodimeric protein described herein, wherein the first antigen binding domain and the second antigen binding domain target different molecules. In some aspects, the first antigen binding domain is conjugated to the heterodimeric protein (e.g., at the N-terminus and / or C-terminus of the first polypeptide or the second polypeptide). In some aspects, the second antigen binding domain is conjugated to the heterodimeric protein (e.g., at the N-terminus and / or C-terminus of the first polypeptide or the second polypeptide). In some aspects, both the first antigen binding domain and the second antigen binding domain are conjugated to the heterodimeric protein.

[0188] In some aspects, the first and second antigen binding domains target different tumor antigens. In some aspects, the first and second antigen binding domains target different antigens on an immune cell. In some aspects, the first antigen binding domain targets a tumor antigen and the second antigen binding domain targets an antigen on an immune cell. Non-limiting examples of tumor antigens and antigens on immune cells are provided elsewhere in the present disclosure.

[0189] In some aspects, the multispecific ligand binding protein comprises (a) a first antigen binding domain, (b) a second antigen binding domain, (c) a third antigen binding domain, and (d) a heterodimeric protein described herein, wherein the first, second, and third antigen binding domains target different molecules. In some aspects, the first antigen binding domain is conjugated to the heterodimeric protein (e.g., at the N-terminus and / or C-terminus of the first polypeptide or the second polypeptide). In some aspects, the second antigen binding domain is conjugated to the heterodimeric protein (e.g., at the N-terminus and / or C-terminus of the first polypeptide or the second polypeptide). In some aspects, the third antigen binding domain is conjugated to the heterodimeric protein (e.g., at the N-terminus and / or C-terminus of the first polypeptide or the second polypeptide). In some aspects, two of the first, second, and third antigen binding domains are conjugated to the heterodimeric protein (e.g., the first antigen binding domain is conjugated to the N-terminus and / or C-terminus of the first polypeptide and the second antigen binding domain is conjugated to the N-terminus and / or C-terminus of the second polypeptide). In some aspects, each of the first, second, and third antigen binding domains is conjugated to the heterodimeric protein.

[0190] In some aspects, the first, second, and third antigen binding domains target different tumor antigens. In some aspects, the first, second, and third antigen binding domains target different antigens expressed on an immune cell. In some aspects, at least one of the antigen binding domains targets a tumor antigen and at least one of the other antigen binding domains targets an antigen expressed on an immune cell. Non-limiting examples of tumor antigens and antigens on immune cells are provided elsewhere in the present disclosure.

[0191] In some aspects, the multispecific ligand binding protein described above further comprises one or more additional antigen binding domains.

[0192] As further described elsewhere in the disclosure, any of the additional moieties (e.g., antigen binding domains) provided herein can be conjugated directly to the heterodimeric protein. In some aspects, the additional moieties (e.g., antigen binding domains) are conjugated to the heterodimeric protein using a linker. Non-limiting examples of useful linkers are provided elsewhere in the disclosure.

[0193] Accordingly, in some aspects, the multispecific ligand binding proteins provided herein comprise (a) a first antigen binding domain, (b) a second antigen binding domain, (c) a third antigen binding domain, and (d) a heterodimeric protein described herein (i.e., comprising a first polypeptide and a second polypeptide), wherein the first antigen binding domain is conjugated to the N-terminus of the first polypeptide via a first linker, the second antigen binding domain is conjugated to the N-terminus of the second polypeptide via a second linker, and the third antigen binding domain is conjugated to the N-terminus of the second polypeptide via a third linker, wherein the first linker, the second linker, and the third linker are selected from an IgGl hinge, an IgG2 hinge, an IgG3 hinge, an IgG4 hinge, an IgD hinge, or a synthetic linker. In some aspects, the multispecific ligand binding proteins provided herein comprise (a) a first antigen binding domain, (b) a second antigen binding domain, (c) a third antigen binding domain, and (d) a heterodimeric protein described herein (i.e., comprising a first polypeptide and a second polypeptide), wherein the first antigen binding domain is conjugated to the N-terminus of the first polypeptide via a first linker, the second antigen binding domain is conjugated to the C-terminus of the first polypeptide via a second linker, and the third antigen binding domain is conjugated to the C-terminus of the second polypeptide via a third linker, and wherein the first linker, the second linker, and the third linker are selected from an IgGl hinge, an IgG2 hinge, an IgG3 hinge, an IgG4 hinge, an IgD hinge, or a synthetic linker.

[0194] IV. Conjugates Some aspects of the disclosure relate to conjugates comprising any of the heterodimeric proteins, fusion proteins, bispecific ligand binding proteins (e.g., bispecific antibodies), or multispecific ligand binding proteins (e.g., multispecific antibodies) described herein linked to one or more conjugate moieties. Accordingly, in some aspects, provided herein are conjugates comprising the heterodimeric proteins described herein linked to a conjugate moiety. In some aspects, provided herein are conjugates comprising the fusion proteins described herein linked to a conjugate moiety. In some aspects, provided herein are conjugates comprising the bispecific ligand binding proteins described herein linked to a conjugate moiety. In some aspects, provided herein are conjugates comprising the multispecific ligand binding proteins described herein linked to a conjugate moiety.

[0195] Non-limiting examples of such conjugating moieties include: clearance modulators, chemotherapeutic agents, toxins, radioisotopes, lanthanides, luminescent labels, fluorescent labels, enzyme substrate labels, DNA alkylating agents, topoisomerase inhibitors, tubulin binding agents, or anti-cancer drugs.

[0196] Conjugates useful in the present disclosure can be prepared by any suitable method known in the art. In some aspects, the conjugation method results in a bond that is substantially (or nearly) non-immunogenic, e.g., a peptide bond (i.e., an amide bond), a sulfur bond (steric hindrance), a disulfide bond, a hydrazone bond, and an ether bond. These bonds are nearly non-immunogenic and show reasonable stability within the serum (see, e.g., Senter, P. D., Wang, Q. C, and Curr. Opin. Chem. Biol. 13 (2009) 235-244; WO 2009 / 059278; WO 95 / 17886, each of which is incorporated by reference herein in its entirety).

[0197] Depending on the biochemical nature of the molecule to be conjugated, different conjugation strategies can be employed (see, e.g., Hackenberger, C. P. R. and Schwarzer, D., Angew. Chem. Int. Ed. Engl. 47 (2008) 10030-10074). In some aspects, site-specific reactions and covalent coupling are based on the conversion of natural amino acids into amino acids with a reactivity that is orthogonal to the reactivity of the other functional groups present. For example, a specific cysteine within a rare sequence stretch can be enzymatically converted into an aldehyde (see Frese, M. A. and Dierks, T., ChemBioChem. 10 (2009) 425-427). It is also possible to obtain the desired amino acid modification by exploiting the specific enzymatic reactivity of certain enzymes with natural amino acids in a given sequence context (see, e.g., Taki, M. et al., Prot. Eng. Des. Sel. 17 (2004) 119-126; Gautier, A. et al., Chem. Biol. 15 (2008) 128-136; and Protease-catalyzed formation of C— N bonds is used by Bordusa, F., Highlights in Bioorganic Chemistry (2004) 389-403).

[0198] Site-specific reactions and covalent coupling can also be achieved by selective reactions of terminal amino acids with suitable modification reagents. The reactivity of N-terminal cysteines with benzonitriles (see Ren, H. et al., Angew. Chem. Int. Ed. Engl.48 (2009) 9658-9662) can be used to achieve site-specific covalent coupling. Natural chemical linkages can also depend on C-terminal cysteine ​​residues (Taylor, E. Vogel; Imperiali, B, Nucleic Acids and Molecular Biology (2009), 22 (Protein Engineering), 65-96).

[0199] The conjugated moiety can also be a synthetic peptide or a peptide mimic. In this case, the polypeptide can be chemically synthesized, during which an amino acid with orthogonal chemical reactivity can be incorporated (see, for example, de Graaf, AJ et al.). Bioconjug. Chem. 20 (2009) 1281-1295). To obtain monolabeled peptides, conjugates with a 1:1 stoichiometry can be separated from other conjugated byproducts by chromatography. This procedure can be facilitated using dye-labeled binding pair members and charged linkers. Utilizing such labeled and highly negatively charged binding pair members, monolabeled peptides can be readily separated from unlabeled peptides and peptides carrying more than one linker, as differences in charge and molecular weight can be used for separation. Fluorescent dyes can be used to purify complexes from unbound components, such as labeled monovalent binders.

[0200] IV. Pharmaceutical Compositions This document also provides compositions comprising any of the heterodimeric proteins, fusion proteins, bispecific ligand-binding proteins, multispecific ligand-binding proteins, conjugates, nucleic acids, carriers, and / or cells described herein, and having the desired purity in physiologically acceptable carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA). Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the doses and concentrations used and include buffers such as phosphates, citrates, and other organic acids; antioxidants (including ascorbic acid and methionine); preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexamethyl diammonium chloride; benzyl ethoxymmonium chloride; phenol, butanol, or benzyl alcohol; alkyl esters of p-hydroxybenzoate, such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 1... Polypeptides (0 residues); proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants, such as TWEEN. ® PLURONICS ® Or polyethylene glycol (PEG).

[0201] In some aspects, the pharmaceutical compositions of this disclosure may be comprised of any of the heterodimeric proteins, fusion proteins, bispecific ligand-binding proteins, multispecific ligand-binding proteins, conjugates, nucleic acids, carriers, and / or cells described herein, in a pharmaceutically acceptable carrier, and optionally one or more additional preventative or therapeutic agents. In some aspects, the heterodimeric proteins, fusion proteins, bispecific ligand-binding proteins, multispecific ligand-binding proteins, conjugates, nucleic acids, carriers, and / or cells described herein are the sole active ingredient contained in the pharmaceutical composition. As described elsewhere in this disclosure, such pharmaceutical compositions may be used to treat a variety of diseases or conditions in subjects of need.

[0202] Pharmaceutically acceptable carriers for parenteral preparations include aqueous media, non-aqueous media, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifiers, sequestering / chelating agents, and other pharmaceutically acceptable substances. Examples of aqueous media include sodium chloride injection, Ringer's solution injection, isotonic dextran injection, sterile water injection, and glucose and lactated Ringer's solution injection. Non-aqueous parenteral media include non-volatile oils of plant origin, cottonseed oil, corn oil, sesame oil, and peanut oil. Antimicrobial agents at concentrations that inhibit bacteria or fungi can be added to parenteral preparations packaged in multi-dose containers, such as phenol or cresol, mercury, benzyl alcohol, chlorobutanol, methylparaben and propylparaben, thimerosal, benzalkonium chloride, and benzylthonium chloride. Isotonic agents include sodium chloride and dextrose. Buffers include phosphates and citrates. Antioxidants include sodium bisulfate. Local anesthetics include procaine hydrochloride. Suspensions and dispersants include sodium carboxymethyl cellulose, hydroxypropyl methylcellulose, and polyvinylpyrrolidone. Emulsifiers include polysorbate 80 (TWEEN). ® 80). Multivalent chelating agents or chelating agents for metal ions include EDTA. Drug carriers also include ethanol, polyethylene glycol, and propylene glycol for water-miscible media; and sodium hydroxide, hydrochloric acid, citric acid, or lactic acid for pH adjustment.

[0203] Pharmaceutical compositions can be formulated for administration to a subject via any route of administration. Specific examples of routes of administration include intramuscular, parenteral, subcutaneous, ocular, intravenous, intraperitoneal, intradermal, intraorbital, intracerebral, intracranial, intraspinal, intraventricular, intrasheath, intracisary, intracystic, or intratumoral administration. Parenteral administration characterized by subcutaneous, intramuscular, or intravenous injection is also contemplated herein. Injectable formulations can be prepared in conventional forms, as liquid solutions or suspensions, as solid forms suitable for dissolving or suspending in a liquid prior to injection, or as emulsions. Injectables, solutions, and emulsions also contain one or more excipients. Suitable excipients are, for example, water, saline, dextran, glycerol, or ethanol. In addition, if desired, the pharmaceutical composition to be administered may also contain small amounts of non-toxic excipients, such as wetting agents or emulsifiers, pH buffers, stabilizers, solubilizers, and other such agents, such as, for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate, and cyclodextrin.

[0204] Preparations for the parenteral administration of the peptides described herein include sterile solutions for injection, sterile dried soluble products (such as lyophilized powders, including subcutaneous tablets) prepared for mixing with a solvent prior to use, sterile suspensions for injection, sterile dried insoluble products prepared for mixing with a medium prior to use, and sterile emulsions. Solutions may be aqueous or non-aqueous.

[0205] If administered intravenously, suitable carriers include physiological saline or phosphate-buffered saline (PBS) and solutions containing thickeners and solubilizers (such as glucose, polyethylene glycol, and polypropylene glycol, and mixtures thereof).

[0206] As described for both local and systemic application, a topical mixture comprising the peptides described herein may be prepared. The resulting mixture may be a solution, suspension, emulsion, etc., and may be formulated as a cream, gel, ointment, emulsion, solution, elixir, lotion, suspension, tincture, paste, foam, aerosol, rinse, spray, suppository, bandage, skin patch, or any other formulation suitable for topical application.

[0207] Pharmaceutical compositions can be formulated as aerosols for external application, such as by inhalation (see, for example, U.S. Patents 4,044,126, 4,414,209, and 4,364,923). These formulations for administration to the respiratory tract can be in the form of aerosols or solutions for nebulizers, or as fine powders for inhalation, alone or in combination with an inert carrier (such as lactose). In this case, the particles of the formulation may have a diameter of less than about 50 micrometers, for example, less than about 10 micrometers.

[0208] Pharmaceutical compositions can be formulated as gels, creams, and lotions for topical or external application, such as for application to the skin and mucous membranes, such as in the eyes, and for application to the eyes or for intracranial or intraspinal applications. Topical application is intended for transdermal delivery and is also intended for application to the eyes or mucous membranes, or for inhalation therapy. Nasal solutions of antibodies can also be administered alone or in combination with other pharmaceutically acceptable excipients.

[0209] Transdermal patches, including iontophoresis and electrophoresis devices, are well known to those skilled in the art and can be used to administer any of the peptides, molecules, nucleic acids, carriers, cells, or protein conjugates described herein. Such patches are disclosed, for example, in U.S. Patent Nos. 6,267,983, 6,261,595, 6,256,533, 6,167,301, 6,024,975, 6,010,715, 5,985,317, 5,983,134, 5,948,433, and 5,860,957.

[0210] In some aspects, the pharmaceutical compositions described herein are lyophilized powders that can be reconstituted for application as solutions, emulsions, and other mixtures. They can also be reconstituted and formulated into solids or gels. Lyophilized powders are prepared by dissolving any of the polypeptides, molecules, nucleic acids, carriers, cell or protein conjugates described herein, or pharmaceutically acceptable derivatives thereof, in a suitable solvent. In some aspects, the lyophilized powder is sterile. The solvent may contain excipients that improve the stability of the powder or a reconstituted solution prepared from the powder, or other pharmacological components. Excipients that can be used include, but are not limited to, dextran, sorbitol, fructose, corn syrup, xylitol, glycerol, glucose, sucrose, or other suitable agents. The solvent may also contain buffers, such as citrate, sodium phosphate, or potassium phosphate, or other such buffers known to those skilled in the art. In some aspects, the buffer is at approximately neutral pH. The solution is then sterilely filtered and then lyophilized under standard conditions known to those skilled in the art to provide the desired formulation. In some aspects, the resulting solution can be dispensed into vials for lyophilization. Each vial may contain a single or multiple doses of a compound (e.g., any of a peptide, molecule, nucleic acid, carrier, cell, or protein conjugate). The lyophilized powder may be stored under suitable conditions, such as from about 4°C to room temperature.

[0211] The lyophilized powder is reconstituted with water for injection to obtain a formulation for parenteral administration. For reconstitution, the lyophilized powder is added to sterile water or another suitable carrier. The precise amount depends on the compound chosen. Such amounts can be determined empirically.

[0212] In some respects, pharmaceutical compositions comprising any of the heterodimeric proteins, fusion proteins, bispecific ligand-binding proteins, multispecific ligand-binding proteins, conjugates, nucleic acids, carriers, and / or cells described herein may also be formulated to target specific tissues, receptors, or other regions of the body of a subject to be treated. For non-limiting examples of targeting methods, see, for example, U.S. Patent Nos. 6,316,652, 6,274,552, 6,271,359, 6,253,872, 6,139,865, 6,131,570, 6,120,751, 6,071,495, 6,060,082, 6,048,736, 6,039,975, 6,004,534, 5,985,307, 5,972,366, 5,900,252, 5,840,674, 5,759,542, and 5,709,874.

[0213] The composition intended for in vivo administration can be sterile. In some respects, this can be achieved through filtration, for example, using a sterile filter membrane.

[0214] V. Nucleic Acids, Vectors, and Host Cells Other aspects described herein relate to one or more nucleic acid molecules (also referred to herein as “nucleic acids” or derivatives thereof) that encode polypeptides or molecules (e.g., heterodimeric proteins, fusion proteins, bispecific ligand-binding proteins, multispecific ligand-binding proteins, conjugates) described herein. Nucleic acids may be present in intact cells, in cell lysates, or in partially purified or substantially pure forms. In some aspects, nucleic acids are DNA sequences and / or RNA sequences (e.g., mRNA). In some aspects, nucleic acids contain modified nucleotide analogs. When purified by standard techniques, including alkaline / SDS treatment, CsCl banding, column chromatography, restriction endonucleases, agarose gel electrophoresis, and other methods well known in the art, from other cellular components or other contaminants such as other cellular nucleic acids (e.g., other chromosomal DNA, e.g., chromosomal DNA linked to DNA isolated in nature) or proteins, nucleic acids are “isolated” or “present substantially pure.” See, F. Ausubel et al., eds. (1987) Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York. In some respects, nucleic acid molecules may or may not contain intron sequences. In some respects, nucleic acids are cDNA molecules. The nucleic acids described herein can be obtained using standard molecular biology techniques known in the art.

[0215] In some aspects, this disclosure provides a vector comprising an isolated nucleic acid molecule encoding a polypeptide or molecule disclosed herein (e.g., a heterodimeric protein, a fusion protein, a bispecific ligand-binding protein, a multispecific ligand-binding protein, or a conjugate). Suitable vectors for use in this disclosure include, but are not limited to, expression vectors, viral vectors, and plasmid vectors. In some aspects, the vector is a viral vector.

[0216] As used herein, “expression vector” refers to any nucleic acid construct containing elements necessary for transcription and translation of the inserted coding sequence, or, in the case of RNA viral vectors, elements necessary for replication and translation when introduced into a suitable host cell. Expression vectors can include plasmids, phage particles, viruses, and their derivatives.

[0217] As used herein, “viral vector” includes, but is not limited to, nucleic acid sequences from the following viruses: retroviruses, such as Moloney murine leukemia virus, Harvey murine sarcoma virus, murine mammary tumor virus, and Rous sarcoma virus; lentiviruses; adenoviruses; adeno-associated viruses; SV40 viruses; polyomaviruses; Epstein-Barr viruses; papillomaviruses; herpesviruses; vaccinia viruses; polioviruses; and RNA viruses, such as retroviruses. Some viral vectors are based on non-cytopathic eukaryotic viruses in which non-essential genes have been replaced by genes of interest. Non-cytopathic viruses include retroviruses, whose life cycle involves reverse transcription of the viral genome RNA into DNA, followed by integration of the provirus into the host cell DNA.

[0218] In some respects, the vector is derived from adeno-associated virus. In other respects, the vector is derived from lentivirus. Examples of lentiviral vectors are disclosed in WO9931251, W09712622, W09817815, W09817816 and WO9818934, each of which is incorporated herein by reference in its entirety.

[0219] Other vectors include plasmid vectors. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, 1989. In recent years, plasmid vectors have been found to be particularly advantageous for in vivo gene delivery to cells because they cannot replicate within and integrate into the host genome. However, these plasmids, with promoters compatible with host cells, can express gene expression peptides that are operatively encoded within the plasmid. Some commonly used plasmids available from commercial vendors include pBR322, pUC18, pUC19, various pcDNA plasmids, pRC / CMV, various pCMV plasmids, pSV40, and pBlueScript. Other examples of specific plasmids include: pcDNA3.1, catalog number V79020; pcDNA3.1 / hygro, catalog number V87020; pcDNA4 / myc-His, catalog number V86320; and pBudCE4.1, catalog number V53220, all of which are from Invitrogen (Carlsbad, CA.). Furthermore, plasmids can be custom-designed using standard molecular biology techniques to remove and / or add specific DNA fragments.

[0220] VI. Kits This document also provides kits containing heterodimeric proteins, fusion proteins, bispecific ligand-binding proteins, multispecific ligand-binding proteins, conjugates, nucleic acids, vectors, any of the cellular and / or pharmaceutical compositions described herein. In some aspects, this document provides pharmaceutical packages or kits comprising one or more containers filled with one or more components of the pharmaceutical compositions described herein (such as one or more peptides provided herein), optionally with instructions for use. In some aspects, the kits contain the pharmaceutical compositions described herein and any preventative or therapeutic agents, such as those described herein.

[0221] VII. Methods of the Disclosure Preparation method This disclosure also includes a method for producing / preparing the heterodimeric protein described herein. In some aspects, this method may include expressing the heterodimeric protein in a cell containing a nucleic acid molecule encoding the protein or a vector containing the nucleic acid molecule. This document covers host cells containing these nucleotide sequences. Non-limiting examples of host cells that may be used include immortalized hybridoma cells, NS / 0 myeloma cells, 293 cells, Chinese hamster ovary (CHO) cells, HeLa cells, human amniotic fluid-derived cells (CapT cells), COS cells, bacterial cells, insect cells, plant cells, yeast cells, or combinations thereof.

[0222] Regarding the methods for producing / preparing heterodimeric proteins described above, some aspects of this disclosure relate to methods for producing fusion proteins. For example, in some aspects, this disclosure provides a method for producing a fusion protein, wherein the method includes conjugating any of the additional portions (e.g., bioactive molecules) described herein to a heterodimeric protein. Any suitable method known in the art can be used to conjugate the additional portion to a heterodimeric protein. In some aspects, the method of producing a fusion protein includes directly conjugating the additional portion to the heterodimeric protein (e.g., fusing to the N-terminus and / or C-terminus of a first polypeptide or a second polypeptide). In some aspects, the method of producing a fusion protein includes conjugating the additional portion to the heterodimeric protein using a linker (e.g., conjugating to the N-terminus and / or C-terminus of a first polypeptide or a second polypeptide via a linker). Non-limiting examples of linkers that can be used are provided elsewhere in this disclosure (e.g., IgG1 hinges, IgG2 hinges, IgG3 hinges, IgG4 hinges, IgD hinges, or synthetic linkers).

[0223] Therapeutic uses It will be apparent to those skilled in the art that the heterodimeric proteins described herein can be specifically used to treat a variety of diseases or disorders in subjects of need. For example, in some aspects, the heterodimeric proteins described herein can be conjugated (e.g., directly or via a linker) to additional portions described herein (e.g., bioactive molecules, such as ligand-binding proteins) to form any of the following: fusion proteins, bispecific ligand-binding proteins, multispecific ligand-binding proteins, conjugates, polynucleotides encoding such proteins, and / or pharmaceutical compositions comprising such proteins and / or polynucleotides (collectively, “therapeutic compositions” herein), which can be administered to a subject to treat a disease or disorder.

[0224] Therefore, some aspects of this disclosure relate to methods of treating a disease or condition in a subject in need, including administering to the subject any of the therapeutic compositions described herein (e.g., fusion proteins, bispecific ligand-binding proteins, multispecific ligand-binding proteins, polynucleotides encoding such proteins, and / or pharmaceutical compositions comprising such proteins and / or polynucleotides). Any suitable disease or condition can be treated using the therapeutic compositions of this disclosure.

[0225] In some respects, diseases or ailments include cancer. Therefore, some aspects of this disclosure relate to methods of treating a subject with cancer in need, which include administering to the subject the therapeutic compositions described herein (e.g., fusion proteins, bispecific ligand-binding proteins, multispecific ligand-binding proteins, polynucleotides encoding such proteins, and / or pharmaceutical compositions comprising such proteins and / or polynucleotides).

[0226] Non-limiting examples of cancers (or tumors) that can be treated with the disclosures provided herein include squamous cell carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer, squamous non-small cell lung cancer (NSCLC), non-squamous NSCLC, gastrointestinal cancers, kidney cancer (e.g., clear cell carcinoma), ovarian cancer, liver cancer (e.g., hepatocellular carcinoma), colorectal cancer, endometrial cancer, kidney cancer (e.g., renal cell carcinoma (RCC)), prostate cancer (e.g., hormone-refractory prostate adenocarcinoma), thyroid cancer, pancreatic cancer, cervical cancer, stomach cancer, bladder cancer, liver cancer, breast cancer, colon cancer and head and neck cancer (or carcinoma), and gastric cancer. Cancer, germ cell tumors, pediatric sarcomas, sinus natural killer cells, melanomas (e.g., metastatic malignant melanomas, such as cutaneous or ocular malignant melanomas), bone cancer, skin cancer, uterine cancer, anal cancer, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, esophageal cancer (e.g., esophagogastric junction cancer), small bowel cancer, endocrine system cancers, parathyroid cancer, adrenal cancer, soft tissue sarcomas, urethral cancer, penile cancer, pediatric solid tumors, ureteral cancer, renal pelvis cancer, tumor angiogenesis, pituitary adenoma, Kaposi's sarcoma. Sarcoma), epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environment-induced cancers (including those induced by asbestos), virus-associated cancers or cancers of viral origin (e.g., human papillomavirus (HPV-associated or derived tumors)), and blood malignancies derived from two major blood cell lineages (i.e., the bone marrow cell lineage (which produces granulocytes, erythrocytes, platelets, macrophages, and mast cells) or the lymphocyte lineage (which produces B cells, T cells, NK cells, and plasma cells)), such as all types of leukemia, lymphoma, and myeloma, such as acute, chronic, lymphocytic, and / or myeloid leukemia, such as acute leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), and chronic myeloid leukemia (CML), undifferentiated AML. (MO), myeloblastic leukemia (M1), myeloblastic leukemia (M2; with cell maturation), promyelocytic leukemia (M3 or M3 variant [M3V]), myelomonocytic leukemia (M4 or M4 variant with eosinophilia [M4E]), monocytic leukemia (M5), erythroleukemia (M6), megakaryocytic leukemia (M7), dissociated granulocytic sarcoma and chloroma; lymphoma, such as Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), B-cell hematologic malignancies (e.g., B-cell lymphoma), T-cell lymphoma, lymphoplasmacytic lymphoma, monocytic B-cell lymphoma, mucosa-associated lymphoid tissue (MALT) lymphoma, anaplastic (e.g., Ki1) +Large cell lymphoma, adult T-cell lymphoma / leukemia, mantle cell lymphoma, angioimmunoblastic T-cell lymphoma, angiocentric lymphoma, intestinal T-cell lymphoma, primary mediastinal B-cell lymphoma, precursor T-lymphoblastic lymphoma, T-lymphoblastic lymphoma / leukemia (T-Lbly / T-ALL), peripheral T-cell lymphoma, lymphoblastic lymphoma, post-transplant lymphoproliferative disorders, true histiocytic lymphoma, primary exudative lymphoma, B-cell lymphoma, lymphoblastic lymphoma (LBL), hematopoietic neoplasms of the lymphoid spectrum, acute lymphoblastic leukemia, diffuse large B-cell lymphoma, Burkitt's lymphoma. Lymphoma, follicular lymphoma, diffuse histiocytic lymphoma (DHL), immunoblastic large cell lymphoma, precursor B lymphoblastic lymphoma, cutaneous T-cell lymphoma (CTLC) (also known as mycosis fungoides or Sezary syndrome), and lymphoplasmacytic lymphoma (LPL) with Waldenstrom's macroglobulinemia. Macroglobulinemia; myeloma, such as IgG myeloma, light chain myeloma, non-secretory myeloma, smoldering myeloma (also known as indolent myeloma), solitary plasmacytoma and multiple myeloma, chronic lymphocytic leukemia (CLL), pilocellular lymphoma; hematopoietic tumors of the bone marrow lineage, mesenchymal tumors (including fibrosarcoma and rhabdomyoscaroma); seminoma, teratoma, mesenchymal tumors (including fibrosarcoma, rhabdomyoscaroma and osteosarcoma); and other tumors, including melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, follicular thyroid carcinoma and teratoma, hematopoietic tumors of the lymphoid lineage, such as T-cell tumors and B-cell tumors, including but not limited to T-cell disorders such as T-prolymphocytic leukemia (T-PLL), including small cell and brain cell types; large granular lymphocytic leukemia (LGL) of the T-cell type; a / d T-NHL hepatosplenomegaly; peripheral / postthymic T-cell lymphoma (pleomorphic and immunoblastic subtypes); angiocentric (nasal) T-cell lymphoma; head and neck cancer, kidney cancer, rectal cancer, thyroid cancer; acute myeloid lymphoma and any combination thereof.

[0227] In some respects, cancers treatable with this disclosure include breast cancer, head and neck cancer, uterine cancer, brain cancer, skin cancer, kidney cancer, lung cancer, colorectal cancer, prostate cancer, liver cancer, bladder cancer, pancreatic cancer, thyroid cancer, esophageal cancer, eye cancer, stomach cancer / gastric cancer, gastrointestinal cancer, carcinoma, sarcoma, leukemia, lymphoma, myeloma, or combinations thereof. In some respects, cancers (tumors) treatable with this disclosure include pancreatic cancer. In some respects, treatable cancers (tumors) include brain cancer. In some respects, brain cancer includes glioma. In some respects, brain cancer includes glioblastoma. In some respects, glioblastoma includes primary glioblastoma. In some respects, glioblastoma includes secondary glioblastoma. In some respects, glioblastomas treatable with this disclosure are refractory (e.g., to standard care including prior cancer therapies such as radiation therapy and / or chemotherapy, such as temozolomide). In some respects, treatable cancers (tumors) include skin cancer. Non-limiting examples of skin cancer include: Merkel cell carcinoma (MCC), basal cell carcinoma (BCC), squamous cell carcinoma of the skin (cSCC), melanoma, or combinations thereof.

[0228] In some respects, a disease or ailment includes an infectious disease. Therefore, in some respects, this disclosure provides a method of treating an infectious disease in a subject in need, comprising administering to the subject a therapeutic composition described herein (e.g., a fusion protein, a bispecific ligand-binding protein, a multispecific ligand-binding protein, a polynucleotide encoding such a protein, and / or a pharmaceutical composition comprising such a protein and / or polynucleotide).

[0229] Non-limiting examples of infectious diseases that can be treated with this disclosure include: acute flaccid myelitis (AFM), chebula disease, anthrax, babesiosis, botulism, brucellosis, campylobacteriosis, carbapenem-resistant infection, chancroid, chikungunya virus infection, chlamydia, fish poisoning, and Clostridium difficile infection. Clostridium difficileClostridium perfringens infection, coccidioidomycosis fungal infection, coronavirus infection, Covid-19 (SARS-CoV-2), Creutzfeldt-Jacob Disease / transmissible spongiform encephalopathy, cryptosporidiosis, coccidioidomycosis, dengue fever 1, 2, 3 or 4, diphtheria, Escherichia coli infection (… E. coliInfections (including Shigella toxin-producing diseases such as STEC, Eastern Equine Encephalitis, Ebola, Ehrlichiosis, Encephalitis, Arbovirus or similar infections, Enterovirus A (non-poliovirus), Enterovirus D68 (EV-D68), Giardiasis, Glandular disease, Gonococcal infection, Granuloma inguinale, Haemophilus influenzae type b (Hib or H-flu), Hantavirus Pulmonary Syndrome (HPS), Hemolytic Uremic Syndrome (HUS), Hepatitis A (Hep A), Hepatitis B (Hep B), Hepatitis C (Hep C), Hepatitis D (Hep D), Hepatitis E (Hep E), Herpes zoster, Histoplasmosis, Human Immunodeficiency Virus (HIV / AIDS). (HIV / AIDS), Human papillomavirus (HPV), Flu, Legionnaires' disease, Leprosy (Hansens disease), Leptospirosis, Listeriosis (Listeria), Lyme disease, Lymphogranuloma venereum infection.LGV, malaria, measles, melioidosis, meningitis (viral), meningococcal disease (meningitis (bacterial)), Middle East Respiratory Syndrome Coronavirus (MERS-CoV), mumps, norovirus, lice, pelvic inflammatory disease (PID), pertussis (whooping cough), plague (lymphoid, septicemic, pneumonia), pneumococcal disease (pneumonia), poliomyelitis (polio), powassan fever, psittacosis, pubic lice, pustular skin diseases (smallpox, monkeypox, cowpox), Q fever (q-fever), rabies, rickettsial disease (rocky mountain spotted fever), rubella (german measles), salmonellosis gastroenteritis. Salmonella, scabies, scombroid, sepsis, severe acute respiratory syndrome (SARS), Shigella gastroenteritis, smallpox, methicillin-resistant Staphylococcus aureus (MRSA), staphylococcal food poisoning, enterotoxin B poisoning, vancomycin intermediate for staphylococcal infection (VISA), vancomycin-resistant Staphylococcus aureus (VRSA), group A streptococcal infection (invasive), group B streptococcal infection (Strep-B), streptococcal toxic shock syndrome, syphilis (primary, secondary, early latent, late latent, congenital), tetanus infection, trichomoniasis, hair follicle infection, tuberculosis (TB), latent tuberculosis (LTBI), tularemia, group D typhoid fever, vaginitis, varicella (chickenpox), Vibrio cholerae. Cholera, vibriosis, Ebola virus hemorrhagic fever, Lhasa virus hemorrhagic fever, Marburg virus hemorrhagic fever, West Nile virus, yellow fever, Yersinia, Zika virus infection, or a combination thereof.

[0230] In some respects, infectious diseases treatable with this disclosure include progressive multifocal leukoencephalopathy (PML; caused by polyomavirus JC). In some respects, infectious diseases treatable with this disclosure include sepsis. In some respects, infectious diseases treatable with this disclosure include HIV. In some respects, infectious diseases treatable with this disclosure include cytomegalovirus (CMV) infection. In some respects, infectious diseases treatable with this disclosure include Epstein-Barr virus (EBV) infection. In some respects, infectious diseases treatable with this disclosure include respiratory infections.

[0231] Any of the treatment methods described herein (e.g., methods for treating cancer or infectious diseases) may also include administration of additional therapeutic agents to the subject. Additional therapeutic agents may include any known medications for treating and / or alleviating one or more symptoms associated with any of the aforementioned diseases or conditions.

[0232] Therefore, in some aspects, this document provides a method for treating cancer in a subject in need, comprising administering to the subject a therapeutic composition described herein (e.g., a fusion protein, a bispecific ligand-binding protein, a multispecific ligand-binding protein, a polynucleotide encoding such a protein, and / or a pharmaceutical composition comprising such a protein and / or polynucleotide) and an additional therapeutic agent. In some aspects, this document provides a method for treating an infectious disease in a subject in need, comprising administering to the subject a therapeutic composition described herein (e.g., a fusion protein, a bispecific ligand-binding protein, a multispecific ligand-binding protein, a polynucleotide encoding such a protein, and / or a pharmaceutical composition comprising such a protein and / or polynucleotide) and an additional therapeutic agent. In some aspects, the therapeutic composition and the additional therapeutic agent of this disclosure are administered to the subject sequentially. For example, in some aspects, the additional therapeutic agent is administered to the subject before the therapeutic composition described herein. In some aspects, the additional therapeutic agent is administered to the subject after the therapeutic composition described herein. In some aspects, the therapeutic composition and the additional therapeutic agent of this disclosure are administered to the subject simultaneously.

[0233] Non-limiting examples of other therapeutic agents that may be used with the therapeutic compositions described herein (e.g., fusion proteins, bispecific ligand-binding proteins, multispecific ligand-binding proteins, polynucleotides encoding such proteins and / or pharmaceutical compositions containing such proteins and / or polynucleotides) include: immune checkpoint inhibitors, immune checkpoint activators, standard care treatments, cytokines, or combinations thereof.

[0234] In some aspects, additional therapeutic agents include immune checkpoint inhibitors. Therefore, some aspects of this disclosure relate to a method of treating a disease or ailment (e.g., cancer and / or infectious disease) in a subject of need, comprising administering to the subject a therapeutic composition provided herein (e.g., a fusion protein, a bispecific ligand-binding protein, a multispecific ligand-binding protein, a polynucleotide encoding such a protein, and / or a pharmaceutical composition comprising such a protein and / or polynucleotide) and an immune checkpoint inhibitor. In some aspects, immune checkpoint inhibitors include CTLA-4 antagonists (e.g., anti-CTLA-4 antibodies), PD-1 antagonists (e.g., anti-PD-1 antibodies, anti-PD-L1 antibodies), TIM3 antagonists (e.g., anti-TIM3 antibodies), or combinations thereof.

[0235] In some aspects, additional therapeutic agents include immune checkpoint activators. Therefore, some aspects of this disclosure relate to a method of treating a disease or ailment (e.g., cancer and / or infectious disease) in a subject of need, comprising administering to the subject a therapeutic composition provided herein (e.g., a fusion protein, a bispecific ligand-binding protein, a multispecific ligand-binding protein, a polynucleotide encoding such a protein, and / or a pharmaceutical composition comprising such a protein and / or polynucleotide) and an immune checkpoint activator. Non-limiting examples of immune checkpoint activators that may be used with this disclosure include: OX40 agonists (e.g., anti-OX40 antibodies), LAG-3 agonists (e.g., anti-LAG3 antibodies), 4-1BB (CD137) (e.g., anti-CD137 antibodies), GITR agonists (e.g., anti-GITR antibodies), or combinations thereof.

[0236] In some respects, additional therapeutic agents include standard of care treatment. In some respects, this article provides a method of treating a disease or ailment (e.g., cancer and / or infectious disease) in a subject in need, comprising administering to the subject a therapeutic composition provided herein (e.g., fusion protein, bispecific ligand-binding protein, multispecific ligand-binding protein, polynucleotide encoding such protein, and / or pharmaceutical composition comprising such protein and / or polynucleotide) and standard of care treatment. In some respects, standard of care treatment includes chemotherapy, radiation therapy, or both.

[0237] In some aspects, additional therapeutic agents include cytokines. Any suitable cytokine known in the art may be used in conjunction with this disclosure. In some aspects, cytokines include interleukin-2 (IL-2), interleukin-7 (IL-7), interleukin-10 (IL-10), interleukin-12 (IL-12), interleukin-15 (IL-15), interleukin-18 (IL-18), interleukin-21 (IL-21), interleukin-23 (IL-23), interferon-α (IFN-α), interferon-β (IFN-β), interferon-γ (IFN-γ), tumor necrosis factor (TNF), or combinations thereof. In some aspects, the cytokines comprise the IL-7 protein. In some aspects, the IL-7 protein is a long-acting IL-7 protein, such as that described in US 2019 / 0106471, which is incorporated herein by reference in its entirety. Therefore, some aspects of this disclosure relate to a method of treating a disease or ailment (e.g., cancer and / or infectious disease) in a subject in need, comprising administering to the subject a therapeutic composition (e.g., fusion protein, bispecific ligand-binding protein, multispecific ligand-binding protein, polynucleotide encoding such protein and / or pharmaceutical composition comprising such protein and / or polynucleotide) and cytokines (e.g., long-acting IL-7 protein).

[0238] In some respects, any of the polypeptides, molecules, nucleic acids, carriers, cells, protein conjugates or compositions described herein may be administered intravenously, orally, parenterally, transmembranely, intrathecally, intraventricularly, intrapulmonaryly, subcutaneously, intradermally, intramuscularly or intraventricularly.

[0239] The following examples are provided by way of illustration rather than limitation.

[0240] Example Example 1: Materials and Methods In order to generate trispecific heterodimer antibodies (such as...) Figure 1 As shown in the diagram, the following methods and materials were used (see also...). Figure 2 ): Plasmid cloning: Antibody chains A and B were transformed into the pEG BacMam vector. As described herein (see, for example, Figure 1Chain A comprises an anti-B7H3 sdAb, an IgD hinge, an IgD / IgG4 CH2 domain, and an IgG4 CH3 domain, containing the amino acid sequence shown in SEQ ID NO: 40 with the following amino acid modifications: T10V, L11Y, F65A, and Y67V (i.e., SEQ ID NO: 24). Chain B comprises an anti-CD3 sdAb, an anti-CD27 scFv, an IgD hinge, an IgD / IgG4 CH2 domain, and an IgG4 CH3 domain, containing the amino acid sequence shown in SEQ ID NO: 40 with the following amino acid modifications: T10V, T26L, K52L, and T54W (i.e., SEQ ID NO: 26). Figure 3 As shown, chain A is encoded as (1) a fusion protein (see construct #1, which also includes a sequence encoding the GFP tag following the CH3 domain with a PreScission cleavage site between the GFP tag and the CH3 domain), or (2) a single polypeptide chain (i.e., without the GFP tag; see construct #4). Chain B is encoded by two different fusion proteins. The vector encoding the first chain B fusion protein also includes a sequence encoding mCherry following the C-terminus of the CH3 domain and a sequence encoding the ALFA tag (LEEELRRRLTE; SEQ ID NO: 14) (see Figure 3 Constructor #2 in the document. As further described herein, the ALFA tag allows for the purification of heterodimers of chain B (encoded by construct #2) and chain A (encoded by construct #1). A PreScission cleavage site is included between the CH3 domain and mCherry, and a thrombin cleavage site is included between mCherry and the ALFA tag. The vector encoding the second-chain B fusion protein also contains a sequence encoding a 6XHis tag (HHHHHH; SEQ ID NO: 17) following the CH3 domain (see [link to SEQ ID NO: 17]). Figure 3 Builder #3 in the middle.

[0241] Generation of recombinant baculovirus The plasmid vector described above was transformed into DH10Bac cells containing 50 μg / ml kanamycin, 7 μg / ml gentamicin, 10 μg / ml tetracycline, 0.17 mM IPTG (isopropyl β-D-1-thiogalactopyranoside), and 20 mg / ml Bluo-gal. The cells were plated on LB (Luria broth) agar plates and incubated at 37°C for 24–48 hours. After white colonies were sorted, colonies were further selected by plate-scraping. From the selected colonies, Taq-PCR was performed to identify colonies that had successfully recombined with the baculovirus genome.

[0242] Selected colonies were inoculated into LB medium containing 50 μg / ml kanamycin, 7 μg / ml gentamicin, and 10 μg / ml tetracycline, and then incubated at 37°C for 16 hours. The recombinant viral genome was purified according to the QIAGEN plasmid purification protocol. The virus was then produced by transfecting the viral genome into SF9 insect cells, and the baculovirus was harvested from the SF9 insect cells.

[0243] Transduction of Expi293F cells Expi293F cells were cultured in 800 ml of Expi293 medium at 37°C, 125 rpm, and 8% CO2. The cell number was increased to 3-5 × 10⁻⁵ cells / year. 6 When the cell count is 1 live cells / mL, passage the cells every 3-4 days.

[0244] For baculovirus infection, at 3×10 6 Cells were prepared at a concentration of 1 live cells / mL and then infected with 80 ml of medium (10% v / v of total medium). The infected cells were then incubated at 37°C with 8% CO2. After 18 hours, enhancers were added to improve transduction, and the cells were further incubated for 6 days to produce the encoded protein.

[0245] Protein purification To purify the resulting protein, cells are first lysed and centrifuged to remove cell debris. The supernatant is then loaded onto an affinity column (anti-His, anti-ALFA, or protein A resin) to capture the labeled antibody protein.

[0246] In summary, an open column for anti-ALFA nanobody was prepared after resin filling but before protein loading by washing the column with 20 mM Tris-HCl, 200 mM NaCl buffer (pH 8.0). The resin passing through the supernatant was washed with 10 column volumes (CV) of 20 mM Tris-HCl, 200 mM NaCl buffer (pH 8.0). Thrombin, corresponding to 1% CV and 1 CV of 20 mM Tris, 200 mM NaCl pH 8.0 solution, was reacted together at 4°C for 16 h to achieve ALFA tag cleavage. The tag and antibody protein were then eluted from the column. The eluted protein was concentrated using an Amicon Ultra-15 centrifuge filter at 100 K (Millipore) and then reacted at 4°C with PreScission protease (2% of protein mass) for 16 h to cleave mCherry and GFP fluorescent protein, yielding purified protein.

[0247] To purify using an anti-His column, the supernatant was loaded onto the column. The resin passing through the supernatant was washed with a solution of 20 mM Tris-HCl, 200 mM NaCl (pH 8.0), and 10 mM imidazole at 10 CV. The concentration of imidazole was gradually increased by 100 mM, 300 mM, and 500 mM using 20 mM Tris-HCl and 200 mM NaCl (pH 8.0), and the protein was eluted by passing twice the volume of solution through the column. The eluted protein was then concentrated using an Amicon Ultra-15 centrifuge filter at 100K (Millipore).

[0248] For purification using a protein A column, the resin through which the supernatant was passed was washed with 10 CV of 20 mM Tris-HCl and 200 mM NaCl (pH 8.0) solution. The antibody proteins attached to the resin were eluted to 100 mM glycine (pH 2.5) solution and collected by neutralizing them in test tubes containing 2 M Tris-HCl (pH 8.0) buffer.

[0249] Example 2: Confirmation of the formation of trispecific heterodimer antibodies As described in Example 1 above, a trispecific heterodimer antibody fused to a fluorescent protein (GFP or mCherry) was expressed in Expi293 cells. The heterodimer antibody bound to the ALFA nanobody, thereby allowing only the trispecific heterodimer to be purified. Because chain A and chain B are fused to different fluorescent proteins (GFP and mCherry, respectively), two distinct bands were observed in an SDS-PAGE gel under reducing conditions (see Example 1).Figure 4A This confirmed the presence of the purified protein as a heterodimer. Next, the purified protein was concentrated and then cleaved using the PreScission protease. Successful cleavage of the fluorescent protein was confirmed by SDS-PAGE analysis (see [link to SDS-PAGE analysis]). Figure 4B ).

[0250] To further confirm the heterodimeric nature of the generated trispecific antibodies, fluorescent proteins (GFP and mCherry) were identified in SDS-PAGE gels under both non-reducing and reducing conditions (see [link to SDS-PAGE gel analysis]). Figure 5 Chain A (B7H3-CH) A ) and chain B (CD3a-CD27-CH B Under reducing conditions, it appears as two distinct bands (see the bands at 44.6 kDa and 72.1 kDa, respectively), but under non-reducing conditions, only a single band is observed.

[0251] To assess the purity of the trispecific heterodimer antibody produced and described above, Chemi-doc analysis was performed using a single band of heterodimer observed in SDS-PAGE under non-reducing conditions. The purity of the trispecific heterodimer antibody was approximately 75% (see [link to Chemi-doc analysis]). Figure 6A and Figure 6B ).

[0252] Example 3: Analysis of the effect of baculovirus infection rate on the formation of trispecific heterodimer antibodies The virus will be used in a 1:1 virus:human cell ratio with chain A (B7H3-CH) A ) and chain B (CD3a-CD27-CH B (As described in Example 1) The culture medium used for baculovirus infection of Expi293 human cells was loaded onto anti-ALFA and anti-GFP nanobody resins, respectively, to measure the yield of heterodimers. Elution columns were used as described in Example 1, and the eluent was analyzed by SDS-PAGE. Figure 7 As shown, an excess of chain A was detected in the eluent of the anti-ALFA nanobody resin, while only a small amount of protein was observed in the anti-GFP nanobody resin.

[0253] Therefore, to increase the production rate of heterodimers, several baculovirus:human cell infection ratios (i.e., 4:1, 8:1, 10:1) were tested and analyzed by SDS-PAGE under non-reducing conditions. The expressed protein was purified using ALFA nanobody resin as previously described (see [link to documentation]). Figure 8 The fluorescent protein was cleaved using PreScission protease. Protein productivity was assessed by SDS-PAGE analysis.

[0254] As shown in Figure 9, more heterodimers formed at infection ratios of 4:1 and 8:1, with the largest formation observed when cells were infected at an 8:1 ratio. Mass spectrometry analysis of the heterodimer antibodies produced in these experiments revealed peak molecular weights similar to the expected molecular weight (115.5 kDa) of the fluorescently inactivated heterodimer (see Figure 9). Figure 10 ).

[0255] The above results further confirm that polypeptide chains A and B described in this paper can be used to produce heterodimeric proteins, such as trispecific heterodimeric antibodies.

[0256] Example 4: Analysis of the effect of baculovirus infection rate on the formation of trispecific heterodimer antibodies To further confirm the results described in the above examples, constructs #3 (encoding strand B, non-fluorescent protein) and #4 (encoding strand A, non-fluorescent protein) were used to produce trispecific heterodimeric antibodies. Briefly, as described in Example 1, cells were infected with recombinant baculovirus particles containing constructs #3 and #4 in a 1:1 ratio, and then the encoded protein was expressed. To purify the resulting protein, culture medium from infected cells was loaded onto an anti-His resin column, and the captured protein was eluted using successive imidazole treatments (concentration increases – i.e., 100 mM, 300 mM, and 500 mM). The eluted protein was analyzed by SDS-PAGE (see [link to example]). Figure 11 ).

[0257] The eluted proteins were further purified using a Protein A column, which captures only antibody proteins. The captured proteins were eluted to 100 mM glycine solution (pH 2.5) and collected by neutralization in tubes containing 2 M Tris-HCl buffer (pH 8.0). Analysis of the eluted proteins by SDS-PAGE showed a total yield of 8.8 mg (0.018 g / L) of protein from 500 mL of the preparation from infected cells, with a purity of approximately 85% (see [link to SDS-PAGE]). Figure 12 ).

[0258] The results show that the removal of fluorescent tags (e.g., GFP and mCherry) not only simplifies the purification process but also helps increase the purity of the produced trispecific heterodimer antibodies.

[0259] Example 5: Additional confirmation of the formation of trispecific heterodimer antibodies To confirm that the purified trispecific antibody in Example 4 was indeed a heterodimer, SDS-PAGE analysis was performed under both reducing and non-reducing conditions to evaluate the eluted proteins (from Example 4).Figure 13 As shown, under reducing conditions, the reaction corresponding to chain A (B7H3-CH) was observed. A ; 43.6 kDa) and chain B (CD3a-CD27-CH B Two distinct bands were observed at approximately 115.5 kDa (72 kDa). However, under non-reducing conditions, only a single band was observed at approximately 115.5 kDa. ChemiDoc analysis confirmed this. Figure 13 The results shown further demonstrate that the purified trispecific heterodimer antibody has a purity of approximately 83% (see [link to original document]). Figure 14 ).

[0260] Next, capillary electrophoresis (CE-SDS) analysis was performed on sodium dodecyl sulfate to confirm the accuracy of the SDS-PAGE results. CE-SDS analysis is an automated assay for quantifying and purifying heterodimeric triple antibodies and reducing monoclonal antibodies, making it an ideal analytical method for analyzing heterodimer formation in samples. Figure 15 As shown, under reducing conditions, the reaction corresponding to chain A (B7H3-CH) was observed. A ) and chain B (CD3a-CD27-CH B The two peaks of the subunit were observed. However, under non-reducing conditions, a single, stronger peak corresponding to the dimer was observed, with a purity of approximately 90%. Figure 16 As shown, the formation of the trispecific heterodimer was also confirmed by Western blot analysis.

[0261] In summary, the above results confirm the heterodimeric protein (e.g., containing...) provided in this paper. Figure 3 Chains A and B described in the text can be used to produce multispecific heterodimer antibodies.

[0262] Example 6: Modification to promote heterodimer formation As described in full in this disclosure, the applicant has identified certain amino acid modifications within the CH3 domain of immunoglobulins that can promote the formation of heterodimers. To better understand such amino acid modifications, polypeptide chains A and B described in Example 1 will be modified to include one or more additional heterodimerization modifications (e.g., the negatively charged amino acid modifications and positively charged amino acid modifications described herein) within the CH3 domain. Exemplary polypeptide chain A and / or polypeptide chain B will contain the amino acid sequence shown in SEQ ID NO: 4, SEQ ID NO: 16 or SEQ ID NO: 20 and one or more of the following positively charged modifications: (a) N188K, (b) N188R, (c) K190R, (d) D197K, (e) D197R, (f) S198K, (g) R207K, (h) F203K and (i) F203R; and / or (2) one or more of the following negatively charged modifications: (a) N188D, (b) N188E, (c) D197E, (d) S198D, (e) S198E, (f) R207D, (g) R207E, (h) F203D, (i) F203E, (j) K190D and (k) K190E. Exemplary chain A polypeptide amino acid sequences containing such positive charge modifications include: SEQ ID NO: 27 (i.e., D197R; "peptide chain A #2"). Exemplary chain B polypeptide amino acid sequences containing such negative charge modifications include: SEQ ID NO: 29 (i.e., R207D; "peptide chain B #2") and SEQ ID NO: 32 (i.e., R207E; "peptide chain B #3"). Exemplary combinations of chain A and chain B polypeptides forming heterodimers include: (1) SEQ ID NO: 27 (positive charge) + SEQ ID NO: 29 (negative charge); and (2) SEQ ID NO: 27 (positive charge) + SEQ ID NO: 32 (negative charge). In some aspects, the control sequence will include a mortar-and-mortar (KiH) modification of the CH3 domain (e.g., chain A contains the amino acid sequence shown in SEQ ID NO: 34, and chain B contains the amino acid sequence shown in SEQ ID NO: 38).

[0263] To produce the heterodimers described above, materials and methods (such as those described in Example 1) will be used. Properties such as heterodimer formation, purity, and stability will then be evaluated.

[0264] Table 3. Exemplary Sequences Example 7: Nano Differential Scanning Fluorescence Assay (Nano DSF) In addition to Example 6 provided above, the following heterodimers were constructed: (1) NIT-Fc-01 (i.e., polypeptide chain A #1 + polypeptide chain B #1), (2) NIT-Fc-02 (i.e., polypeptide chain A #2 + polypeptide chain B #2), (3) NIT-Fc-03 (i.e., polypeptide chain A #2 + polypeptide chain B #3), (4) NIT-Fc-04 (i.e., control polypeptide chain A + control polypeptide chain B), and (5) NIT-Fc-05 (i.e., control polypeptide chain C + control polypeptide chain D). NIT-Fc-04 and NIT-Fc-05 did not contain the specific CH3 domain modifications shown in Table 4 and were used as controls. To further characterize these heterodimers, nano differential scanning fluorometry (nano DSF) measurements were performed using a Prometheus NT.48 instrument (NanoTemper Technologies GmbH, München, Germany) to evaluate the thermal stability of the different heterodimers. In summary, the heterodimer was prepared in 1xPBS (pH 7.4) at a concentration of 2.0 mg / ml. Each experimental condition was set and measured in a standard capillary tube (NanoTemperTechnologies GmbH). Experiments were conducted at 40% sensitivity, with temperatures ranging from 20°C to 90°C. Figure 21 The heating rate was 1 °C / min. The antibody melting temperature (Tm) was determined by monitoring the temperature-dependent changes in tryptophan and tyrosine fluorescence at emission wavelengths of 350 nm and 330 nm, respectively. The Tm value was determined by identifying the peak of the first derivative of the fluorescence ratio (F350 / 330). Figure 22 As summarized in Table 4, each heterodimer containing the CH3 domain modification described herein (i.e., NIT-Fc-01, NIT-Fc-02, and NIT-Fc-03) exhibits comparable thermal stability compared to the control heterodimers (i.e., NIT-Fc-04 and NIT-Fc-05).

[0265] Table 4. Comparison of temperatures measured by Nano differential scanning fluorometry for heterodimers NIT-Fc-01 and NIT-Fc-05. Example 8: Dynamic Light Scattering (DLS) To assess the tendency of the heterodimers to form aggregates as described in Example 7, DLS measurements were performed using a WYATT-493-WPR3 instrument with the detector at a 150° angle relative to the incident light. The heterodimer samples contained in 96-well plates were irradiated using a monochromatic laser (auto-attenuation activated) with a wavelength set to 825.1 nm. The experimental procedure involved increasing the temperature from 25°C to 85°C to observe the temperature-dependent changes in antibody sample size. Figure 23 The data show that the heterodimers of NIT-Fc-01, NIT-Fc-02, and NIT-Fc-03 have thermal stability comparable to the control heterodimer.

[0266] Example 9: Reheterodimerization As described at least in Example 6, the specific CH3 modification described herein was designed to promote heterodimerization. Therefore, the ability of the heterodimer described in Example 7 to re-heterodimerize upon denaturation was evaluated. Briefly, the initially prepared heterodimer at a concentration of 2.0 mg / ml was reduced by adding 10 mM β-mercaptoethanol (BME) at room temperature for a duration of 1 hour. This reduction step was designed to disrupt any disulfide bonds present in the antibody molecule. Following reduction, the denatured polypeptide chain underwent heterodimerization by dialyzing in PBS buffer for 18 hours. This extended dialyzing duration ensured complete equilibration of the reduced antibody molecules, thereby promoting heterodimer formation. Finally, re-heterodimerization was confirmed by analyzing the samples using SDS-PAGE gel chromatography.

[0267] As shown in Table 5, the heterodimers containing the CH3 modification described herein (i.e., NIT-Fc-01, NIT-Fc-02, and NIT-Fc-03) are much better at reheterodimerization than the controls (i.e., NIT-Fc-04 and NIT-Fc-05).

[0268] Table 5. Comparison of reheterodimation on heterodimers Table 6. Information on expression yield and purity of heterodimers Example 10: Thermal stability assessment and further characterization To further characterize the heterodimer described in Example 6, its stability at 37°C was evaluated. Briefly, the heterodimer sample was prepared in phosphate-buffered saline (PBS) at a concentration of 2.0 mg / ml. The prepared heterodimer sample was incubated at 37°C for 7 days. Changes in concentration were monitored by measuring the absorbance at 280 nm (A280).Figure 24 As shown, the heterodimers all appear to possess similar stability. Table 6 provides data showing the expression yield and purity of the heterodimers during in vitro expression. Compared to each control in the control group, the heterodimers containing the CH3-modified material described herein (i.e., NIT-Fc-01, NIT-Fc-02, and NIT-Fc-03) exhibit improved purity and / or improved total yield. Unbound by any single theory, these data highlight the improved preparability of the heterodimers containing the CH3-modified material described herein.

[0269] In summary, the data presented in this paper highlight the usefulness of the CH3 domain modifications described herein for the production of multispecific heterodimeric proteins (e.g., antibodies).

[0270] It should be understood that the claims are intended to be interpreted using the detailed description section rather than the summary and abstract section. The summary and abstract section may illustrate one or more, but not all, exemplary aspects of this disclosure as conceived by the inventors, and therefore is not intended to limit this disclosure and the appended claims in any way.

[0271] This disclosure has been described above using functional structural units that illustrate the implementation of specific functions and their relationships. For ease of description, the boundaries of these functional building blocks have been arbitrarily defined. Alternative boundaries may also be defined, provided that the specified functions and their interrelationships are properly performed.

[0272] The foregoing description of specific aspects will fully reveal the general characteristics of this disclosure, enabling others to readily modify and / or alter such specific aspects for various applications by applying knowledge in the art, without excessive experimentation and without departing from the general conception of this disclosure. Therefore, based on the teachings and guidance presented herein, such changes and modifications are intended within the meaning and scope of equivalents of the disclosed aspects. It should be understood that the wording or terminology herein is for descriptive rather than limiting purposes, and that the terminology or terminology of this specification should be interpreted by a skilled person based on the teachings and guidance.

[0273] The breadth and scope of this disclosure should not be limited by any of the foregoing exemplary aspects, but should be defined solely by the following claims and their equivalents.

[0274] All publications, patents, patent applications, websites, and accession / database sequences (including polynucleotide and polypeptide sequences) cited herein are incorporated herein by reference in their entirety for all purposes, as if each individual publication, patent, patent application, website, or accession / database sequence were specifically and individually indicated to be incorporated by reference.

Claims

1. A heterodimeric protein comprising a first polypeptide and a second polypeptide, Wherein (a) the first polypeptide contains the amino acid sequence shown in SEQ ID NO: 27, and (b) the second polypeptide contains the amino acid sequence shown in SEQ ID NO: 32 or SEQ ID NO:

29.

2. The heterodimeric protein of claim 1, wherein the second polypeptide comprises the amino acid sequence shown in SEQ ID NO:

32.

3. The heterodimeric protein of claim 1, wherein the second polypeptide comprises the amino acid sequence shown in SEQ ID NO:

29.

4. A heterodimeric protein comprising a first polypeptide and a second polypeptide, Wherein (a) the first polypeptide contains the amino acid sequence shown in SEQ ID NO: 16, and (b) the second polypeptide contains the amino acid sequence shown in SEQ ID NO:

20.

5. A heterodimeric protein comprising a first polypeptide and a second polypeptide, The first polypeptide and the second polypeptide are different. The first polypeptide contains a first CH3 domain, and the second polypeptide contains a second CH3 domain. Wherein (a) the first CH3 domain contains the amino acid sequence shown in SEQ ID NO: 24, and (b) the second CH3 domain contains the amino acid sequence shown in SEQ ID NO:

26. in: (1) The amino acid residues N50, S60 or both of the first CH3 domain are not modified or are modified by positive charge, and the amino acid residues N50, S60 or both of the second CH3 domain are not modified or are modified by negative charge, such that the first CH3 domain and the second CH3 domain have opposite charges. (2) The amino acid residues N50, S60 or both of the first CH3 domain are not modified or are modified by negative charge, and the amino acid residues N50, S60 or both of the second CH3 domain are not modified or are modified by positive charge, such that the first CH3 domain and the second CH3 domain have opposite charges. (3) The amino acid residues D59, R69 or both of the first CH3 domain are not modified or are modified by positive charge, and the amino acid residues D59, R69 or both of the second CH3 domain are not modified or are modified by negative charge, such that the first CH3 domain and the second CH3 domain have opposite charges. (4) The amino acid residues D59, R69 or both of the first CH3 domain are not modified or are modified by negative charge, and the amino acid residues D59, R69 or both of the second CH3 domain are not modified or are modified by positive charge, such that the first CH3 domain and the second CH3 domain have opposite charges. (5) The amino acid residue K52 of the first CH3 domain is either unmodified or modified with a positive charge, and the amino acid residue F65 of the second CH3 domain is either unmodified or modified with a negative charge, such that the first CH3 domain and the second CH3 domain have opposite charges; and / or (6) The amino acid residue K52 of the first CH3 domain is not modified or is modified by negative charge, and the amino acid residue F65 of the second CH3 domain is not modified or is modified by positive charge, so that the first CH3 domain and the second CH3 domain have opposite charges.

6. The heterodimeric protein of claim 5, wherein the modification in the first CH3 domain comprises (a) N50K or N50R, (b) K52R, (c) D59K or D59R, (d) S60K or S60R, (e) R69K or (f) any combination thereof; and wherein the modification in the second CH3 domain comprises (a) N50D or N50E, (b) D59E, (c) S60D or S60E, (d) R69D or R69E, (e) F65D or F65E or (f) any combination thereof.

7. The heterodimeric protein of claim 5, wherein the modification in the first CH3 domain comprises (a) N50D or N50E, (b) K52D or K52E, (c) D59E, (d) S60D or S60E, (e) R69D or R69E, or (f) any combination thereof; and wherein the modification in the second CH3 domain comprises (a) N50K or N50R, (b) D59K or D59R, (c) S60K or S60R, (d) R69K, (e) F65K or F65R, or (f) any combination thereof.

8. The heterodimeric protein according to any one of claims 5 to 7, wherein: (a) The amino acid residue N50 in the first CH3 domain is modified to N50K or N50R, and the amino acid residue S60 in the second CH3 domain is modified to S60D or S60E. (b) The amino acid residue S60 in the first CH3 domain is modified to S60D or S60E, and the amino acid residue N50 in the second CH3 domain is modified to N50K or N50R. (c) The amino acid residue K52 in the first CH3 domain is either unmodified or modified to K52R, and the amino acid residue F65 in the second CH3 domain is modified to F65D or F65E. (d) The amino acid residue D59 in the first CH3 domain is modified to D59K or D59R, and the amino acid residue R69 in the second CH3 domain is modified to R69D or R69E. (e) The amino acid residue R69 in the first CH3 domain is modified to R69D or R69E, and the amino acid residue D59 in the second CH3 domain is modified to D59K or D59R, or (f) Any combination of (a) to (e).

9. The heterodimeric protein according to any one of claims 5 to 8, wherein the first CH3 domain or the second CH3 domain comprises the amino acid sequence shown in SEQ ID NO: 28, SEQ ID NO: 31 or SEQ ID NO:

33.

10. The heterodimeric protein according to any one of claims 5 to 8, wherein the first CH3 domain comprises the amino acid sequence shown in SEQ ID NO: 28, and the second CH3 domain comprises the amino acid sequence shown in SEQ ID NO: 31 or SEQ ID NO:

33.

11. The heterodimeric protein according to any one of claims 5 to 8, wherein: (a) The first polypeptide further comprises a first hinge domain, a first CH2 domain, or both; (b) The second polypeptide further comprises a second hinge domain, a second CH2 domain, or both; or (c) both (a) and (b).

12. The heterodimeric protein of claim 11, wherein the first hinge domain and the second hinge domain are each selected from IgG1 hinge, IgG2 hinge, IgG3 hinge, IgG4 hinge, IgD hinge or synthetic linker.

13. The heterodimeric protein of claim 12, wherein the synthetic linker comprises a glycine-serine linker, a glycine-alanine linker, an alanine-serine linker, or a combination thereof.

14. The heterodimeric protein of any one of claims 11 to 13, wherein the first hinge domain and the second hinge domain are the same or different.

15. The heterodimeric protein according to any one of claims 11 to 14, wherein: (a) the first hinge domain contains the amino acid sequence shown in SEQ ID NO: 22, (b) the second hinge domain contains the amino acid sequence shown in SEQ ID NO: 22, or (c) both (a) and (b).

16. The heterodimeric protein of any one of claims 11 to 15, wherein the first CH2 domain and the second CH2 domain are the same or different.

17. The heterodimeric protein of claim 16, wherein: (a) the first CH2 domain contains the amino acid sequence shown in SEQ ID NO: 25, (b) the second CH2 domain contains the amino acid sequence shown in SEQ ID NO: 25, or (c) both (a) and (b).

18. The heterodimeric protein according to any one of claims 1 to 17, further comprising a first bioactive molecule.

19. The heterodimeric protein of any one of claims 18, wherein the first bioactive molecule is attached to the N-terminus of the first polypeptide, the C-terminus of the first polypeptide, the N-terminus of the second polypeptide, the C-terminus of the second polypeptide, or any combination thereof.

20. The heterodimeric protein of any one of claims 19, wherein the first bioactive molecule is directly attached or attached via a linker.

21. The heterodimeric protein of claim 20, wherein the adapter is selected from IgG1 hinges, IgG2 hinges, IgG3 hinges, IgG4 hinges, IgD hinges, or synthetic adapters.

22. The heterodimeric protein according to any one of claims 18 to 21, further comprising a second bioactive molecule.

23. The heterodimeric protein of claim 22, wherein the second bioactive molecule is attached to the N-terminus of the first polypeptide, the C-terminus of the first polypeptide, the N-terminus of the second polypeptide, the C-terminus of the second polypeptide, the first bioactive molecule, or any combination thereof.

24. The heterodimeric protein of any one of claims 23, wherein the second bioactive molecule is directly attached or attached via a linker.

25. The heterodimeric protein of claim 24, wherein the adapter is selected from IgG1 hinges, IgG2 hinges, IgG3 hinges, IgG4 hinges, IgD hinges, or synthetic adapters.

26. The heterodimeric protein of any one of claims 18 to 25, further comprising one or more additional bioactive molecules.

27. The heterodimeric protein of claim 26, wherein one or more additional bioactive molecules are attached to the N-terminus of the first polypeptide, the C-terminus of the first polypeptide, the N-terminus of the second polypeptide, the C-terminus of the second polypeptide, the first bioactive molecule, the second bioactive molecule, or any combination thereof.

28. The heterodimeric protein of any one of claims 18 to 27, wherein the first bioactive molecule, the second bioactive molecule, the one or more other bioactive molecules, or combinations thereof comprise ligand-binding proteins, cytokines, or both.

29. The heterodimeric protein of claim 28, wherein the ligand-binding protein comprises a T-cell receptor, an antibody, or both.

30. The heterodimeric protein of claim 29, wherein the antibody comprises Ig NAR, Fab fragment, Fab′ fragment, F(ab)′2 fragment, F(ab)′3 fragment, Fv, single-chain variable fragment (scFv), bis-scFv, (scFv)2, microantibody, biantibody, triantibody, tetraantibody, intracellular antibody, disulfide-stabilized Fv protein (dsFv), monoclonal antibody, nanobody, aptamer, single-domain antibody (sdAB), or combinations thereof.

31. The heterodimeric protein of claim 29, wherein the antibody comprises a T-cell conjugate (e.g., a bispecific T-cell conjugate (BiTE) antibody), an amphiphilic heavy-targeting molecule (DART), a CrossMAb antibody, a DutaMab™ antibody, a DuoBody antibody, a Triomab, a TandAb, a bispecific nanobody, a tandem scFv, a biantibody, a single-chain biantibody, an HSA antibody, a (scFv)2 HSA antibody, a scFv-IgG antibody, a docking and locking bispecific antibody, a DVD-IgG antibody, a TBTI DVD-IgG, an IgG-fynomer, a tetravalent bispecific tandem IgG antibody, a dual-targeting domain antibody, a chemically linked bispecific (Fab')2 molecule, a cross-linked mAb, a bifunctional Fab IgG (DAF-IgG), an ortho-Fab-IgG, a bispecific CovX-Body, a bispecific hexavalent trimer, a 2 scFv linked to diphtheria toxin, an ART-Ig, an IgM T-cell conjugate, a Humabody™ human heavy-chain-only antibody (HCAb), or a UniAb™ HCAb, shark heavy chain antibody (VNAR), or a combination thereof.

32. The heterodimeric protein of any one of claims 28 to 31, wherein the ligand-binding protein binds to the tumor antigen.

33. The heterodimeric protein of any one of claims 28 to 32, wherein the ligand-binding protein binds to an antigen expressed on an immune cell.

34. The heterodimeric protein of claim 32 or 33, wherein the tumor antigen comprises guanylate cyclase C (GC-C), epidermal growth factor receptor (EGFR or erbB-1), or human epidermal growth factor receptor 2. (HER2 or erbB2), erbB-3, erbB-4, MUC-1, melanoma-associated chondroitin sulfate proteoglycan (MCSP), mesothelin (MSLN), folate receptor 1 (FOLR1), CD4, CD19, CD20, CD22, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD123, CD138, CD171, CEA, CSPG4, CXCR5, c-Met, HERV-capsule protein, eriostin, Biggs3, SPARC, BCR, CD79, CD37, EGFRvin, EGP2, EGP40, IGFr, LICAM, AXL, tissue factor (TF), CD74, EpCAM, EphA2, MRP3 cadherin 19 (CDH19), epidermal growth factor 2 (FIER2), 5T4, 8H9 Integrins, BCMA, B7-H3, B7-H6, CAIX, CA9, FAP, FBP, fetal AchR, FRcc, GD2, GD3, phosphatidylinositol proteoglycan-1 (GPC1), phosphatidylinositol proteoglycan-2 (GPC2), phosphatidylinositol proteoglycan-3 (GPC3), HLA-A1+MAGE1, HLA-A1+NY-ESO-1, IL-13Rcc2, Lewis-Y, KDR, MCSP, mesothelin, Mud, Mucl6, NCAM, NKG2D ligand, NY-ESO-1, PRAME, PSC1, PSCA, PSMA, ROR1, ROR2, SP17, survivin, TAG72, TEM, carcinoembryonic antigen, HMW-MAA, VEGF, CLDN18.2, programmed death-ligand 1 (PDL-1), or combinations thereof.

35. The heterodimeric protein of claim 33 or 34, wherein the antigen expressed on immune cells comprises CD3, CD27, B7H3, CD2, CD4, CD5, CD8, CD11b, CD14, CD16, CD19, CD28, CD32, CD45, CD56, CD64, KLRG-1, NKG2D, NKp30, DNAM-1, TIM-3, LAG-3, TIGIT, PD-1, CTLA-4, ILT2, ILT3, ILT4, LAIR1, VISTA, 4-1BB, OX40, CD40L, ICOS, LIGHT, or combinations thereof.

36. The heterodimeric protein of any one of claims 29 to 35, wherein the cytokines comprise IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21, IL-23, IFNα, IFNβ, IFNγ, TNF, combinations thereof, IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21, IL-23, IFNα, IFNβ, IFNγ, TNF, combinations thereof.

37. A fusion protein comprising a heterodimeric protein as described in any one of claims 1 to 36.

38. A bispecific antibody comprising (a) a first antigen-binding domain, (b) a second antigen-binding domain, and (c) a heterodimeric protein as described in any one of claims 1 to 36.

39. The bispecific antibody of claim 38, wherein the first antigen-binding domain, the second antigen-binding domain, or both are conjugated to the heterodimeric protein.

40. The bispecific antibody as described in claim 39, wherein: (a) the first antigen-binding domain is directly conjugated, (b) the first antigen-binding domain is conjugated via a first linker, (c) the second antigen-binding domain is directly conjugated, (d) the second antigen-binding domain is conjugated via a second linker, or (e) any combination of (a) to (d).

41. The bispecific antibody of claim 40, wherein the first adapter, the second adapter, or both are selected from IgG1 hinges, IgG2 hinges, IgG3 hinges, IgG4 hinges, IgD hinges, or synthetic adapters.

42. The bispecific antibody according to any one of claims 38 to 41, wherein the first antigen-binding domain, the second antigen-binding domain, or both bind to a tumor antigen.

43. The bispecific antibody of any one of claims 38 to 42, wherein the first antigen-binding domain, the second antigen-binding domain, or both bind to an antigen expressed on an immune cell.

44. The bispecific antibody of claim 42 or 43, wherein the first antigen-binding domain binds to a tumor antigen, and the second antigen-binding domain binds to an antigen expressed on an immune cell.

45. The bispecific antibody according to any one of claims 42 to 44, wherein the tumor antigen comprises guanylate cyclase C (GC-C), epidermal growth factor receptor (EGFR or erbB-1), human epidermal growth factor receptor 2 (HER2 or erbB2), erbB-3, erbB-4, MUC-1, melanoma-associated chondroitin sulfate proteoglycan (MCSP), mesothelin (MSLN), and folate receptor 1. (FOLR1), CD4, CD19, CD20, CD22, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD123, CD138, CD171, CEA, CSPG4, CXCR5, c-Met, HERV-enveloping protein, eriostin, Biggs3, SPARC, BCR, CD79, CD37, EGFRvin, EGP2, EGP40, IGFr, LICAM, AXL, tissue factor (TF), CD74, EpCAM, EphA2, MRP3 cadherin 19 (CDH19), epidermal growth factor 2 (FIER2), 5T4, 8H9 Integrins, BCMA, B7-H3, B7-H6, CAIX, CA9, FAP, FBP, fetal AchR, FRcc, GD2, GD3, phosphatidylinositol proteoglycan-1 (GPC1), phosphatidylinositol proteoglycan-2 (GPC2), phosphatidylinositol proteoglycan-3 (GPC3), HLA-A1+MAGE1, HLA-A1+NY-ESO-1, IL-13Rcc2, Lewis-Y, KDR, MCSP, mesothelin, Mud, Mucl6, NCAM, NKG2D ligand, NY-ESO-1, PRAME, PSC1, PSCA, PSMA, ROR1, ROR2, SP17, survivin, TAG72, TEM, carcinoembryonic antigen, HMW-MAA, VEGF, CLDN18.2, programmed death-ligand 1 (PDL-1), or combinations thereof.

46. ​​The bispecific antibody according to any one of claims 43 to 45, wherein the antigen expressed on immune cells comprises CD3, CD27, B7H3, CD2, CD4, CD5, CD8, CD11b, CD14, CD16, CD19, CD28, CD32, CD45, CD56, CD64, KLRG-1, NKG2D, NKp30, DNAM-1, TIM-3, LAG-3, TIGIT, PD-1, CTLA-4, ILT2, ILT3, ILT4, LAIR1, VISTA, 4-1BB, OX40, CD40L, ICOS, LIGHT, or combinations thereof.

47. The bispecific antibody according to any one of claims 38 to 46, comprising Ig NAR, Fab fragment, Fab′ fragment, F(ab)′2 fragment, F(ab)′3 fragment, Fv, single-chain variable fragment (scFv), bis-scFv, (scFv)2, microantibody, biantibody, triantibody, tetraantibody, intracellular antibody, disulfide-stabilized Fv protein (dsFv), monoclonal antibody, nanobody, aptamer, Humabody™ human heavy chain-only antibody (HCAb), UniAb™ HCAb, shark heavy chain-only antibody (VNAR), or combinations thereof.

48. A multispecific antibody comprising (a) a first antigen-binding domain, (b) a second antigen-binding domain, (c) a third antigen-binding domain, and (d) a heterodimeric protein as described in any one of claims 1 to 36.

49. The multispecific antibody of claim 48, wherein the first antigen-binding domain, the second antigen-binding domain, the third antigen-binding domain, or a combination thereof are conjugated to the heterodimeric protein.

50. The multispecific antibody as described in claim 49, wherein: (a) The first antigen-binding domain is directly conjugated, (b) The first antigen-binding domain is conjugated via a first linker, (c) The second antigen-binding domain is directly conjugated, (d) The second antigen-binding domain is conjugated via a second linker, (e) The third antigen-binding domain is directly conjugated, (f) The third antigen-binding domain is conjugated via a third linker, or (g) Any combination of (a) to (f).

51. The multispecific antibody of claim 50, wherein the first adapter, the second adapter, the third adapter, or a combination thereof are selected from IgG1 hinges, IgG2 hinges, IgG3 hinges, IgG4 hinges, IgD hinges, or synthetic adapters.

52. The multispecific antibody of any one of claims 48 to 51, wherein the first antigen-binding domain is attached to the N-terminus of the first polypeptide via a first adapter, the second antigen-binding domain is attached to the N-terminus of the second polypeptide via a second adapter, and the third antigen-binding domain is attached to the N-terminus of the second polypeptide via a third adapter, wherein the first adapter, the second adapter, and the third adapter are selected from IgG1 hinges, IgG2 hinges, IgG3 hinges, IgG4 hinges, IgD hinges, or synthetic adapters.

53. The multispecific antibody of any one of claims 48 to 51, wherein the first antigen-binding domain is attached to the N-terminus of the first polypeptide via a first adapter, the second antigen-binding domain is attached to the C-terminus of the first polypeptide via a second adapter, and the third antigen-binding domain is attached to the C-terminus of the second polypeptide via a third adapter, wherein the first adapter, the second adapter, and the third adapter are selected from IgG1 hinges, IgG2 hinges, IgG3 hinges, IgG4 hinges, IgD hinges, or synthetic adapters.

54. The multispecific antibody of any one of claims 48 to 53, wherein the first antigen-binding domain, the second antigen-binding domain, the third antigen-binding domain, or a combination thereof binds to a tumor antigen.

55. The multispecific antibody of any one of claims 48 to 54, wherein the first antigen-binding domain, the second antigen-binding domain, the third antigen-binding domain, or a combination thereof binds to an antigen expressed on an immune cell.

56. The multispecific antibody according to any one of claims 48 to 55, comprising an additional antigen-binding domain.

57. The multispecific antibody of claim 56, wherein the additional antigen-binding domain binds to a tumor antigen or an antigen expressed on an immune cell.

58. The multispecific antibody according to any one of claims 54 to 57, wherein the tumor antigen comprises guanylate cyclase C (GC-C), epidermal growth factor receptor (EGFR or erbB-1), human epidermal growth factor receptor 2 (HER2 or erbB2), erbB-3, erbB-4, MUC-1, melanoma-associated chondroitin sulfate proteoglycan (MCSP), mesothelin (MSLN), folate receptor 1 (FOLR1), CD4, CD19, CD20, CD22, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD123, CD138, CD171, CEA, CSPG4, CXCR5, c-Met, HERV-enveloping protein, eriostin, Big3, SPARC, BCR, CD79, CD37, EGFRvin, EGP2, EGP40, IGFr, LI CAM, AXL, Tissue Factor (TF), CD74, EpCAM, EphA2, MRP3 cadherin 19 (CDH19), Epidermal Growth Factor 2 (FIER2), 5T4, 8H9 Integrins, BCMA, B7-H3, B7-H6, CAIX, CA9, FAP, FBP, fetal AchR, FRcc, GD2, GD3, phosphatidylinositol proteoglycan-1 (GPC1), phosphatidylinositol proteoglycan-2 (GPC2), phosphatidylinositol proteoglycan-3 (GPC3), HLA-A1+MAGE1, HLA-A1+NY-ESO-1, IL-13Rcc2, Lewis-Y, KDR, MCSP, mesothelin, Mud, Mucl6, NCAM, NKG2D ligand, NY-ESO-1, PRAME, PSC1, PSCA, PSMA, ROR1, ROR2, SP17, survivin, TAG72, TEM, carcinoembryonic antigen, HMW-MAA, VEGF, CLDN18.2, programmed death-ligand 1 (PDL-1), or combinations thereof.

59. The multispecific antibody according to any one of claims 55 to 58, wherein the antigen expressed on immune cells comprises CD3, CD27, B7H3, CD2, CD4, CD5, CD8, CD11b, CD14, CD16, CD19, CD28, CD32, CD45, CD56, CD64, KLRG-1, NKG2D, NKp30, DNAM-1, TIM-3, LAG-3, TIGIT, PD-1, CTLA-4, ILT2, ILT3, ILT4, LAIR1, VISTA, 4-1BB, OX40, CD40L, ICOS, LIGHT, or combinations thereof.

60. A conjugate comprising a heterodimeric protein as claimed in any one of claims 1 to 36, a fusion protein as claimed in claim 37, a bispecific antibody as claimed in any one of claims 38 to 47, or a multispecific antibody as claimed in any one of claims 48 to 59, linked to one or more conjugate portions.

61. The conjugate of claim 60, wherein one or more conjugate portions comprise a scavenging modulator, a chemotherapeutic agent, a toxin, a radioisotope, a lanthanide, a luminescent label, a fluorescent label, an enzyme substrate label, a DNA alkylating agent, a topoisomerase inhibitor, a tubulin binder, an anticancer drug, or a combination thereof.

62. An isolated nucleic acid encoding a first polypeptide and / or a second polypeptide of a heterodimeric protein as described in any one of claims 1 to 36.

63. A vector comprising the isolated nucleic acid as described in claim 62.

64. An isolated nucleic acid encoding a fusion protein as described in claim 37, a bispecific antibody as described in any one of claims 38 to 47, a multispecific antibody as described in any one of claims 48 to 59, or a conjugate as described in claim 60 or 61.

65. A vector comprising the isolated nucleic acid as described in claim 64.

66. A pharmaceutical composition comprising a heterodimeric protein as described in any one of claims 1 to 36, a fusion protein as described in claim 37, a bispecific antibody as described in any one of claims 37 to 47, a multispecific antibody as described in any one of claims 48 to 59, a conjugate as described in claim 60 or 61, an isolated nucleic acid as described in claim 62 or 64, or a carrier as described in claim 63 or 65, and a pharmaceutically acceptable carrier.

67. A kit comprising a heterodimeric protein as claimed in any one of claims 1 to 36, a fusion protein as claimed in claim 37, a bispecific antibody as claimed in any one of claims 38 to 47, a multispecific antibody as claimed in any one of claims 48 to 59, a conjugate as claimed in claim 60 or 61, an isolated nucleic acid as claimed in claim 62 or 64, a vector as claimed in claim 63 or 65, or a pharmaceutical composition as claimed in claim 66, and instructions for use.

68. A method for preparing a heterodimeric protein as described in any one of claims 1 to 36, comprising culturing cells containing the isolated nucleic acid as described in claim 62 or the vector as described in claim 63, and optionally recovering the produced heterodimeric protein.

69. A method for producing a fusion protein, comprising conjugating one or more bioactive molecules to a heterodimeric protein as described in any one of claims 1 to 36.

70. The method of claim 69, wherein the one or more bioactive molecules are directly conjugated or conjugated via a linker.

71. The method of claim 70, wherein the connector is selected from IgG1 hinges, IgG2 hinges, IgG3 hinges, IgG4 hinges, IgD hinges, or synthetic connectors.

72. A method of treating a disease or ailment of a subject in need, comprising administering to the subject a heterodimeric protein as described in any one of claims 1 to 36, a fusion protein as described in claim 37, a bispecific antibody as described in any one of claims 38 to 47, a multispecific antibody as described in any one of claims 48 to 59, a conjugate as described in claim 60 or 61, an isolated nucleic acid as described in claim 62 or 64, a carrier as described in claim 63 or 65, or a pharmaceutical composition as described in claim 66.

73. The method of claim 72, wherein the disease or ailment includes cancer, an infectious disease, or both.

74. The method of claim 73, wherein the cancer includes breast cancer, head and neck cancer, uterine cancer, brain cancer, skin cancer, kidney cancer, lung cancer, colorectal cancer, prostate cancer, liver cancer, bladder cancer, kidney cancer, pancreatic cancer, thyroid cancer, esophageal cancer, eye cancer, stomach cancer, gastrointestinal cancer, carcinoma, sarcoma, leukemia, lymphoma, myeloma, or combinations thereof.

75. The method of claim 73 or 74, wherein the infectious disease includes progressive multifocal leukoencephalopathy (PML; caused by polyomavirus JC), sepsis, HIV, cytomegalovirus (CMV) infection, Epstein-Barr virus (EBV) infection, respiratory infectious diseases, or combinations thereof.

76. The method of any one of claims 72 to 75, wherein the heterodimeric protein, fusion protein, bispecific antibody, multispecific antibody, conjugate, isolated nucleic acid, carrier, or pharmaceutical composition is administered to the subject intramuscularly, parenterally, subcutaneously, ocularly, intravenously, intraperitoneally, intradermally, intraorbitally, intracerebrally, intracranially, intraspinally, intravenously, intracardiaclysm, intravenously, intracranially, intracysticly, intraspinally, intracardiaclysm, intrasheath, intracisternally, intracysticly, or intratumorally.

77. The method of any one of claims 72 to 76, further comprising administering an additional therapeutic agent to the subject.

78. The method of claim 77, wherein the additional therapeutic agent is administered to the subject intramuscularly, parenterally, subcutaneously, ocularly, intravenously, intraperitoneally, intradermally, intraorbitally, intracranially, intraspinally, intraventricularly, intrasheathically, intracisionally, intracysticly, or intratumorally.

79. The method of claim 77 or 78, wherein the additional therapeutic agent comprises an immune checkpoint inhibitor, an immune checkpoint activator, standard care treatment, a cytokine, or a combination thereof.

80. The method of claim 79, wherein the immune checkpoint inhibitor comprises a CTLA-4 antagonist (e.g., an anti-CTLA-4 antibody), a PD-1 antagonist (e.g., an anti-PD-1 antibody, an anti-PD-L1 antibody), a TIM3 antagonist (e.g., an anti-TIM3 antibody), or a combination thereof.

81. The method of claim 79 or 80, wherein the immune checkpoint activator comprises an OX40 agonist (e.g., an anti-OX40 antibody), a LAG-3 agonist (e.g., an anti-LAG3 antibody), 4-1BB (CD137) (e.g., an anti-CD137 antibody), a GITR agonist (e.g., an anti-GITR antibody), or a combination thereof.

82. The method of any one of claims 79 to 81, wherein the standard care treatment includes chemotherapy, radiation therapy, or both.

83. The method of any one of claims 79 to 82, wherein the cytokine comprises IL-7.

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