WO2025010272A1

WO2025010272A1 - Heterodimeric fc molecules and uses thereof

Info

Publication number: WO2025010272A1
Application number: PCT/US2024/036568
Authority: WO
Inventors: Sunkyoung IM; Minji Lee; Donghoon Choi; Taekyung Yu; Jie-Oh Lee; Hye Jin Jeong; Geun Young Cho
Original assignee: POSTECH Academy Industry Foundation; Neoimmunetech Inc
Current assignee: POSTECH Academy Industry Foundation; Neoimmunetech Inc
Priority date: 2023-07-03
Filing date: 2024-07-02
Publication date: 2025-01-09
Anticipated expiration: 2026-01-03

Abstract

The present disclosure provides heteromultimeric complexes comprising a first polypeptide and a second polypeptide, wherein the first and second polypeptides are not the same and comprise a heteromultimerization modification that reduces the formation of homomultimers and promotes the formation of heteromultimers. The present disclosure further provides fusion proteins, multispecific ligand-binding proteins, conjugates, and pharmaceutical compositions comprising such heteromultimeric complexes, including methods of making and uses to treat a wide range of diseases or conditions.

Description

HETERODIMERIC FC MOLECULES AND USES THEREOF

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This PCT application claims the priority benefit of U.S. Provisional Application No. 63/511,726, filed July 3, 2023, which is incorporated herein by reference in its entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0002] The content of the electronically submitted sequence listing in .XML file (4241_042PC01_SequenceListing_ST26.xml; 85,846 bytes; July 2, 2024) filed with the application is incorporated herein by reference in its entirety.

FIELD OF THE DISCLOSURE

[0003] The present disclosure provides heteromultimeric complexes (e.g., heterodimeric proteins) comprising a modified Fc and uses of such complexes.

BACKGROUND OF THE DISCLOSURE

[0004] Antibody-based therapeutics have been used successfully to treat a variety of diseases, including cancer and autoimmune/inflammatory disorders. Yet, improvements are still needed, particularly with respect to enhancing their clinical efficacy. The development of multispecific antibodies (e.g., bispecific antibodies) has great clinical potential (e.g., can recognize two different antigens, recruit different types of effector cells, and/or modulate multiple signaling pathways). However, production of multispecific antibodies can be challenging. The broad application of multispecific antibodies has been hindered by the difficulties of developing a platform for producing such antibodies that exhibit favorable half-life, high stability, lack of immunogenicity, and feasibilities for large scale manufacturing and purification. Therefore, there remains a need for a new and improved platform for developing heteromultimeric complexes (e.g., multispecific antibodies) that can be used in treating a wide range of diseases and conditions. BRIEF SUMMARY OF THE DISCLOSURE

[0005] 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: 16, and (b) the second polypeptide comprises the amino acid sequence set forth 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 present disclosure is related to a heterodimeric protein comprising a first polypeptide and a second polypeptide, wherein the first polypeptide and second polypeptide are not the same, 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: 24, and (b) the second CH3 domain comprises the amino acid sequence set forth in SEQ ID NO: 26, wherein: (1) amino acid residue N50, S60, or both of the first CH3 domain are not modified or modified with a positive charge modification, and amino acid residue N50, S60, or both of the second CH3 domain are not modified 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 not modified or modified with a negative charge modification, and amino acid residue N50, S60, or both of the second CH3 domain are not modified 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 not modified or modified with a positive charge modification, and amino acid residue D59, R69, or both of the second CH3 domain are not modified 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 not modified or modified with a negative charge modification, and amino acid residue D59, R69, or both of the second CH3 domain are not modified 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 not modified or modified with a positive charge modification, and amino acid residue F65 of the second CH3 domain is not modified 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 not modified or modified with a negative charge modification, and amino acid residue F65 of the second CH3 domain is not modified 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 comain 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) the amino acid residue N50 of the first CH3 domain is modified to N50K or N50R and the amino acid residue S60 of the second CH3 domain is modified to S60D or S60E; (b) the amino acid residue S60 of the first CH3 domain is modified to S60D or S60E and the amino acid residue N50 of the second CH3 domain is modified toN50K orN50R; (c) the amino acid residue K52 of the first CH3 domain is not modified or modified to K52R and the amino acid residue F65 of the second CH3 domain is modified to F65D or F65E; (d) the amino acid residue D59 of the first CH3 domain is modified to D59K or D59R and the amino acid residue R69 of the second CH3 domain is modified to R69D or R69E; (e) the amino acid residue R69 of the first CH3 domain is modified R69D or R69E and the amino acid residue D59 of the second CH3 domain is modified 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 glycinealanine linker, an alanine-serine linker, or combinations 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 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 biologically active molecule. In some aspects, the first biologically active 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 biologically active 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 a first biologically active molecule further comprises a second biologically active molecule. In some aspects, the second biologically active 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 biologically active molecule, or any combination thereof. In some aspects, the second biologically active 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 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 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 Ig NAR, 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 combinations thereof. In some aspects, the antibody comprises a T cell engager (e.g., bispecific T- cell engager (BiTE) antibody), dual-affinity retargeting molecule (DART), CrossMAb antibody, DutaMab™ antibody, DuoBody antibody, Triomab, TandAb, bispecific NanoBody, Tandem scFv, diabody, single chain diabody, HSA body, (scFv)2 HSA Antibody, scFv-IgG antibody, Dock and Lock bispecific antibody, DVD-IgG antibody, TBTI DVD-IgG, IgG-fynomer, Tetravalent bispecific tandem IgG antibody, dual targeting domain antibody, chemically linked bispecific (Fab')2 molecule, crosslinked mAb, Dual-action Fab IgG (DAF-IgG), orthoFab-IgG, bispecific CovX-Body, bispecific hexavalent trimerbody, 2 scFv linked to diphtheria toxin, ART-Ig, IgM T- cell engager, Humabody™ human heavy-chain only antibody (HCAb), UniAb™ HCAb, shark heavy chain-only antibody (VNAR), or combinations thereof.

[0018] In some aspects, the ligand-binding protein binds to a tumor antigen. In some aspects, wherein the ligand-binding protein binds to an antigen expressed on an immune cell. In some aspects, the tumor antigen comprises a 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, MRP3cadherin 19 (CDH19), epidermal growth factor 2 (FIER2), 5T4, 8H9, anbo 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, Mucl6, NCAM, NKG2D ligands, NY-ESO-1, PRAME, PSC1, PSCA, PSMA, ROR1, ROR2, SP17, survivin, TAG72, TEMs, 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 a CD3, CD27, B7H3, CD2, CD4, CD5, CD8, CDl lb, 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, 0X40, 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 an IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21, IL-23, IFNa, IFNp. IFNy, TNF, combinations thereof an IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21, IL-23, IFNa, IFNp. IFNy, TNF, combinations 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 conjugated directly, (b) the first antigen-binding domain is conjugated via a first linker, (c) the second antigen binding domain is conjugated directly, (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 a 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, MRP3cadherin 19 (CDH19), epidermal growth factor 2 (FIER2), 5T4, 8H9, anbo 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-ES0-1, IL-13Rcc2, Lewis-Y, KDR, MCSP, Mesothelin, Mud, Mucl6, NCAM, NKG2D ligands, NY-ESO-1, PRAME, PSC1, PSCA, PSMA, R0R1, R0R2, SP17, survivin, TAG72, TEMs, 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 a CD3, CD27, B7H3, CD2, CD4, CD5, CD8, CDl lb, 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, 0X40, CD40L, ICOS, LIGHT, or combinations thereof

[0024] In some aspects, a bispecific antibody described herein comprises an Ig NAR, 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, Humabody™ human heavy-chain only antibody (HCAb), UniAb™ HCAb, shark heavy chain-only antibody (VNAR), or combinations thereof.

[0025] Also provided herein is a multispecific antibody comprising (a) a first antigenbinding 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 combinations thereof are conjugated to the heterodimeric protein. In some aspects, (a) the first antigen-binding domain is conjugated directly, (b) the first antigen-binding domain is conjugated via a first linker, (c) the second antigen-binding domain is conjugated directly, (d) the second antigen-binding domain is conjugated via a second linker, (e) the third antigen-binding domain is conjugated directly, (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 combinations thereof are 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 the first polypeptide via a first linker, the second antigen-binding domain is attached to the N terminus of the 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, second linker, and third linker are selected from IgGl hinge, IgG2 hinge, IgG3 hinge, IgG4 hinge, IgD hinge, or synthetic linker. In some aspects, the first antigen-binding domain is attached to the N terminus of the 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 the second polypeptide via a third linker, and wherein the first linker, second linker, and third linker are selected from IgGl hinge, IgG2 hinge, IgG3 hinge, IgG4 hinge, IgD hinge, or synthetic linker.

[0028] In some aspects, the first antigen-binding domain, the second antigen-binding domain, the third antigen-binding domain, or combinations thereof bind to a tumor antigen. In some aspects, the first antigen-binding domain, the second antigen-binding domain, the third antigen-binding domain, or combinations thereof bind 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 a 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, LI CAM, AXL, Tissue Factor (TF), CD74, EpCAM, EphA2, MRP3cadherin 19 (CDH19), epidermal growth factor 2 (FIER2), 5T4, 8H9, anbo 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, Mucl6, NCAM, NKG2D ligands, NY-ESO-1, PRAME, PSC1, PSCA, PSMA, ROR1, ROR2, SP17, survivin, TAG72, TEMs, 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 a CD3, CD27, B7H3, CD2, CD4, CD5, CD8, CDl lb, 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, 0X40, CD40L, ICOS, LIGHT, or combinations thereof [0031] Some aspects of the present disclosure relates to a conjugate comprising any of the heterodimeric proteins, fusion proteins, bispecific antibodies, or multispecific antibodies provided herein, which is linked to one or more conjugate moieties. In some aspects, the one or more conjugate moieties comprise a clearance-modifying agent, a chemotherapeutic agent, a toxin, a radioactive isotope, a lanthanide, a luminescent label, a fluorescent label, an enzyme-substrate label, a DNA-alkylators, a topoisomerase inhibitor, a tubulin-binders, an anticancer drug, or combinations thereof.

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

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

[0034] 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 provided herein, and instructions for use.

[0035] Some aspects of the present disclosure relates to a method of preparing 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 heterodimeric protein that is produced.

[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 any of the heterodimeric proteins, fusion proteins, bispecific antibodies, multispecific antibodies, conjugates, isolated nucleic acids, vectors, or pharmaceutical compositions provided herein to the subject. [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, head and neck cancer, uterine cancer, brain cancer, skin cancer, renal cancer, lung cancer, colorectal cancer, prostate cancer, liver cancer, bladder cancer, kidney cancer, pancreatic cancer, thyroid cancer, esophageal cancer, eye cancer, stomach (gastric) cancer, gastrointestinal cancer, carcinoma, sarcoma, leukemia, lymphoma, myeloma, or a combination thereof. In some aspects, the infectious disease comprises a progressive multifocal leukoencephalopathy (PML; caused by polyomavirus JC), a sepsis, a HIV, a cytomegalovirus (CMV) infection, an Epstein-Barr virus (EBV) infection, a respiratory infectious disease, or combinations thereof.

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

[0040] In some aspects, the method comprises administering an additional therapeutic agent to the subject. In some aspects, the additional therapeutic agent is administered to the subject intramuscularly, parenthetically, subcutaneously, ophthalmic, intravenously, intraperitoneally, intradermally, intraorbitally, intracerebrally, intracranially, intraspinally, intraventricular, intrathecally, intraci stemally, intracapsularly, or intratumorally.

[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 combinations thereof. In some aspects, the immune checkpoint inhibitor comprises a CTLA-4 antagonist (e.g., anti-CTLA-4 antibody), a PD-1 antagonist (e.g., anti-PD-1 antibody, anti-PD-Ll antibody), a TIM3 antagonist (e.g., anti-TIM3 antibody), or combinations thereof. In some aspects, the immune checkpoint activator comprises an 0X40 agonist (e.g., anti-OX40 antibody), a LAG- 3 agonist (e.g., anti-LAG3 antibody), a 4-1BB (CD137) (e.g., anti-CD137 antibody), a GITR agonist (e.g., anti-GITR antibody), or combinations thereof. In some aspects, the standard of care treatment comprises a chemotherapy, a radiation, or both. In some aspects, the cytokine comprises an IL-7. BRIEF DESCRIPTION OF THE FIGURES

[0042] FIG. 1 provides a predictive structural model of an exemplary trispecific antibody comprising a heterodimeric protein described herein (also referred to herein as "trispecific heterodimer antibody"). In both the first polypeptide (CH3 A; left chain; "chain A") and the second polypeptide (CH3B; right chain; and "chain B"), the heterodimeric Fc portion 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 first polypeptide chain further comprises an anti-B7H3 single domain antibody (sdAb) attached to the N-terminus of the IgD hinge. 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 comprised the amino acid sequence set forth in SEQ ID NO: 40 with the following amino acid modifications: T10V, LI 1 Y, F65A, and Y67V (i.e., T148V, L149Y, F203A, and Y205V in SEQ ID NO: 4). The CH3 domain of chain B comprised 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] FIG. 2 provides a summary of an exemplary process used in producing the multispecific ligand-binding proteins conjugated to a heterodimeric protein described herein (e.g., trispecific heterodimer antibody shown in FIG. 1). As further described in Example 1, the coding sequences were first coded into plasmids that included common affinity tags at the C-terminal portion of the insertion site, along with a cleavage site to facilitate removal ("Step 1"). These plasmids were then transformed into baculoviral vectors, which were then transfected into insect cells and the virus was produced over the course of 2 weeks ("Step 2"). The resulting viral particles were used to transduce human cells (e.g., Expi293F cells), which were enhanced and further cultured ("Step 3"). The resulting antibodies were then purified via affinity chromatography ("Step 4").

[0044] FIG. 3 provides a schematic of the different constructs used in producing a trispecific heterodimer antibody (e.g., shown in FIG. 1). The constructs shown include: (a) Construct #1 comprising (from N-terminus to C-terminus): (i) an anti-B7H3 sdAb, (ii) the first polypeptide CH3A (i.e., comprising an IgD hinge, IgD/IgG4 CH2, and IgG4 CH3), (iii) a cleavage site, and (iv) GFP tag; (b) Construct #2 comprising (from N-terminus to C-terminus): (i) an anti- CD3a sdAb, (ii) an anti-CD27 sdAb, (iii) the second polypeptide CH3B (i.e., comprising an 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-terminus to C-terminus): (i) an anti-CD3 sdAb, (ii) an anti-CD27 sdAb, (iii) the second polypeptide CH3B, and (iv) a His tag; and (d) Construct #4 comprising (from N-terminus to C-terminus): (i) an anti-B7H3 sdAb and the first polypeptide CH3A.

[0045] FIGs. 4A and 4B provide purification analysis of a trispecific heterodimer antibody (comprising chain A encoded in Construct #1 and chain B encoded in Construct #2; see FIG. 3 for description of Construct #1 and #2) under non-reducing conditions using ALFA nanobody resin. FIG. 4A provides a SDS-PAGE analysis of the anti-ALFA column fractions using fluorescence imaging (left) and Coomasie staining (right). The lanes for these gels are labeled as follows: (i) "M": molecular weight marker, (ii) "R": protein bound to ALFA nanobody resin, and (iii) "El-4": protein eluted by thrombin. Expected molecular weights for CD3a-CD27-CHB-mCherry polypeptide chain (chain B+mCherry) and B7H3-CHA-GFP polypeptide chain (chain A+GFP) were 103.2 kDa and 72 kDa, respectively. FIG. 4B provides a SDS-PAGE analysis before and after cleavage of the preScission site. The lanes for these gels are labeled as follows: (i) "M": molecular weight marker, (ii) "pre-cut": prior to cleavage, and (iii) "post-cut": post cleavage.

[0046] FIG. 5 provides SDS-PAGE analysis confirming the successful formation of the trispecific heterodimer antibody described in FIGs. 4A and 4B (z.e., comprising chain A and chain B encoded in Constructs #1 and #2, respectively). The lanes for these gels are labeled as follows: (i) "R": P-mercaptoethnol containing (reducing), (ii) "M": molecular weight marker, and (iii) "NR": P-mercaptoethnol free (non-reducing).

[0047] FIGs. 6A and 6B provide analysis showing the purity of the trispecific heterodimer antibody described in FIGs. 4A and 4B (z.e. , comprising chain A and chain B encoded in Constructs #1 and #2, respectively) after purification under non-reducing conditions. FIG. 6A provides a SDS-PAGE gel confirming the molecular weight of the purified trispecific heterodimer antibody (see band at 116.7 kDa). FIG. 6B provides ChemiDoc readout showing the estimated purity of the trispecific heterodimer antibody (see band no. 2; purity of about 75%).

[0048] FIG. 7 provides SDS-PAGE analysis showing the relative amounts of chain A (B7H3-CHA) (encoded in Construct #1) and chain B (CD3a-CD27-CHB) (encoded in Construct #2) present after purification using an anti-ALFA column and an anti-GFP column, respectively. The gel to the left was developed using fluorescence, and the gel to the right was developed using Coomasie staining. Lanes for both gels were labeled as follows: (i) "sup": supernatant, (ii) "FT": flow through, (iii) "R": protein bound to ALFA nanobody or GFP nanobody resins, and (iv) "M": molecular weight marker. Expected molecular weights for chain B+mCherry and chain A+GFP were 103.2 kDa and 72 kDa, respectively.

[0049] FIG. 8 provides SDS-PAGE analysis showing the relative amounts of chain A (B7H3-CHA) (encoded in Construct #1) and chain B (CD3a-CD27-CHn) (encoded in Construct #2) after purification using baculovirus:human cell infection ratios (4: 1, 8: 1, and 10:1). As in FIG. 7, chain A (B7H3-CHA) and chain B (CD3a-CD27-CHn) were purified using an anti-ALFA column and an anti-GFP column, respectively. Lanes for all gels were labeled as follows: (i) "M": molecular weight marker, (ii) "sup": supernatant, (iii) "FT": flow through, (iv) "R": protein bound to ALFA nanobody or GFP nanobody resin, and (v) "E": protein eluted with thrombin. The gel to the left was developed using fluorescence, and the gel to the right was developed using Coomasie staining.

[0050] FIG. 9A provides SDS-PAGE analysis showing the presence of chain A and chain B (z.e., encoded in Constructions #1 and #2, respectively) under non-reducing conditions and at the three different infection ratios: (i) 4: 1, (ii) 8: 1, and (iii) 10-1. Expected molecular weights for Chain B (i.e., CD3a-CD27-CH_B) and Chain A i.e., B7H3-CHA) were 72 kDa and 43.6 kDa, respectively. FIG. 9B provides SDS-PAGE analysis confirming the successful formation of a trispecific heterodimer antibody comprising chain A and B shown in FIG. 9A at the different infection ratios.

[0051] FIG. 10 provides a mass photometry analysis of the heterodimeric trispecific antibody described in FIG. 9B, with a peak at approximately 115.5 kDa.

[0052] FIG. 11 provides a SDS-PAGE analysis of the fractions eluted after purification of the trispecific heterodimer antibody (comprising chain A and chain B encoded in Constructs #4 and #3, respectively) using an anti-His column. The lanes for this gel are labeled as follows: (i) "M": molecular weight marker, (ii) "Sup": supernatant, (iii) "FT": flow through, (iv) "W": wash, and (v) "El -3": eluted protein.

[0053] FIG. 12 provides a SDS-PAGE analysis of the fractions eluted after further purifying the eluted protein from FIG. 11 using a Protein A column. The lanes for this gel are labeled as follows: (i) "input": imidazole-eluted protein, (ii) "FT": flow through, (iii) "W": wash, (iv) "El -5": eluted protein, and (v) "M": marker.

[0054] FIG. 13 provides a SDS-PAGE analysis confirming the heterodimeric formation of chain A (encoded in Construct #4) and chain B (encoded in Construct #3) to produce a trispecific heterodimer antibody (such as that illustrated in FIG. 1) under both reducing and non-reducing conditions. The lanes for this gel are labeled 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). Expected molecular weights for chain B (z.e., CD3a-CD27-CHn), chain A (z.e., B7H3-CHA), and the trispecific heterodimer antibody were 72 kDa, 43.6 kDa, and 115.5 kDa, respectively.

[0055] FIG. 14 provides ChemiDoc readout showing the estimated purity of the purified trispecific heterodimer antibody described in FIG. 13. Band no. 1 corresponds to chain A (encoded in Construct #4). Band no. 4 corresponds to chain B (encoded in Construct #3). The final purified protein had a purity of 83%.

[0056] FIG. 15 provides CE-SDS analysis of the purified trispecific heterodimer antibody described in FIG. 9B, under reducing (top, in blue) and non-reducing (bottom, in black) conditions. [0057] FIG. 16 provides Western blot analysis confirming the heterodimeric formation of chain A (encoded in Construct #4) and chain B (encoded in Construct #3) to produce the trispecific heterodimer antibody, such as that described in FIG. 1. For comparison, homodimers of chain B and monomers of chain A and chain B are also shown. The lanes for this gel are labeled as follows: (i) "NR": non-reducing, (ii) "R": reducing conditions, and (iii) "M": molecular weight ladder. Expected molecular weights for chain B (CD3a-CD27-CHn), chain A (B7H3-CHA), the trispecific heterodimer protein, and the chain B (CD3a-CD27-CHn) homodimer were 72 kDa, 43.6 kDa, 115.5 kDa, and 144 kDa, respectively.

[0058] FIGs. 17A-17C provide list of exemplary positive charge amino acid modifications and negative charge amino acid modifications with the CH3 domain of first and second polypeptide described herein (chains A and B, respectively). For the 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 an arginine (R) or a lysine (K). As described herein, these amino acid residues correspond to amino acid residues N50, K52, D59, S60, and R69, respectively, of a CH3 domain described herein (e.g., comprising the amino acid sequence set forth in SEQ ID NO: 24. For the negative charge modifications of chain B, amino acid residues N188, DI 97, SI 98, R207, and/or F203 of chain B (e.g., comprising the amino acid sequence set forth in SEQ ID NO: 20 are modified to an aspartic acid (D) or a glutamic acid (E). As also described herein, these amino acid residues correspond to amino acid residues N50, D59, S60, R69, and F65, respectively, of a CH3 domain described herein (e.g., comprising the amino acid sequence set forth in SEQ ID NO: 26. FIG. 17A shows exemplary positive charge combinations #l-#8 and exemplary negative charge combinations #I-#I2. As is apparent from the present disclosure, based on the positive and negative charge combinations provided in FIG. 17A, 96 different combinations of chain A (having a positive charge) and chain B (having a negative charge) heterodimers can be produced (e.g., positive charge combination #1 + negative charge combination #1; positive charge combination #1 + negative charge combination #2; and so on). FIG. 17B shows exemplary positive charge combinations #9-#28 and exemplary negative charge combinations #13-#35. FIG. 17C shows exemplary positive charge combinations #29-#38 and exemplary negative charge combinations #36-#60. Based on the positive and negative charge combinations provided in FIG. 17B and 17C, 1,536 different combinations of chain A (having a positive charge) and chain B (having a negative charge) heterodimers can be produced.

[0059] FIGs. 18A-18D provide list of exemplary negative charge amino acid modifications and positive charge amino acid modifications with the CH3 domain of first and second polypeptides described herein (chains A and B, respectively). For the 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 an aspartic acid (D) or a glutamic acid (E). For the positive charge modification 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 an arginine (R) or a lysine (K). FIG. 18A shows exemplary negative charge combinations #1-#I2 and exemplary positive charge combinations #1-#I2. Based on such positive and negative charge combinations, 144 different combinations of chain A (having a negative charge) and chain B (having a positive charge) heterodimers can be produced. FIG. 18B shows exemplary negative charge combinations #13-#33 and exemplary positive charge combinations #13-#33. FIG. 18C shows exemplary negative charge combinations #34-#54 and exemplary positive charge combinations #34-#54. FIG. 18D shows exemplary negative charge combinations #55-#60 and exemplary positive charge combinations #55-#60. Based on the negative and positive charge combinations provided in FIGs. 18B-18D, 2,304 different combinations of chain A (having a negative charge) and chain B (having a positive charge) heterodimers can be produced.

[0060] FIG. 19 provides a schematic 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 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 each other to form a heterodimeric protein (such as that described herein). As shown, amino acid residue N188 on the first polypeptide is capable of interacting with amino acid residue SI 98 on the second polypeptide. Similarly, amino acid residue SI 98 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 DI 97 on the first polypeptide is capable of interacting with amino acid residue R207 of the second polypeptide. Amino acid residue R207 of the first polypeptide is capable of interacting with the amino acid residue DI 97 of the second polypeptide. As further described 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] FIG. 20 provides a schematic 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 either prevent interaction ("repulsion between same chain") or promote interaction ("attraction between different chain").

[0062] FIG. 21 provides melting temperature (T_m) profile as determined using a nano differential scanning fluorimetry assay for the following heterodimers: (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 ratios (F350/330)

[0063] FIG. 22 provides individual melting temperature profile for each of the heterodimers described in FIG. 21.

[0064] FIG. 23 provides comparison of aggregation temperatures (T_agg) as determined using DLS experiment for the following heterodimers (1) NIT-Fc-01, (2) NIT-Fc-02, (3) NIT -Fc- 03, (4) NIT-Fc-04, and (5) NIT-Fc-05. The DLS-T_agg was determined as the point at which the radius of the sample increased to three times its initial value, indicating the onset of denaturation and aggregation. [0065] FIG. 24 provides 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 OF THE DISCLOSURE

[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 not the same, wherein: (a) the first polypeptide comprises a first CH3 domain comprising a heterodimerization modification (e.g., positive or negative charge modifications described herein), and (b) the second polypeptide comprises a second CH3 domain comprising a heterodimerization modification (e.g., negative or positive charge modifications described herein), and wherein the heterodimerization modification of the first CH3 domain and the heterodimerization modification of the second CH3 domain promotes the interaction of the first and second CH3 domains to produce the heteromultimeric protein. As further described herein, the heteromultimeric proteins described herein are capable of increasing productivity and/or stability of a protein. Additional aspects of the present disclosure are provided throughout the present 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.

I. Definitions

[0068] Throughout this disclosure, the term "a" or "an" entity refers to one or more of that entity; for example, "a polypeptide," is understood to represent one or more polypeptides. As such, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

[0069] Furthermore, "and/or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and/or" as used in a phrase such as "A and/or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Likewise, the term "and/or" as used in a phrase such as "A, B, and/or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0070] It is understood that wherever aspects are described herein with the language "comprising," otherwise analogous aspects described in terms of "consisting of' and/or "consisting essentially of" are also provided. [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, 2000, Oxford University Press, provide one of skill with a general dictionary of many of the terms used in this disclosure.

[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 immediately below are more fully defined by reference to the specification in its entirety.

[0073] The term "about" is used herein to mean approximately, roughly, around, or in the regions of. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term "about" can modify a numerical value above and below the stated value by a variance of, e.g., 10 percent, up or down (higher or lower).

[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 instance, in some aspects, the first polypeptide and the second polypeptide differ in their amino acid sequences by at least one amino acid residue. Accordingly, in some aspects, the amino acid sequence of the first polypeptide and the amino acid sequence of the second polypeptide share a sequence identity of 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%. 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, heteromultimers can comprise a "heterodimer" formed by the first and second polypeptides. In some aspects, heteromultimers can comprise higher order tertiary structures where polypeptides in addition to the first and second polypeptides are present. The polypeptides of the heteromultimer can interact with each other by various means known in the art, e.g., a non-peptidic, covalent bond (e.g., disulfide bond) and/or a non-covalent interaction (e.g., hydrogen bonds, ionic bonds, van der Waals forces, and/or hydrophobic interactions). Unless indicated otherwise, heteromultimer and heterodimer (and variants thereof) are used interchangeably.

[0075] As used herein, the term "heteromultimerization modification" (or grammatical variants thereof) refer to alterations that promote heteromultimer formation and reduce/inhibit homomultimer formation. Examples of such modifications are known in the art, e.g., WO 2013/063702, which is incorporated herein by reference in its entirety. Additional non-limiting examples of such modifications are provided throughout the present disclosure. In some aspects, a heteromultimerization modification comprises an amino acid modification. For instance, in some aspects, a first polypeptide comprises a first amino acid modification and a second polypeptide comprises a second amino acid modification, wherein the first and second amino acid modifications are such that they promote the interaction of the first and second polypeptides. In some aspects, the first amino acid modification and the second amino acid modification results in opposite charges (e.g., positive charge modification and 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 an amino acid insertion, deletion, substitution, and rearrangement.

[0077] As used herein, a "first polypeptide" refers to any polypeptide that is to be associated with a second polypeptide. As used herein, a "second polypeptide" refers to any polypeptide that is to be associated with a first polypeptide. Unless indicated otherwise, the first polypeptide and the second polypeptide are not the same (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 those skilled in the arts that any heteromultimerization modifications (e.g., amino acid modifications described herein) can be on either of the first polypeptide and the second polypeptide, as long as the heteromultimerization modifications promote the interaction of the first and second polypeptides. In some aspects, 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 the C-terminal region of the heavy chain of an antibody that mediates the binding of the 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, a Fc region comprises the constant region of an antibody excluding the first constant region immunoglobulin domain (e.g., CHI or CL). For instance, in some aspects, a Fc useful for the present disclosure comprises a CH3 domain. In some aspects, the Fc further comprises: (a) a hinge domain, (b) a CH2 domain, or (c) both (a) and (b). For instance, in some aspects, the Fc comprises a hinge domain and a CH3 domain. In some aspects, the Fc comprises a CH2 domain and a CH3 domain. In some aspects, the Fc comprises a hinge domain, CH2 domain, and CH3 domain. In some aspects, the Fc is a human IgG Fc. In some aspects, the human IgG Fc is a human IgGl, IgG2, IgG3, or IgG4 Fc. In some aspects, the Fc is from an immunoglobulin selected from an 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 antibody's two heavy chains; 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 the hinge between CHI and CH2 domains. Although the definition of the boundaries of the Fc region of an immunoglobulin heavy chain might vary, as defined herein, the human IgG heavy chain Fc region is defined to stretch from an amino acid residue D221 for IgGl, V222 for IgG2, L221 for IgG3 and P224 for IgG4 to the carboxy -terminus of the heavy chain, wherein the numbering is 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 positioned on C-terminal side of a CH2 domain in an Fc region, z.e., it extends from amino acid 341 to amino acid 447 or 446 (if the C-terminal lysine residue is absent) or 445 (if the C-terminal glycine and lysine residues are absent) of an IgG. As used herein, the 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 an Fc-comprising protein polypeptide such as a "binding protein comprising an Fc region," also referred to as a "Fc fusion protein" (e.g., an antibody or immunoadhesion). Exemplary Fc sequences are provided elsewhere in the present disclosure (see, e.g., Table 3).

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

[0081] As used herein, the term "hinge domain" refers to any moiety that is capable of linking two or more molecules. In some aspects, a 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 sequence of the heavy chains linking the antigen-binding fragment (Fab) to the Fc region. As further described elsewhere in the present disclosure, in some aspects, a hinge domain comprises a linker (e.g., synthetic linker). Examples of linkers that can be used as a hinge domain are known in the art. See, e.g., WO 2014/087248, which is incorporated herein by reference in its entirety. Accordingly, unless indicated otherwise, the terms "hinge domain" and "linker" can be used interchangeably in the present disclosure.

[0082] As used herein, the term "CH2 domain" refers to the heavy chain immunoglobulin constant chain that is located between the hinge and the CH3 domain. A 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 the desired biological properties. A desired biological activity can be a natural biological activity, an enhanced biological activity or a reduced biological activity relative to that of the naturally occurring domain.

[0083] As used herein, the term "CH3 domain" refers to the heavy chain immunoglobulin constant chain that is located C-terminally of the CH2 domain and spans approximately 110 residues from the N-terminus of the CH2 domain, e.g., about positions 341-446b (EU numbering system). A 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 the desired biological properties. A desired biological activity can be a natural biological activity, an enhanced biological activity or a reduced biological activity relative to that of the naturally occurring domain. In some aspects, a CH3 domain described herein comprises a C-terminal lysine. In some aspects, a CH3 domain described herein does not comprise a C-terminal lysine.

[0084] "EU" indicates that amino acid positions, e.g., in a heavy chain constant region, such as within the hinge, CH2, and/or CH3 domains, are 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 edition, 1991).

[0085] The term "fusion protein" refers to proteins created through the joining of two or more genes that originally coded for separate proteins. Translation of this fusion gene results in a single polypeptide or multiple polypeptides with functional properties derived from each of the original proteins. In some aspects, the two or more genes can comprise a substitution, a deletion, and / or an addition in its nucleotide sequence.

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

[0087] As used herein, "attached," "conjugated, " and "linked can be used interchangeably and refer to the joining of a first moiety to a second moiety (e.g., 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 and second moi eties are fused together). In some aspects, the first moiety can be attached the second moiety via a linker. Non-limiting examples of useful linkers are described elsewhere in the present disclosure.

[0088] The terms "antibody" and "antibodies" are terms of art and can be used interchangeably herein and refer to a molecule with an antigen binding site that specifically binds an antigen. The terms as used herein include whole antibodies and any antigen binding fragments (z.e., "antigen-binding portions" or "antigen-binding fragments") or single chains thereof. An "antibody" refers, in some aspects, to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen-binding portion thereof. In some aspects, an "antibody" refers to a single chain antibody comprising a single variable domain, e.g., VHH domain. Each heavy chain is comprised of 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 is comprised of three domains, CHI, CH2 and CH3. In some naturally- occurring antibodies, each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL.

[0089] Unless indicated otherwise, antibodies 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, which differ from each other in at most a few amino acids. An antibody disclosed herein can be from any of the commonly known isotypes, classes, subclasses, or allotypes. In some aspects, the antibodies described herein are of the IgGl, IgG2, IgG3 or IgG4 subclass or any hybrid thereof. In some aspects, the antibodies are of the human IgGl subclass, human IgG2 subclass, or human IgG4 subclass.

[0090] The term "naturally-occurring" or "naturally-existing," as used herein, refers to the fact that an object (e.g., protein) can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally-occurring. As further described elsewhere in the present disclosure, polypeptides useful for the present disclosure are not naturally-occurring.

[0091] A "polypeptide" refers to a chain comprising at least two consecutively linked amino acid residues, with no upper limit on the length of the chain. One or more amino acid residues in the protein can contain a modification 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" can be used interchangeably.

[0092] The term "nucleic acids," "nucleic acid molecule," or "polynucleotide" (or variants thereof) as used herein, refers to polymers of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, analogs thereof, or mixtures thereof. This term refers to the primary structure of the molecule. Thus, the term includes triple-, double- and single-stranded deoxyribonucleic acid ("DNA"), as well as triple-, double- and single-stranded ribonucleic acid ("RNA"). It also includes modified, for example by alkylation, and/or by capping, and unmodified forms of the polynucleotide. More particularly, 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 normucleotidic backbones, for example, polyamide (e.g., peptide nucleic acids "PNAs") and polymorpholino polymers, and other synthetic sequence-specific nucleic acid polymers providing that the polymers contain nucleobases in a configuration which allows for base pairing and base stacking, such as is found in DNA and RNA

[0093] For nucleic acids, the term "substantial homology" indicates that two nucleic acids, or designated sequences thereof, when optimally aligned and compared, are identical, with appropriate nucleotide insertions or deletions, in at least about 80% of the nucleotides, at least about 90% to 95%, or at least about 98% to 99.5% of the nucleotides. Alternatively, substantial homology exists when the segments will hybridize under selective hybridization conditions, to the complement of the strand. For polypeptides, the term "substantial homology" indicates that two polypeptides, or designated sequences thereof, when optimally aligned and compared, are identical, with appropriate amino acid insertions or deletions, in at least about 80% of the amino acids, at least about 90% to 95%, or at least about 98% to 99.5% of the amino acids.

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

[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 gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. 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)) which has been incorporated into the ALIGN program (version 2.0), 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 Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at worldwideweb.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight 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 NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 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 worl d wi de web . neb i . nl m . ni h . gov .

[0097] The term "vector," as used herein, 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 techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" can be used interchangeably as the plasmid is the most commonly used form of vector. However, also included are other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

[0098] The term "recombinant host cell" (or simply "host cell"), as used herein, is intended to refer to a cell that comprises 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 refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications can occur in succeeding generations due to either mutation or environmental influences, such progeny cannot, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein. [0099] As used herein, "administering" refers to the physical introduction of an agent (e.g. , polypeptide or molecule described herein) or a composition comprising the agent to a subj ect, using any of the various methods and delivery systems known to those skilled in the art. Nonlimiting examples of routes of administration that can be used include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal or other parenteral routes of administration, for example by injection or infusion. The phrase "parenteral administration" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, as well as 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, for example, intranasally, orally, vaginally, rectally, sublingually or topically. Administering can also be performed, for example, once, a plurality of times, and/or over one or more extended periods.

[0100] As used herein, the term "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, dog, cow, chickens, amphibians, reptiles, etc.

[0101] The term "therapeutically effective amount" as used herein refers to an amount of a substance, alone or in combination with another therapeutic agent, effective to "treat" a disease or disorder in a subject or reduce the risk, potential, possibility or occurrence of a disease or disorder (e.g, a cancer and/or infectious disease described herein). A "therapeutically effective amount" includes an amount of a substance or a therapeutic agent that provides some improvement or benefit to a subject having or at risk of having a disease or disorder. Thus, a "therapeutically effective" amount is an amount that reduces the risk, potential, possibility or occurrence of a disease or provides disorder or some alleviation, mitigation, and/or reduces at least one indicator, and/or decrease in at least one clinical symptom of a disease or disorder.

[0102] The terms "treat," "treating," and "treatment," as used herein, refer to any type of intervention or process performed on, or administering an active agent to, the subject with the objective of reversing, alleviating, ameliorating, inhibiting, or slowing down or preventing the progression, development, severity or recurrence of a symptom, complication, condition or biochemical indicia associated with a disease. Treatment can be of a subject having a disease or a subject who does not have 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 further detail in the following subsections.

IL Polypeptides

[0105] 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 heteromultimeric formation). In some aspects, a polypeptide comprises multiple heteromultimerization modifications (e.g., two or more amino acid modifications that promote heteromultimeric formation). Where multiple heteromultimerization modifications are involved, in some aspects, each of the heteromultimerization modifications are the same type of modification (e.g., all amino acid modifications). In some aspects, one or more of the multiple heteromultimerization modifications are different types of modifications.

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

Heterodimeric Fc Proteins

[0107] As demonstrated herein, the heteromultimerization modifications described herein are particularly useful in producing heteromultimeric proteins. 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 not the same, and wherein the first polypeptide, the second polypeptide, or both the first and second polypeptides 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 and second polypeptides comprise a heteromultimerization modification.

CH 3 Domain

[0108] In some aspects, the heterodimeric protein comprises a Fc protein or a fragment thereof. For instance, 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 not the same, 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). 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 are such that the first CH3 domain and the second CH3 domain are capable of interacting with each other to form a heterodimer.

[0109] For instance, in some aspects, the heteromultimerization modification of the first CH3 domain comprises substituting 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 substituting 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 the interaction of the first and second CH3 domains. Unless indicated otherwise, positive and negative charge modifications useful for the present disclosure includes any amino acid modifications (e.g., substitutions) that create a positive and negative charge, respectively, and thereby, promoting the interaction between a polypeptide comprising the positive charge modification and a polypeptide comprising the negative charge modification.

[0110] In some aspects, a positive charge modification comprises the substitution of an amino acid with either a lysine (K) or arginine (R). In some aspects, a negative charge modification comprises the substitution of an amino acid with either an aspartic acid (D) or a glutamic acid (E). It will be apparent to those skilled in the arts that where a CH3 domain is modified to comprise a positive charge and an amino acid residue within the CH3 domain already has a positive charge (e.g., amino acid residue KI 90 in SEQ ID NO: 4 or SEQ ID NO: 16, in some aspects, the amino acid residue with the positive charge is not further modified. Similarly, wherein a CH3 domain is modified to comprise a negative charge and an amino acid residue within the CH3 domain already has a negative charge (e.g., amino acid residue DI 97 in SEQ ID NO: 4, SEQ ID NO: 16, or SEQ ID NO: 20), in some aspects, the amino acid residue with the negative charge is not further modified. Non-limiting examples of positive charge and negative charge modifications that can be made within a CH3 domain of a polypeptide provided herein (e.g., comprising the amino acid sequence set forth in SEQ ID NO: 4) are provided in Table 1 (below). Exemplary combinations of positive and negative charge modifications that can be introduced into a CH3 domain of a polypeptide described herein are provided in FIGs. 17A-17C and 18A-18D. Each of the different combinations of a first polypeptide (chain A; comprising a first CH3 domain) and second polypeptide (chain B; comprising a second CH3 domain) that are possible based on the combinations of positive and negative charge combinations provided in FIGs. 17A-17C and 18A- 18D are within the scope of the present disclosure. Accordingly, 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 and second CH3 domains comprise a combination of positive and negative charge modifications provided in any of FIGs. 17A-17C and 18A-18D, wherein the combination of the positive and negative charge modifications promote the interaction of the first and second CH3 domains.

Table 1. Exemplary Positive and Negative Charge Modifications

[0111] In some aspects, a 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 skilled in the arts, in some aspects, the first CH3 domain can comprise the negative charge modification and the second CH3 domain can comprise the positive charge modification. For instance, in some aspects, a 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. Accordingly, any heteromultimerization modifications described herein for a first polypeptide (e.g. , positive charge modification or negative charge modification) can be present on a second polypeptide, and any heteromultimerization modifications described herein for a second polypeptide (e.g., negative charge modification or positive charge modification) can be present on a first polypeptide, as long as the heteromultimerization modifications of the first and second polypeptides promote the interaction of the first polypeptide and the second polypeptide to form a heterodimer.

[0112] As described elsewhere in the present disclosure, a CH3 domain useful for the present disclosure can be derived from the heavy chain of any suitable antibody. In some aspects, the CH3 domain is that of an IgG antibody, IgA antibody, IgD antibody, IgE antibody, or IgM antibody. In some aspects, the CH3 domain is that of an IgG antibody (e.g., IgGl, IgG2, IgG3, or IgG4). For instance, in some aspects, the CH3 domain is that of an IgG4 antibody (referred to herein as "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, a 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., amino acid modification described herein) and the second polypeptide comprises a second IgG4 CH3 domain comprising a heteromultimerization domain (e.g., 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 not the same. For instance, 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 and negative charge modifications promote the interaction of the first IgG4 CH3 domain with the second IgG4 CH3 domain, and thereby, forming a 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 and negative charge modifications promote the interaction of the first IgG4 CH3 domain with the second IgG4 CH3 domain, and thereby, forming a heterodimeric protein.

[0114] In some aspects, a 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 not the same, wherein the first polypeptide comprises a first CH3 domain, which comprises 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, which comprises 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 the interaction of the first and second CH3 domains.

[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 residues T10, Li l, F65, Y67, or combinations thereof have been modified. For instance, 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). 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 T10V, L11Y, F65A, Y67V, or combinations 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, LI 1 Y, F65A, and Y67V. As is apparent from the present disclosure, amino acid residues T10, LI 1, F65, and Y67 of SEQ ID NO: 40 correspond to amino acid residues T148, L149, F203, and Y205 of SEQ ID NO: 4, respectively. Accordingly, in some aspects, a polypeptide of a heterodimeric protein (e.g., first polypeptide and/or 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 T148V, L149Y, F203A, Y205V, or combinations 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 residues T10, T26, K52, T54, or combinations thereof have 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). 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 T10V, T26L, K52L, T54W, or combinations 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. As is apparent from the present disclosure, amino acid residues T10, T26, K52, and T54 correspond to amino acid residues T148, T164, K190, and T192 of SEQ ID NO: 4, respectively. Accordingly, in some aspects, a polypeptide of a heterodimeric protein (e.g., first polypeptide and/or 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 T148V, T164L, K190L, T192W, or combinations 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 is a heterodimeric protein comprising a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide are not the same, 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, LI 1 Y, 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 promotes the interaction of the first and second CH3 domains. In some aspects, a heterodimeric protein comprises a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide are not the same, 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, LI 1 Y, 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, a heterodimeric protein provided herein comprises a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide are not the same, 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, F203 A, 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 that has a sequence identity of 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% to the amino acid sequence set forth in SEQ ID NO 24 or SEQ ID NO: 26. 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 not the same, 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, any of the first and second CH3 domains described above can comprise one or more additional modifications that help further promote heteromultimer formation. For instance, 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 combinations thereof have been modified.

[0120] In some aspects, the amino acid residue N50, K52, D59, S60, R69, or combinations thereof have been modified to comprise a positive charge (referred to herein as "positive charge modification"). For instance, in some aspects, the asparagine (N) at amino acid residue 50 has been modified to a lysine (K) or an arginine (R). In some aspects, the lysine (K) at amino acid residue 52 has not been modified or has been modified to an arginine (R). In some aspects, the aspartic acid (D) at amino acid residue 59 has been modified to a lysine (K) or an arginine (R). In some aspects, the serine (S) at amino acid residue 60 has been modified to a lysine (K) or an arginine (R). In some aspects, the arginine (R) at amino acid residue 69 has not been modified or has been modified to a 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) 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: 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) 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: 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) 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: 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 is apparent from the present disclosure, amino acid residues N50, K52, D59, S60, and R69 correspond to amino acid residues N188, K190, D197, S198, and R207 of SEQ ID NOs: 4, 16, or 20, respectively. Accordingly, in some aspects, a polypeptide of a heterodimeric protein comprises the amino acid sequence set forth in SEQ ID NO: 4 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). In some aspects, a polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 4 with 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, a polypeptide of a heterodimeric protein comprises the amino acid sequence set forth in SEQ ID NO: 16 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). In some aspects, a polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 16 with 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, a polypeptide of a heterodimeric protein 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). In some aspects, a polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 16 with 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 combinations thereof have been modified to comprise a negative charge (referred to herein as "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 has not been modified 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) 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: 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) 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: 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) 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: 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, a polypeptide of a heterodimeric protein described herein comprises the amino acid sequence set forth in SEQ ID NO: 4 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, a polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 4 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. In some aspects, a polypeptide of a heterodimeric protein described herein 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, a 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. In some aspects, a polypeptide of a heterodimeric protein described herein comprises the amino acid sequence set forth in SEQ ID NO: 20 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, a polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 20 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.

[0126] 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 not the same, 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 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) to (e), and (2) 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) 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 promotes the interaction of the first and second CH3 domains. In some aspects, (1) the first CH3 domain comprises the amino acid sequence set forth in SEQ ID NO: 40 with the following amino acid modifications: (a) N50K orN50R, (b) K52R, (c) D59K or D59R, (d) S60K or S60R, and (e) R69K„ and (2) 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, (e) R69D or R69E.

[0127] 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 not the same, 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 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) to (e), and (2) 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) 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 promotes the interaction of the first and second CH3 domains. In some aspects, (1) the first CH3 domain comprises the amino acid sequence set forth in SEQ ID NO: 24 with the following amino acid modifications: (a) N50K orN50R, (b) K52R, (c) D59K or D59R, (d) S60K or S60R, and (e) R69K„ and (2) 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.

[0128] 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 not the same, 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 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) to (e), and (2) 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) 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 promotes the interaction of the first and second CH3 domains. In some aspects, (1) the first 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, and (2) 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. [0129] In some aspects, the first CH3 domain or the second CH3 domain useful for the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 40, wherein amino acid residues T10, Li l, F65, Y67, T26, K52, T54, D59, R69, or combinations thereof have been modified. 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 D59R amino acid modification and one or more the following amino acid modifications: T10V, L11Y, F65A, Y67V, or combinations 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, 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 R69D amino acid modification and one or more of the following amino acid modifications: T10V, T26L, K52L, T54W, or combinations 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, a heterodimeric protein described herein comprises a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide are not the same, 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 D59R amino acid modification and one or more the following amino acid modifications: 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 R69D amino acid modification and one or more of the following amino acid modifications: T10V, T26L, K52L, T54W, or combinations 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 promotes the interaction of the first and second CH3 domains. In some aspects, a heterodimeric protein comprises a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide are not the same, 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 combinations 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, a heterodimeric protein described herein comprises a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide are not the same, 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 D59R amino acid modification and one or more the following amino acid modifications: 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 R69E amino acid modification and one or more of the following amino acid modifications: T10V, T26L, K52L, T54W, or combinations 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 promotes the interaction of the first and second CH3 domains. 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) the amino acid residue N50 of the first CH3 domain is modified to N50K or N50R and the amino acid residue S60 of the second CH3 domain is modified to S60D or S60E; (b) the amino acid residue S60 of the first CH3 domain is modified to S60D or S60E and the amino acid residue N50 of the second CH3 domain is modified toN50K orN50R; (c) the amino acid residue K52 of the first CH3 domain is not modified or modified to K52R and the amino acid residue F65 of the second CH3 domain is modified to F65D or F65E; (d) the amino acid residue D59 of the first CH3 domain is modified to D59K or D59R and the amino acid residue R69 of the second CH3 domain is modified to R69D or R69E; (e) the amino acid residue R69 of the first CH3 domain is modified R69D or R69E and the amino acid residue D59 of the second CH3 domain is modified D59K or D59R; or (f) any combination of (a) to (e). In some aspects, the amino acid residue N50 of the first CH3 domain is modified to N50K or N50R and the amino acid residue S60 of the second CH3 domain is modified to S60D or S60E. In some aspects, the amino acid residue S60 of the first CH3 domain is modified to S60D or S60E and the amino acid residue N50 of the second CH3 domain is modified to N50K or N50R. In some aspects, the amino acid residue K52 of the first CH3 domain is not modified or modified to K52R and the amino acid residue F65 of the second CH3 domain is modified to F65D or F65E. In some aspects, the amino acid residue D59 of the first CH3 domain is modified to D59K or D59R and the amino acid residue R69 of the second CH3 domain is modified to R69D or R69E. In some aspects, the amino acid residue R69 of the first CH3 domain is modified R69D or R69E and the amino acid residue D59 of the second CH3 domain is modified D59K or D59R.

[0135] In some aspects, the first CH3 domain or the second CH3 domain comprises an amino acid sequence that has a sequence identity of 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% 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 not the same, 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 not the same, 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.

Hinge Domain

[0136] As further described elsewhere in the present disclosure, in some aspects, a first polypeptide and/or second polypeptide further comprises a hinge domain. Accordingly, 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 not the same, and wherein: (a) the first polypeptide comprises (i) a first hinge domain and (ii) a first CH3 domain comprising a heteromultimerization modification (e.g., amino acid modifications 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., amino acid modifications described herein).

[0137] In some aspects, a hinge domain e.g., first hinge domain and/or second hinge domain) useful for the present disclosure can be derived from the heavy chain of any suitable antibody. In some aspects, the hinge domain is that of an IgG antibody, IgA antibody, IgD antibody, IgE antibody, or IgM antibody. In some aspects, the hinge domain is that of an IgG antibody (referred to herein as "IgG hinge domain"). For example, in some aspects, the hinge domain is that of an IgGl antibody ("IgGl hinge domain"). In some aspects, the hinge domain is that of an IgG2 antibody ("IgG2 hinge domain"). In some aspects, the hinge domain is that of an IgG3 antibody ("IgG3 hinge domain"). In some aspects, the hinge domain is that of an IgG4 antibody ("IgG4 hinge domain"). In some aspects, the hinge domain is that of an IgD antibody (referred to herein as "IgD hinge domain"). Exemplary amino acid sequences for 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., first hinge domain and/or second hinge domain) comprises an amino acid sequence that has a sequence identity of 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% to the amino acid sequence set forth in SEQ ID NO 22.. In some aspects, a hinge domain comprises the amino acid sequence set forth in SEQ ID NO: 22.

Linker

[0139] In some aspects, a hinge domain useful for the present disclosure comprises a linker, e.g., synthetic linker. Non-limiting examples of linkers include: m- maleimidobenzoyl-N- hydroxysuccinimide ester (MBS) (see, e.g., Ramakrishnan etal., Cancer Research 44(1): 201-208 (1984), which is incorporated herein by reference in its entirety); (i) EDC (l-ethyl-3-(3- dimethylamino -propyl) carbodiimide hydrochloride; (ii) SMPT (4- succinimidyloxycarbonyl- alpha-methyl-alpha-(2-pridyl-dithio)-toluene (Pierce Chem. Co., Cat. (21558G); (iii) SPDP (succinimidyl-6 [3-(2-pyridyldithio) propionamido]hexanoate (Pierce Chem. Co., Cat #21651G); (iv) Sulfo-LC-SPDP (sulfosuccinimidyl 6 [3-(2- pyridyldithio)-propianamide] hexanoate (Pierce Chem. Co. Cat. #2165-G); and (v) sulfo- NHS (N-hydroxysulfo-succinimide: Pierce Chem. Co., Cat. #24510) conjugated to EDC. [0140] In some aspects, a linker comprises a glycine, serine, alanine, or combinations thereof. Accordingly, in some aspects, a linker useful for the present disclosure comprises glycine polymers (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, a linker comprises glycine-serine polymers (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, a linker comprises glycine-alanine polymers (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, a linker comprises alanine-serine polymers (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 herein by reference in its entirety.

[0141] In some aspects, a hinge domain comprises a glycine/ serine linker according to the formula [(Gly)n-Ser]m (SEQ ID NO: 44) where n is any integer from 1 to 100 and m is any integer from 1 to 100. In some aspects, the glycine/serine linker is according to the formula [(Gly)x- (Ser)y]z (SEQ ID NO: 45) wherein x in an integer from 1 to 4, y is 0 or 1, and z is an integers from 1 to 50. In some aspects, the optional linker comprises the sequence (G)n (SEQ ID NO: 46), where 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 for the present disclosure are described, e.g., in US 2021/0206806 and US 2017/0114151, each of which is incorporated herein by reference 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 inker 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 specific aspects, the hinge domain (e.g., linker) is between 1 and 10 amino acids in length. In some aspects, the hinge domain (e.g., linker) is between about 5 and about 10, between about 10 and about 20, between about 20 and about 30, between about 30 and about 40, between about 40 and about 50, between about 50 and about 60, between about 60 and about 70, between about 70 and about 80, between about 80 and about 90, or between about 90 and about 100 amino acids in length.

[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, an 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, a linker useful for the present disclosure comprises an Internal Ribosome Entry Site (IRES), such that separate polypeptides encoded by the first and second genes are produced during translation. Additional description of linkers that can be used with the present disclosure are provided, e.g., in WO 2020/223625 Al and US 2019/0276801 Al, each of which is incorporated herein by reference in its entirety.

Table 2: Linker Sequences

[0147] 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 not the same, wherein: (a) the first polypeptide comprises (i) a first hinge domain selected from an IgGl hinge domain, IgG2 hinge domain, IgG3 hinge domain, IgG4 hinge domain, IgD hinge domain, synthetic linker, or combinations thereof, and (ii) a first CH3 domain comprising a heteromultimerization modification (e.g., amino acid modifications described herein); and (b) the second polypeptide comprises (i) a second hinge domain selected from an IgGl hinge domain, IgG2 hinge domain, IgG3 hinge domain, IgG4 hinge domain, IgD hinge domain, synthetic linker, or combinations thereof, and (ii) a second CH3 domain comprising a heteromultimerization modification e.g., amino acid modifications 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 not the same.

[0148] 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 not the same, 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, LI 1 Y, F65A, Y67V, or combinations 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 combinations thereof. In some aspects, a heterodimeric protein comprises a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide are not the same, 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, LI 1 Y, 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 is a heterodimeric protein comprising a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide are not the same, 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) 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: 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) 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 promotes the interaction of the first and second CH3 domains. 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 not the same, 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 is a heterodimeric protein comprising a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide are not the same, 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 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) 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 with one or more amino acid modifications selected from: (a) N50D or N50E, (b) D59E, (c) S60D or S60E, (d) F65D or F65 E, (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 promotes the interaction of the first and second CH3 domains. 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 not the same, 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 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: 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.

[0151] 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 not the same, 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 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) 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 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) 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 promotes the interaction of the first and second CH3 domains. 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 not the same, 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 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: 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.

[0152] In some aspects, a 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 with D59R amino acid modification and one or more the following amino acid modifications: T10V, LI 1 Y, F65A, Y67V, or combinations 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 R69D amino acid modification and one or more of the following amino acid modifications: T10V, T26L, K52L, T54W, or combinations thereof. In some aspects, a 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 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, a 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 with D59R amino acid modification and one or more the following amino acid modifications: T10V, LI 1 Y, F65A, Y67V, or combinations 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 R69E amino acid modification and one or more of the following amino acid modifications: T10V, T26L, K52L, T54W, or combinations thereof. In some aspects, a 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 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: R69E, T10V, T26L, K52L, and T54W. CH 2 Domain

[0154] In some aspects, a first polypeptide and/or second polypeptide provided herein (e.g. , comprising a CH3 domain with one or more heteromultimerization modifications) further comprises a CH2 domain. The CH2 domain useful for the present disclosure can be derived from the heavy chain of any suitable antibody. For example, in some aspects, the CH2 domain is that of an IgG antibody ("IgG CH2 domain"), IgA antibody ("IgA CH2 domain"), IgD antibody ("IgD CH2 domain"), IgE antibody ("IgE CH2 domain"), or IgM antibody ("IgM CH2 domain"). In some aspects, the CH2 domain comprises CH2 domains of multiple antibodies. For example, in some aspects, the CH2 domain comprises a part of human IgD CH2 domain and a part of human IgG4 CH2 domain (referred to herein as "IgD/IgG4 CH2 domain"). An exemplary amino acid sequence of the 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 that has a sequence identity of 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% 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] As is apparent from the present disclosure, 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 not the same, wherein: (a) the first polypeptide comprises (i) a first CH2 domain (e.g., IgD/IgG4 CH2 domain), and (ii) a first CH3 domain comprising a heteromultimerization modification (e.g., an amino acid modification described herein, e.g., positive and/or negative charge modifications described herein); and (b) the second polypeptide comprises (i) a second CH2 domain (e.g., IgD/IgG4 CH2 domain), and (ii) a second CH3 domain comprising a heteromultimerization modification (e.g., an amino acid modification described herein, e.g., negative and/or positive charge modifications 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 not the same.

[0157] 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 not the same, wherein: (a) the first polypeptide comprises (i) a first hinge domain (e.g., selected from an IgGl hinge domain, IgG2 hinge domain, IgG3 hinge domain, IgG4 hinge domain, IgD hinge domain, synthetic linker, or combinations thereof), (ii) a first CH2 domain (e.g., IgD/IgG4 CH2 domain), and (iii) a first CH3 domain comprising a heteromultimerization modification (e.g., an amino acid modification described herein, e.g., positive and/or negative charge modifications described herein); and (b) the second polypeptide comprises (i) a second hinge domain (e.g., selected from an IgGl hinge domain, IgG2 hinge domain, IgG3 hinge domain, IgG4 hinge domain, IgD hinge domain, synthetic linker, or combinations thereof), (ii) a second CH2 domain (e.g., IgD/IgG4 CH2 domain), and (iii) a second CH3 domain comprising a heteromultimerization modification (e.g., an amino acid modification described herein, e.g., negative and/or positive charge modifications described herein).

[0158] 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 not the same, 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, LI 1 Y, 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 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.

[0159] 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 not the same, 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) to (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) 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 promotes the interaction of the first and second CH3 domains. 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 is a heterodimeric protein comprising a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide are not the same, 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) to (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 F65 E, (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 promotes the interaction of the first and second CH3 domains. 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 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.

[0161] 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 not the same, 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) to (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) 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 promotes the interaction of the first and second CH3 domains. 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 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.

[0162] In some aspects, a heterodimeric 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 with D59R amino acid modification and one or more the following amino acid modifications: T10V, LI 1 Y, 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 R69D amino acid modification and one or more of the following amino acid modifications: 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: 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 with the following amino acid modifications: R69D, T10V, T26L, K52L, and T54W. 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.

[0163] In some aspects, a heterodimeric 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 with D59R amino acid modification and one or more the following amino acid modifications: T10V, LI 1 Y, 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 R69E amino acid modification and one or more of the following amino acid modifications: 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: 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 with the following amino acid modifications: R69E, T10V, T26L, K52L, and T54W. 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.

[0164] 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 not the same, 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 modification selected from 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 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, a heterodimeric protein provided herein comprises a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide are not the same, wherein: (1) the first polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 4 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: 4 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: 4 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: 4 with the following amino acid modifications: (a) N188D orN188E, (b) D197E, (c) S198D or S198E, (d) F203D orF203E, and (e) R207D orR207E. [0166] 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 not the same, wherein: (1) the first polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 16 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: 20 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: 16 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: 20 with the following amino acid modifications: (a) N188D orN188E, (b) D197E, (c) S198D or S198E, (d) F203D orF203E, and (e) R207D orR207E. [0167] 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 not the same, 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 orN188E, (b) D197E, (c) S198D or S198E, (d) F203D orF203E, and (e) R207D orR207E. [0168] In some aspects, heterodimeric protein provided herein comprises a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide are not the same, wherein: (1) the first polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 4 with D197R amino acid modification and one or more the following amino acid modifications: 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 R207D amino acid modification and one or more of the following amino acid modifications: T148V, T164L, K190L, T192W, or combinations 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, heterodimeric protein provided herein comprises a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide are not the same, wherein: (1) the first polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 4 with D197R amino acid modification and one or more the following amino acid modifications: 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 R207E amino acid modification and one or more of the following amino acid modifications: T148V, T164L, K190L, T192W, or combinations 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, a heterodimeric protein provided herein comprises a first polypeptide and a second polypeptide, wherein (a) the first polypeptide comprises an amino acid sequence that has a sequence identity of 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% to the amino acid sequence set forth in SEQ ID NO 16; and (b) the second polypeptide comprises an amino acid sequence that has a sequence identity of 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% to the amino acid sequence set forth in SEQ ID NO 20. 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: 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 that has a sequence identity of 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% to the amino acid sequence set forth in SEQ ID NO 27; and (b) the second polypeptide comprises an amino acid sequence that has a sequence identity of 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% 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 that has a sequence identity of 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% to the amino acid sequence set forth in SEQ ID NO 27; and (b) the second polypeptide comprises an amino acid sequence that has a sequence identity of 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% 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.

Additional Moiety

[0173] 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 comprises any molecules 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, the one or more additional moieties can be conjugated directly to the heterodimeric protein (e.g., fused directly to the heterodimeric protein). In some aspects, the one or more additional moieties can be conjugated to the heterodimeric protein via a linker (e.g., synthetic linkers described herein). Unless indicated otherwise, the expression "conjugated to the heterodimeric protein" (or variants thereof) comprises both direct conjugation (e.g., fused directly to the heterodimeric protein) and conjugation via a linker.

[0175] For instance, 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 not the same, wherein: (a) the first polypeptide comprises a first CH3 domain (e.g., IgG4 CH3 domain) comprising a heterodimerization modification (e.g., an amino acid modification described herein), and (b) the second polypeptide comprise a second CH3 domain (e.g., 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). Where multiple (e.g., two or more) additional moieties are attached, in some aspects, (a) the 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) the second additional moiety is conjugated to the first additional moiety (directly or via a linker). It will be apparent to those skilled in the arts 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, a 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 not the same, wherein: (a) the first polypeptide comprises (i) a first hinge domain (e.g., selected from an IgGl hinge domain, IgG2 hinge domain, IgG3 hinge domain, IgG4 hinge domain, IgD hinge domain, synthetic linker, or combinations thereof), and (ii) a first CH3 domain (e.g., 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, IgG2 hinge domain, IgG3 hinge domain, IgG4 hinge domain, IgD hinge domain, synthetic linker, or combinations thereof), and (ii) a second CH3 domain (e.g., 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) the second additional moiety is conjugated to the first additional moiety (directly or via a linker).

[0177] In some aspects, a 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 not the same, wherein: (a) the first polypeptide comprises (i) a first CH2 domain (e.g, IgD/IgG4 CH2 domain) and (ii) a first CH3 domain (e.g., 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., IgD/IgG4 CH2 domain) and (ii) a second CH3 domain (e.g., 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) the second additional moiety is conjugated to the first additional moiety (directly or via a linker).

[0178] In some aspects, a 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 not the same, wherein: (a) the first polypeptide comprises (i) a first hinge domain (e.g., selected from an IgGl hinge domain, IgG2 hinge domain, IgG3 hinge domain, IgG4 hinge domain, IgD hinge domain, synthetic linker, or combinations thereof), (ii) a first CH2 domain (e.g., IgD/IgG4 CH2 domain), and (iii) a first CH3 domain (e.g., 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, IgG2 hinge domain, IgG3 hinge domain, IgG4 hinge domain, IgD hinge domain, synthetic linker, or combinations thereof), (ii) a second CH2 domain (e.g., IgD/IgG4 CH2 domain), and (iii) a second CH3 domain (e.g., 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) the second additional moiety is conjugated to the first additional moiety (directly or via a linker).

[0179] For any of the aspects described above involving multiple (e.g., two or more) additional moieties, in some aspects, each of the additional moieties are the same. In some aspects, one or more of the additional moieties are different. In some aspects, a 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 the C-terminus). In some aspects, all of the additional moi eties are conjugated to the second polypeptide (e.g., at the N-terminus and/or the C-terminus). In some aspects, some (e.g., at least one) of the additional moi eties are conjugated to the first polypeptide e.g., at the N-terminus and/or the C-terminus) and some e.g., at least one) of the additional moi eties are conjugated to the second polypeptide e.g., at the N-terminus and/or the 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) the second additional moiety is conjugated to the first additional moiety (directly or 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., the N-terminus and/or the C-terminus) and four of the additional moieties are attached to the second polypeptide (e.g., the N-terminus and/or the C-terminus).

[0180] In some aspects, an 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 has activity 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 part thereof, such as a Fv fragment, which antibody or part 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, cDNA, genomic DNA, an artificial or natural chromosome (e.g. a yeast artificial chromosome) or a part thereof, RNA, including mRNA, tRNA, rRNA or a ribozyme, or a peptide nucleic acid (PNA); a virus or viruslike particles; a nucleotide or ribonucleotide or synthetic analogue thereof, which may be modified or unmodified; an amino acid or analogue thereof, which may be modified or unmodified; a nonpeptide (e.g., 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, a heterodimeric protein provided herein is conjugated to multiple ligand-binding proteins. For instance, in some aspects, a 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. Wherein a heterodimeric protein is conjugated to multiple ligand-binding proteins, the multiple-ligand binding proteins can specifically bind to the same molecule (e.g., antigen), which can help improve the binding affinity. In some aspects, one or more of the multiple-ligand binding proteins can bind to different molecules (e.g., different antigens), allowing the heterodimeric protein to exhibit multispecificity.

[0182] In some aspects, a ligand-binding protein useful for the present disclosure is an antibody. Non-limiting examples of such antibodies include: a single domain antibody (sdAB), an Ig NAR, 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, or combinations thereof. In some aspects, the ligand-binding protein is a sdAb. Nonlimiting examples of sdAb that are useful for the present disclosure include: human heavy chain only antibody, rabbit single domain antibody, camelid VHH, shark heavy chain only antibody (VNAR), or combinations thereof.

[0183] In some aspects, a ligand-binding protein comprises a T cell engager (e.g., bispecific T-cell engager (BiTE) antibody), dual-affinity retargeting molecule (DART), CrossMAb antibody, DutaMab™ antibody, DuoBody antibody, Triomab, TandAb, bispecific NanoBody, Tandem scFv, diabody, single chain diabody, HSA body, (scFv)2 HSA Antibody, scFv-IgG antibody, Dock and Lock bispecific antibody, DVD-IgG antibody, TBTI DVD-IgG, IgG-fynomer, Tetravalent bispecific tandem IgG antibody, dual targeting domain antibody, chemically linked bispecific (Fab')2 molecule, crosslinked mAb, Dual-action Fab IgG (DAF-IgG), orthoFab-IgG, bispecific CovX-Body, bispecific hexavalent trimerbody, 2 scFv linked to diphtheria toxin, ART- Ig, IgM T-cell engager, Humabody™ human heavy-chain only antibody (HCAb), UniAb™ HCAb, shark heavy chain-only antibody (VNAR), or combinations thereof.

[0184] As will be apparent from the present disclosure, a ligand-binding protein useful for the present disclosure can bind to various molecules known in the art. In some aspects, a ligandbinding protein binds to a tumor antigen. Non-limiting examples of such tumor antigens include a 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 (F0LR1), CD4, CD 19, 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, LI CAM, AXL, Tissue Factor (TF), CD74, EpCAM, EphA2, MRP3cadherin 19 (CDH19), epidermal growth factor 2 (FIER2), 5T4, 8H9, anbo 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, Mucl6, NCAM, NKG2D ligands, NY-ESO-1, PRAME, PSC1, PSCA, PSMA, R0R1, R0R2, SP17, surviving, TAG72, TEMs, carcinoembryonic antigen, HMW-MAA, VEGF, CLDN18.2, programmed death-ligand 1 (PDL-1), or combinations thereof.

[0185] In some aspects, a ligand binding protein binds to an antigen expressed on an immune cell. In some aspects, an antigen expressed on an immune cell comprises a checkpoint molecule. In some aspects, an antigen expressed on an immune cell comprises a stimulatory molecule. Non-limiting examples of such antigens include CD3, CD27, B7H3, CD2, CD4, CD5, CD8, CDl lb, 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, 0X40, CD40L, ICOS, LIGHT, or combinations thereof.

[0186] Accordingly, 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 not the same, 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., negative and/or positive charge modifications 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 further described herein, in some aspects, the first ligand-binding protein (i.e., binds to a tumor antigen) can be instead conjugated to the second polypeptide and the second ligand-binding protein (z.e., binds to an antigen on an immune cell) can be conjugated to the first polypeptide.

III. Bispecific/Multispecific Ligand-Binding Proteins

[0187] As is apparent from at least the above, some aspects of the present disclosure is directed to a bispecific ligand-binding protein (e.g., bispecific antibody). In some aspects, the bispecific ligand-binding protein comprises: (a) first antigen-binding domain, (b) a second antigenbinding 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 antigen-binding domain and the second antigen-binding domain target different tumor antigens. In some aspects, the first antigen-binding domain and the second antigen-binding domain target different antigens on immune cells. 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] Some aspects of the present disclosure relates to a multispecific ligand-binding protein (e.g., multispecific antibody). For instance, in some aspects, a multispecific ligand-binding protein of the present disclosure comprises (a) a first antigen-binding domain, (b) a second antigenbinding domain, (c) a third antigen-binding domain, and (d) a heterodimeric protein described herein, wherein the first antigen-binding domain, the second antigen-binding domain, and the third 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, 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 conjugated to the N-terminus and/or C-terminus of the first polypeptide, and the second antigen — binding domain 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 are conjugated to the heterodimeric protein.

[0190] In some aspects, the first antigen-binding domain, the second antigen-binding domain, and the third antigen-binding domain target different tumor antigens. In some aspects, the first antigen-binding domain, the second antigen-binding domain, and the third antigen-binding domain target different antigens expressed on immune cells. 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 immune cells. 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 present disclosure, any of the additional moi eties provided herein (e.g., antigen-binding domain) can be conjugated directly to the heterodimeric protein. In some aspects, the additional moi eties (e.g., antigen-binding domain) are conjugated to the heterodimeric protein using a linker. Non-limiting examples of useful linkers are provided elsewhere in the present disclosure.

[0193] Accordingly, in some aspects, a multispecific ligand-binding protein provided herein 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 (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 by a first linker, the second antigen-binding domain is conjugated to the N-terminus of the second polypeptide by a second linker, and the third antigenbinding domain is conjugated to the N-terminus of the second polypeptide by a third linker, wherein the first linker, second linker, and third linker are selected from an IgGl hinge, IgG2 hinge, IgG3 hinge, IgG4 hinge, IgD hinge, or synthetic linker. In some aspects, a multispecific ligandbinding protein provided herein comprises (a) a first antigen-binding domain, (b) a second antigenbinding 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 antigenbinding domain is conjugated to the N-terminus of the first polypeptide by a first linker, the second antigen-binding domain is conjugated to the C-terminus of the first polypeptide by a second linker, and the third antigen-binding domain is conjugated to the C-terminus of the second polypeptide by a third linker, and wherein the first linker, second linker, and third linker are selected from an IgGl hinge, IgG2 hinge, IgG3 hinge, IgG4 hinge, IgD hinge, or synthetic linker.

IV Conjugates

[0194] Some aspects of the present disclosure relates to a conjugate comprising any of the heterodimeric proteins, fusion proteins, bispecific ligand-binding proteins (e.g., bispecific antibody), or multispecific ligand-binding proteins (e.g., multispecific antibody) linked to one or more conjugate moieties. Accordingly, in some aspects, provided herein is a conjugate comprising a heterodimeric protein described herein linked to a conjugate moiety. In some aspects, provided herein is a conjugate comprising a fusion protein described herein linked to a conjugate moiety. In some aspects, provided herein is a conjugate comprising a bispecific ligand-binding protein described herein linked to a conjugate moiety. In some aspects, provided herein is a conjugate comprising a multispecific ligand-binding protein described herein linked to a conjugate moiety.

[0195] Non-limiting examples of such conjugate moieties include: a clearance-modifying agent, a chemotherapeutic agent, a toxin, a radioactive isotope, a lanthanide, a luminescent label, a fluorescent label, an enzyme-substrate label, a DNA-alkylators, a topoisomerase inhibitor, a tubulin-binders, or an anticancer drug.

[0196] Conjugates useful for the present disclosure can be prepared by any suitable methods known in the art. In some aspects, conjugation methods result in linkages which are substantially (or nearly) non-immunogenic, e.g., peptide- (z.e., amide-), sulfide-, (sterically hindered), disulfide-, hydrazone-, and ether linkages. These linkages are nearly non-immunogenic and show reasonable stability within serum (see, e.g., Senter, P. D., Curr. Opin. Chem. Biol. 13 (2009) 235-244; WO 2009/059278; WO 95/17886, each of which is incorporated herein by reference in its entirety).

[0197] Depending on the biochemical nature of the molecules 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 reaction and covalent coupling is based on transforming a natural amino acid into an amino acid with a reactivity which is orthogonal to the reactivity of the other functional groups present. For example, a specific cysteine within a rare sequence context can be enzymatically converted in an aldehyde see Frese, M. A., and Dierks, T., ChemBioChem. 10 (2009) 425-427). It is also possible to obtain a desired amino acid modification by utilizing the specific enzymatic reactivity of certain enzymes with a natural amino acid in a given sequence context (see, e.g., Taki, M. et al., Prot. Eng. Des. Sei. 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 reaction and covalent coupling can also be achieved by the selective reaction of terminal amino acids with appropriate modifying reagents. The reactivity of an N- terminal cysteine with benzonitrils (see Ren, H. et al. , Angew. Chem. Int. Ed. Engl. 48 (2009) 9658- 9662) can be used to achieve a site-specific covalent coupling. Native chemical ligation can also rely on C-terminal cysteine residues (Taylor, E. Vogel; Imperiali, B, Nucleic Acids and Molecular Biology (2009), 22 (Protein Engineering), 65-96).

[0199] The conjugate moiety can also be a synthetic peptide or peptide mimic. In such cases, a polypeptide can be chemically synthesized, amino acids with orthogonal chemical reactivity can be incorporated during such synthesis (see, e.g., de Graaf, A. J. et al., Bioconjug. Chem. 20 (2009) 1281-1295). To obtain a mono-labeled polypeptide, the conjugate with 1 : 1 stoichiometry can be separated by chromatography from other conjugation side-products. This procedure can be facilitated using a dye labeled binding pair member and a charged linker. With this kind of labeled and highly negatively charged binding pair member, mono conjugated polypeptides are easily separated from non-labeled polypeptides and polypeptides which carry more than one linker, since the difference in charge and molecular weight can be used for separation. The fluorescent dye can be useful for purifying the complex from un-bound components, like a labeled monovalent binder.

IV Pharmaceutical Compositions

[0200] Also provided herein are compositions comprising any of the heterodimeric proteins, fusion proteins, bispecific ligand-binding proteins, multispecific ligand-binding proteins, conjugates, nucleic acids, vectors, and/or cells described herein and having the desired degree of purity in a physiologically acceptable carrier, excipient or stabilizer (Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA). Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; 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, di saccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionic surfactants such as TWEEN®, PLURONICS® or polyethylene glycol (PEG).

[0201] In some aspects, a pharmaceutical composition useful for the present disclosure comprises any of the heterodimeric proteins, fusion proteins, bispecific ligand-binding proteins, multispecific ligand-binding proteins, conjugates, nucleic acids, vectors, and/or cells described herein, and optionally one or more additional prophylactic or therapeutic agents, in a pharmaceutically acceptable carrier. In some aspects, the heterodimeric proteins, fusion proteins, bispecific ligand-binding proteins, multispecific ligand-binding proteins, conjugates, nucleic acids, vectors, and/or cells described herein are the only active ingredient included in the pharmaceutical composition. As described elsewhere in the present disclosure, such pharmaceutical compositions can be useful in treating a wide range of diseases or conditions in a subject in need thereof.

[0202] Pharmaceutically acceptable carriers used in parenteral preparations include aqueous vehicles, nonaqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, sequestering or chelating agents and other pharmaceutically acceptable substances. Examples of aqueous vehicles include sodium chloride injection, ringers injection, isotonic dextrose injection, sterile water injection, dextrose and lactated ringers injection. Nonaqueous parenteral vehicles include fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil and peanut oil. Antimicrobial agents in bacteriostatic or fungistatic concentrations can be added to parenteral preparations packaged in multiple-dose containers which include phenols or cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride and benzethonium chloride. Isotonic agents include sodium chloride and dextrose. Buffers include phosphate and citrate. Antioxidants include sodium bisulfate. Local anesthetics include procaine hydrochloride. Suspending and dispersing agents include sodium carboxymethylcelluose, hydroxypropyl methylcellulose and polyvinylpyrrolidone. Emulsifying agents include Polysorbate 80 (TWEEN® 80). A sequestering or chelating agent of metal ions includes EDTA. Pharmaceutical carriers also include ethyl alcohol, polyethylene glycol and propylene glycol for water miscible vehicles; and sodium hydroxide, hydrochloric acid, citric acid or lactic acid for pH adjustment.

[0203] A pharmaceutical composition can be formulated for any route of administration to a subject. Specific examples of routes of administration include intramuscularly, parenthetically, subcutaneously, ophthalmic, intravenously, intraperitoneally, intradermally, intraorbitally, intracerebrally, intracranially, intraspinally, intraventricular, intrathecally, intraci stemally, intracapsularly, or intratumorally. Parenteral administration, characterized by either subcutaneous, intramuscular or intravenous injection, is also contemplated herein. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. The injectables, solutions and emulsions also contain one or more excipients. Suitable excipients are, for example, water, saline, dextrose, glycerol or ethanol. In addition, if desired, the pharmaceutical compositions to be administered can also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers, and other such agents, such as for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate and cyclodextrins.

[0204] Preparations for parenteral administration of a polypeptide described herein include sterile solutions ready for injection, sterile dry soluble products, such as lyophilized powders, ready to be combined with a solvent just prior to use, including hypodermic tablets, sterile suspensions ready for injection, sterile dry insoluble products ready to be combined with a vehicle just prior to use and sterile emulsions. The solutions can be either aqueous or nonaqueous.

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

[0206] Topical mixtures comprising a polypeptide described herein are prepared as described for the local and systemic administration. The resulting mixture can be a solution, suspension, emulsions or the like and can be formulated as creams, gels, ointments, emulsions, solutions, elixirs, lotions, suspensions, tinctures, pastes, foams, aerosols, irrigations, sprays, suppositories, bandages, dermal patches or any other formulations suitable for topical administration.

[0207] A pharmaceutical composition can be formulated as an aerosol for topical application, such as by inhalation (see, e.g., U.S. Patent Nos. 4,044,126, 4,414,209 and 4,364,923). These formulations for administration to the respiratory tract can be in the form of an aerosol or solution for a nebulizer, or as a microfine powder for insufflations, alone or in combination with an inert carrier such as lactose. In such a case, the particles of the formulation can, in some aspects, have diameters of less than about 50 microns, e.g., less than about 10 microns.

[0208] A pharmaceutical composition can be formulated for local or topical application, such as for topical application to the skin and mucous membranes, such as in the eye, in the form of gels, creams, and lotions and for application to the eye or for intracistemal or intraspinal application. Topical administration is contemplated for transdermal delivery and also for administration to the eyes or mucosa, or for inhalation therapies. Nasal solutions of the antibody alone or in combination with other pharmaceutically acceptable excipients can also be administered.

[0209] Transdermal patches, including iontophoretic and electrophoretic devices, are well known to those of skill in the art, and can be used to administer any of the polypeptides, molecules, nucleic acids, vectors, cells, or protein-conjugates described herein. For example, such patches are disclosed in U.S. Patent Nos. 6,267,983, 6,261,595, 6,256,533, 6,167,301, 6,024,975, 6,010715, 5,985,317, 5,983,134, 5,948,433, and 5,860,957.

[0210] In some aspects, a pharmaceutical composition described herein is a lyophilized powder, which can be reconstituted for administration as solutions, emulsions and other mixtures. It can also be reconstituted and formulated as solids or gels. The lyophilized powder is prepared by dissolving any of the polypeptides, molecules, nucleic acids, vectors, cells, or proteinconjugates described herein, or a pharmaceutically acceptable derivative thereof, in a suitable solvent. In some aspects, the lyophilized powder is sterile. The solvent can contain an excipient which improves the stability or other pharmacological component of the powder or reconstituted solution, prepared from the powder. Excipients that can be used include, but are not limited to, dextrose, sorbitol, fructose, com syrup, xylitol, glycerin, glucose, sucrose or other suitable agent. The solvent can also contain a buffer, such as citrate, sodium, or potassium phosphate or other such buffer known to those of skill in the art. In some aspects, the buffer is at about neutral pH. Subsequent sterile filtration of the solution followed by lyophilization under standard conditions known to those of skill in the art provides the desired formulation. In some aspects, the resulting solution can be apportioned into vials for lyophilization. Each vial can contain a single dosage or multiple dosages of the compound (e.g., any of the polypeptides, molecules, nucleic acids, vectors, cells, or protein-conjugates). The lyophilized powder can be stored under appropriate conditions, such as at about 4°C to room temperature. [0211] Reconstitution of this lyophilized powder with water for injection provides a formulation for use in parenteral administration. For reconstitution, the lyophilized powder is added to sterile water or other suitable carrier. The precise amount depends upon the selected compound. Such amount can be empirically determined.

[0212] In some aspects, a pharmaceutical composition comprising any of the heterodimeric proteins, fusion proteins, bispecific ligand-binding proteins, multispecific ligand-binding proteins, conjugates, nucleic acids, vectors, and/or cells described herein can also be formulated to be targeted to a particular tissue, receptor, or other area of the body of the subject to be treated. For non-limiting examples of targeting methods, see, e.g., 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 compositions to be used for in vivo administration can be sterile. In some aspects, this can be accomplished by filtration through, e.g., sterile filtration membranes.

V. Nucleic Acids, Vectors, Host Cells

[0214] Further aspect described herein pertains to one or more nucleic acid molecules (also referred to herein as "nucleic acids" or derivatives thereof) that encode a polypeptide or a molecule described herein (e.g., heterodimeric proteins, fusion proteins, bispecific ligand-binding proteins, multispecific ligand-binding proteins, conjugates). The nucleic acids can be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form. In some aspects, the nucleic acid is a DNA sequence and/or an RNA sequence (e.g., mRNA). In some aspects, the nucleic acids comprise a modified nucleotide analog. A nucleic acid is "isolated" or "rendered substantially pure" when purified away from other cellular components or other contaminants, e.g., other cellular nucleic acids (e.g., other chromosomal DNA, e.g., the chromosomal DNA that is linked to the isolated DNA in nature) or proteins, by standard techniques, including alkaline/SDS treatment, CsCl banding, column chromatography, restriction enzymes, agarose gel electrophoresis and others well known in the art. See, F. Ausubel, et al. , ed. (1987) Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York. In some aspects, a nucleic acid molecule can or cannot contain intronic sequences. In some aspects, the nucleic acid is a cDNA molecule. Nucleic acids described herein can be obtained using standard molecular biology techniques known in the art.

[0215] In some aspects, the present disclosure provides a vector comprising an isolated nucleic acid molecule encoding a polypeptide or a molecule disclosed herein (e.g., heterodimeric proteins, fusion proteins, bispecific ligand-binding proteins, multispecific ligand-binding proteins, conjugates). Suitable vectors for the 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, an "expression vector" refers to any nucleic acid construct which contains the necessary elements for the transcription and translation of an inserted coding sequence, or in the case of a RNA viral vector, the necessary elements for replication and translation, when introduced into an appropriate host cell. Expression vectors can include plasmids, phagemids, viruses, and derivatives thereof.

[0217] As used herein, "viral vectors" include, but are not limited to, nucleic acid sequences from the following viruses: retrovirus, such as Moloney murine leukemia virus, Harvey murine sarcoma virus, murine mammary tumor virus, and Rous sarcoma virus; lentivirus; adenovirus; adeno-associated virus; SV40-type viruses; polyomaviruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus. Certain viral vectors are based on non-cytopathic eukaryotic viruses in which non-essential genes have been replaced with the gene of interest. Non-cytopathic viruses include retroviruses, the life cycle of which involves reverse transcription of genomic viral RNA into DNA with subsequent proviral integration into host cellular DNA.

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

[0219] Other vectors include plasmid vectors. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989. In the last few years, plasmid vectors have been found to be particularly advantageous for delivering genes to cells in vivo because of their inability to replicate within and integrate into a host genome. These plasmids, however, having a promoter compatible with the host cell, can express a peptide from a gene operably encoded within the plasmid. Some commonly used plasmids available from commercial suppliers include pBR322, pUC18, pUC19, various pcDNA plasmids, pRC/CMV, various pCMV plasmids, pSV40, and pBlueScript. Additional 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 from Invitrogen (Carlsbad, CA.). Additionally, plasmids can be custom designed using standard molecular biology techniques to remove and/or add specific fragments of DNA.

VI. Kits

[0220] Also provided herein are kits comprising any of the heterodimeric proteins, fusion proteins, bispecific ligand-binding proteins, multispecific ligand-binding proteins, conjugates, nucleic acids, vectors, cells, and/or pharmaceutical compositions described herein. In some aspects, provided herein is a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions described herein, such as one or more polypeptides provided herein, optional an instruction for use. In some aspects, the kits contain a pharmaceutical composition described herein and any prophylactic or therapeutic agent, such as those described herein.

VII. Methods of the Disclosure

Methods of Making

[0221] Also encompassed by the present disclosure is a method for producing/making a heterodimeric protein described herein. In some aspects, such a method can comprise expressing the heterodimeric protein in a cell comprising a nucleic acid molecule encoding the protein or a vector comprising the nucleic acid molecule. Host cells comprising these nucleotide sequences are encompassed herein. Non-limiting examples of host cell that can be used include immortal hybridoma cell, NS/0 myeloma cell, 293 cell, Chinese hamster ovary (CHO) cell, HeLa cell, human amniotic fluid-derived cell (CapT cell), COS cell, bacterial cell, insect cell, plant cell, yeast cell, or combinations thereof.

[0222] Related to the above methods of producing/making a heterodimeric protein, some aspects of the present disclosure is related to methods of producing a fusion protein. For instance, in some aspects, the present disclosure provides a method of producing a fusion protein, wherein the method comprises conjugating any of the additional moi eties described herein (e.g., biologically active molecules) to a heterodimeric protein described herein. Any suitable methods known in the art can be used in conjugating the additional moieties to the heterodimeric protein. In some aspects, a method of producing a fusion protein, comprises directly conjugating the additional moieties to the heterodimeric protein (e.g., fused to the N-terminus and/or C-terminus of the first polypeptide or the second polypeptide). In some aspects, a method of producing a fusion protein, comprises conjugating the additional moieties to the heterodimeric protein using a linker (e.g., conjugated via a linker to the N-terminus and/or C-terminus of the first polypeptide or the second polypeptide). Non-limiting examples of linkers that can be used are provided elsewhere in the present disclosure (e.g., IgGl hinge, IgG2 hinge, IgG3 hinge, IgG4 hinge, IgD hinge, or synthetic linker).

Therapeutic Uses

[0223] As will be apparent to those skilled in the arts, a heterodimeric protein described herein can be particularly useful in treating a wide range of diseases or conditions in a subject in need thereof. For example, in some aspects, a heterodimeric protein described herein can be conjugated (e.g., directly or via a linker) to an additional moiety described herein (e.g., biologically active molecule, e.g., ligand-binding protein) to form any of the fusion proteins, bispecific ligandbinding proteins, multispecific ligand-binding proteins, conjugates, polynucleotides encoding such proteins, and/or pharmaceutical compositions comprising such proteins and/or polynucleotides (collectively referred to herein as "therapeutic composition"), which can be administered to a subject to treat a disease or condition.

[0224] Accordingly, some aspects of the present disclosure relates to a method of treating a disease or condition in a subject in need thereof, comprising administering to the subject any of the therapeutic compositions described herein (e.g., fusion protein, bispecific ligand-binding protein, multispecific ligand-binding protein, polynucleotide encoding such proteins, and/or pharmaceutical composition comprising such proteins and/or polynucleotides). Any suitable diseases or conditions can be treated using the therapeutic compositions of the present disclosure. [0225] In some aspects, the disease or condition comprises a cancer. Accordingly, some aspects of the present disclosure is related to a method of treating a cancer in a subject in need thereof, comprising administering to the subject a therapeutic composition described herein (e.g., fusion protein, bispecific ligand-binding protein, multispecific ligand-binding protein, polynucleotide encoding such proteins, and/or pharmaceutical composition 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), nonsmall cell lung cancer, squamous non-small cell lung cancer (NSCLC), nonsquamous NSCLC, gastrointestinal cancer, renal 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, hepatoma, breast cancer, colon carcinoma, and head and neck cancer (or carcinoma), gastric cancer, germ cell tumor, pediatric sarcoma, sinonasal natural killer, melanoma (e.g, metastatic malignant melanoma, such as cutaneous or intraocular malignant melanoma), bone cancer, skin cancer, uterine cancer, cancer of the anal region, testicular cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, cancer of the esophagus (e.g., gastroesophageal junction cancer), cancer of the small intestine, cancer of the endocrine system, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, solid tumors of childhood, cancer of the ureter, carcinoma of the renal pelvis, tumor angiogenesis, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, T-cell lymphoma, environmentally-induced cancers including those induced by asbestos, virus-related cancers or cancers of viral origin (e.g., human papilloma virus (HPV-related or -originating tumors)), and hematologic malignancies derived from either of the two major blood cell lineages, i.e., the myeloid cell line (which produces granulocytes, erythrocytes, thrombocytes, macrophages and mast cells) or lymphoid cell line (which produces B, T, NK and plasma cells), such as all types of leukemias, lymphomas, and myelomas, e.g., acute, chronic, lymphocytic and/or myelogenous leukemias, such as acute leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), and chronic myelogenous leukemia (CML), undifferentiated AML (MO), myeloblastic leukemia (Ml), 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), megakaryoblastic leukemia (M7), isolated granulocytic sarcoma, and chloroma; lymphomas, such as Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), B cell hematologic malignancy, e.g., B-cell lymphomas, T-cell lymphomas, lymphoplasmacytoid lymphoma, monocytoid B-cell lymphoma, mucosa-associated lymphoid tissue (MALT) lymphoma, anaplastic (e.g, Ki 1⁺) large-cell lymphoma, adult T-cell lymphoma/leukemia, mantle cell lymphoma, angio immunoblastic T-cell lymphoma, angiocentric lymphoma, intestinal T-cell lymphoma, primary mediastinal B-cell lymphoma, precursor T-lymphoblastic lymphoma, T- lymphoblastic; and lymphoma/leukemia (T-Lbly/T-ALL), peripheral T- cell lymphoma, lymphoblastic lymphoma, post-transplantation lymphoproliferative disorder, true histiocytic lymphoma, primary effusion lymphoma, B cell lymphoma, lymphoblastic lymphoma (LBL), hematopoietic tumors of lymphoid lineage, acute lymphoblastic leukemia, diffuse large B-cell lymphoma, Burkitt's lymphoma, follicular lymphoma, diffuse histiocytic lymphoma (DHL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, cutaneous T-cell lymphoma (CTLC) (also called mycosis fungoides or Sezary syndrome), and lymphoplasmacytoid lymphoma (LPL) with Waldenstrom's macroglobulinemia; myelomas, such as IgG myeloma, light chain myeloma, nonsecretory myeloma, smoldering myeloma (also called indolent myeloma), solitary plasmocytoma, and multiple myelomas, chronic lymphocytic leukemia (CLL), hairy cell lymphoma; hematopoietic tumors of myeloid lineage, tumors of mesenchymal origin, including fibrosarcoma and rhabdomyoscarcoma; seminoma, teratocarcinoma, tumors of mesenchymal origin, including fibrosarcoma, rhabdomyoscaroma, and osteosarcoma; and other tumors, including melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, thyroid follicular cancer and teratocarcinoma, hematopoietic tumors of lymphoid lineage, for example T-cell and B- cell tumors, including but not limited to T-cell disorders such as T-prolymphocytic leukemia (T- PLL), including of the small cell and cerebriform cell type; large granular lymphocyte leukemia (LGL) of the T-cell type; a/d T-NHL hepatosplenic lymphoma; peripheral/post-thymic T cell lymphoma (pleomorphic and immunoblastic subtypes); angiocentric (nasal) T-cell lymphoma; cancer of the head or neck, renal cancer, rectal cancer, cancer of the thyroid gland; acute myeloid lymphoma, and any combinations thereof.

[0227] In some aspects, a cancer that can be treated with the present disclosure comprises a breast cancer, head and neck cancer, uterine cancer, brain cancer, skin cancer, renal cancer, lung cancer, colorectal cancer, prostate cancer, liver cancer, bladder cancer, kidney cancer, pancreatic cancer, thyroid cancer, esophageal cancer, eye cancer, stomach (gastric) cancer, gastrointestinal cancer, carcinoma, sarcoma, leukemia, lymphoma, myeloma, or a combination thereof. In some aspects, a cancer (tumor) that can be treated with the present disclosure comprises a pancreatic cancer. In some aspects, a cancer (tumor) that can be treated comprises a brain cancer. In some aspects, brain cancer comprise a glioma. In some aspects, the brain cancer comprises a glioblastoma. In some aspects, glioblastoma comprises primary glioblastoma. In some aspects, glioblastoma comprises secondary glioblastoma. In some aspects, a glioblastoma that can be treated with the present disclosure is refractory (e.g., to previous cancer therapy, e.g., a standard of care comprising radiotherapy and/or chemotherapy such as temozolomide). In some aspects, a cancer (tumor) that can be treated comprises a skin cancer. Non-limiting examples of skin cancers include: a Merkel cell carcinoma (MCC), basal cell carcinoma (BCC), cutaneous squamous cell carcinoma (cSCC), melanoma, or a combination thereof. [0228] In some aspects, the disease or condition comprises an infectious disease. Accordingly, in some aspects, the present disclosure provides a method of treating an infectious disease in a subject in need thereof, comprising administering to the subject a therapeutic composition described herein (e.g., fusion protein, bispecific ligand-binding protein, multispecific ligand-binding protein, polynucleotide encoding such proteins, and/or pharmaceutical composition comprising such proteins and/or polynucleotides).

[0229] Non-limiting examples of infectious diseases that can be treated with the present disclosure include: an Acute Flaccid Myelitis (AFM), anaplasmosis, anthrax, babesiosis, botulism, brucellosis, campylobacteriosis, carbapenem- resistant infection, chancroid, Chikungunya virus infection, chlamydia, ciguatera, Clostridium difficile infection, perfringens, coccidioidomycosis fungal infection, coronavirus infection, Covid-19 (SARS-CoV-2), Creutzfeldt-Jacob Disease/transmissible spongiform encephalopathy, cryptosporidiosis (crypto), cyclosporiasis, Dengue 1,2,3 or 4, diphtheria, E. coli infection/Shiga toxin-producing (STEC), Eastern Equine Encephalitis, Hemorrhagic Fever (Ebola), ehrlichiosis, encephalitis, arboviral or parainfectious, non-polio enterovirus, D68 enterovirus (EV-D68), giardiasis, glanders, gonococcal infection, granuloma inguinale, haemophilus influenza disease 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, herpes zoster (shingles), histoplasmosis infection, human immunodeficiency virus/ AIDS (HIV/AIDS), human papillomavirus (HPV), influenza (Flu), Legionellosis (Legionnaires Disease), peprosy (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, pediculosis, pelvic inflammatory disease (PID), pertussis (whooping cough), plague (bubonic, septicemic, pneumonic), pneumococcal disease (pneumonia), poliomyelitis (polio), powassan, psittacosis, pthiriasis, pustular rash diseases (small pox, monkeypox, cowpox), q-fever, rabies, rickettsiosis (rocky mountain spotted fever), rubella (german measles), salmonellosis gastroenteritis (salmonella), scabies, scombroid, sepsis, severe acute respiratory syndrome (sars), shigellosis gastroenteritis (shigella), smallpox, staphyloccal infection methicillin-resistant (mrsa), staphylococcal food poisoning enterotoxin b poisoning (staph food poisoning), saphylococcal infection vancomycin intermediate (visa), staphylococcal infection vancomycin resistant (vrsa), streptococcal disease group a (invasive) (strep a (invasive), streptococcal disease, group b (strep- b), streptococcal toxic-shock syndrome stss toxic shock, syphilis (primary, secondary, early latent, late latent, congenital), tetanus infection, trichomoniasis, trichonosis infection, tuberculosis (tb), tuberculosis latent (Itbi), tularemia, typhoid fever group d, vaginosis, varicella (chickenpox), vibrio cholerae (cholera), vibriosis (vibrio), ebola virus hemorrhagic fever, lasa virus hemorrhagic fever, marburg virus hemorrhagic fever, west nile virus, yellow fever, yersenia, zika virus infection, or combinations thereof.

[0230] In some aspects, an infectious disease that can be treated with the present disclosure comprises a progressive multifocal leukoencephalopathy (PML; caused by polyomavirus JC). In some aspects, an infectious disease that can be treated with the present disclosure comprises a sepsis. In some aspects, an infectious disease that can be treated with the present disclosure comprises a HIV. In some aspects, an infectious disease that can be treated with the present disclosure comprises a cytomegalovirus (CMV) infection. In some aspects, an infectious disease that can be treated with the present disclosure comprises an Epstein-Barr virus (EB V) infection. In some aspects, an infectious disease that can be treated with the present disclosure comprises a respiratory infectious disease.

[0231] Any of the treatment methods provided herein (e.g., method of treating a cancer or an infectious disease) can further comprise administering an additional therapeutic agent to the subject. The additional therapeutic agent can include any known agents to treat and/or alleviate one or more symptoms associated with any of the above-described diseases or conditions.

[0232] Accordingly, in some aspects, provided herein is a method of treating a cancer in a subject in need thereof, comprising administering to the subject a therapeutic composition described herein (e.g., fusion protein, bispecific ligand-binding protein, multispecific ligandbinding protein, polynucleotide encoding such proteins, and/or pharmaceutical composition comprising such proteins and/or polynucleotides) and an additional therapeutic agent. In some aspects, provided herein is a method of treating an infectious disease in a subject in need thereof, comprising administering to the subject a therapeutic composition described herein (e.g., fusion protein, bispecific ligand-binding protein, multispecific ligand-binding protein, polynucleotide encoding such proteins, and/or pharmaceutical composition comprising such proteins and/or polynucleotides) and an additional therapeutic agent. In some aspects, the therapeutic composition of the present disclosure and the additional therapeutic agent are administered to the subject sequentially. For example, in some aspects, the additional therapeutic agent is administered to the subject prior to the administration of the therapeutic composition described herein. In some aspects, the additional therapeutic agent is administered to the subject after the administration of the therapeutic composition described herein. In some aspects, the therapeutic composition of the present disclosure and the additional therapeutic agent are administered to the subject concurrently. [0233] Non-limiting examples of additional therapeutic agents that can be used with the therapeutic compositions described herein (e.g., fusion protein, bispecific ligand-binding protein, multispecific ligand-binding protein, polynucleotide encoding such proteins, and/or pharmaceutical composition comprising such proteins and/or polynucleotides) include: an immune checkpoint inhibitor, an immune checkpoint activator, a standard of care treatment, a cytokine, or combinations thereof.

[0234] In some aspects, the additional therapeutic agent comprises an immune checkpoint inhibitor. Accordingly, some aspects of the present disclosure is related to a method of treating a disease or condition (e.g., cancer and/or infectious disease) in a subject in need thereof, 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 proteins, and/or pharmaceutical composition comprising such proteins and/or polynucleotides) and an immune checkpoint inhibitor. In some aspects, the immune checkpoint inhibitor comprises a CTLA-4 antagonist (e.g., anti-CTLA-4 antibody), a PD-1 antagonist (e.g., anti-PD-1 antibody, anti-PD-Ll antibody), a TIM3 antagonist (e.g., anti-TIM3 antibody), or combinations thereof.

[0235] In some aspects, the additional therapeutic agent comprises an immune checkpoint activator. Accordingly, some aspects of the present disclosure is related to a method of treating a disease or condition (e.g, cancer and/or infectious disease) in a subject in need thereof, 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 proteins, and/or pharmaceutical composition comprising such proteins and/or polynucleotides) and an immune checkpoint activator. Non-limiting examples of immune checkpoint activators that can be used with the present disclosure include: an 0X40 agonist (e.g., anti-OX40 antibody), a LAG-3 agonist (e.g., anti-LAG3 antibody), a 4-1BB (CD137) (e.g., anti- CD137 antibody), a GITR agonist (e.g., anti-GITR antibody), or combinations thereof.

[0236] In some aspects, the additional therapeutic agent comprises a standard of care treatment. In some aspects, provided herein is a method of treating a disease or condition (e.g., cancer and/or infectious disease) in a subject in need thereof, 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 proteins, and/or pharmaceutical composition comprising such proteins and/or polynucleotides) and a standard of care treatment. In some aspects, the standard of care treatment comprises a chemotherapy, a radiation, or both.

[0237] In some aspects, the additional therapeutic agent comprises a cytokine. Any suitable cytokines known in the art can be used with the present disclosure. In some aspects, the cytokine comprises an 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-alpha (IFN-a), interferon-beta (IFN-[3), interferon-gamma (IFN-y), tumor necrosis factor (TNF), or combinations thereof. In some aspects, the cytokine comprises an 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. Accordingly, some aspects of the present disclosure is related to a method of treating a disease or condition (e.g., cancer and/or infectious disease) in a subject in need thereof, 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 proteins, and/or pharmaceutical composition comprising such proteins and/or polynucleotides) and a cytokine (e.g., a long-acting IL-7 protein).

[0238] In some aspects, any of the polypeptides, molecules, nucleic acids, vectors, cells, protein conjugates, or compositions described herein is administered intravenously, orally, parenterally, transthecally, intrathecally, intra-cerebroventricularly, pulmonarily, subcutaneously, intradermally, intramuscularly, or intraventricularly.

[0239] The following examples are offered by way of illustration and not by way of limitation.

EXAMPLES

Example 1: Materials and Methods

[0240] To produce a trispecific heterodimer antibody (such as that shown in FIG. 1), the following methods and materials were used (see, also FIG. 2):

Plasmid Cloning: [0241] Antibody chain A and chain B were transformed into the pEG BacMam vector. As described herein (see, e.g., FIG. 1), chain A included an anti-B7H3 sdAb, IgD hinge, IgD/IgG4 CH2 domain, and IgG4 CH3 domain comprising the amino acid sequence set forth in SEQ ID NO: 40 with the following amino acid modifications: T10V, LI 1 Y, F65A, and Y67V (i.e., SEQ ID NO: 24). Chain B included an anti-CD3 sdAb, anti-CD27 scFv, IgD hinge, IgD/IgG4 CH2 domain, and IgG4 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 (i.e., SEQ ID NO: 26). As shown in FIG. 3, chain A was encoded as either (1) a fusion protein (see Construct #1, which further comprised a sequence encoding a GFP tag after the CH3 domain with a PreScission cleavage site in between the GFP tag and the CH3 domain, or (2) chain A polypeptide alone (i.e., without the GFP tag; see Construct #4). Chain B was encoded as two different fusion proteins. The vector encoding the first chain B fusion protein further comprised, after the C-terminal end of the CH3 domain, a sequence encoding mCherry and a sequence encoding an ALFA tag (LEEELRRRLTE; SEQ ID NO: 14) (see Construct #2 in FIG. 3). As further described herein, the ALFA tag allowed for the purification of the heterodimer of chain B (encoded by Construct #2) and chain A (encoded by Construct #1). A PreScission cleavage site was included in between the CH3 domain and mCherry, and a thrombin cleavage site was included in between mCherry and the ALFA tag. The vector encoding the second chain B fusion protein further comprised a sequence encoding a 6XHis tag (HHHHHH; SEQ ID NO: 17) after the CH3 domain (see Construct #3 in FIG. 3).

Recombinant Baculovirus Generation

[0242] The above described plasmid vectors were transformed into DHIOBac cells with 50 pg/ml kanamycin, 7 pg/ml gentamicin, 10 pg/ml tetracycline, 0.17 mM IPTG (isopropyl P-D-l- thiogalactopyranoside), and 20 mg/ml Bluo-gal. LB (Luria broth) agar plates were smeared and incubated at 37 °C for 24-48 hours. After the white colonies were sorted, the colonies were selected once more by smearing them on board. From the selected colonies, taq-PCR was performed to identify colonies in which recombination with the baculovirus genome was successful.

[0243] The selected colonies were inoculated in LB medium with 50 pg/ml kanamycin, 7 pg/ml gentamicin, and 10 pg/ml tetracycline, 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 transfection of the viral genome to SF9 insect cells, from which the baculovirus was harvested. Transduction of Expi293F Cells

[0244] Expi293F cells were cultured at 37°C, 125 rpm, 8% CO2 using 800 ml of Expi293 medium. Cell passage was performed every 3-4 days when the number of cells reached 3-5 x 10⁶ viable cells/mL.

[0245] For baculovirus infection, cells were prepared at a concentration of 3 x 10⁶ viable cells/mL and then infected using 80 ml of culture medium (which is 10% v/v of the total culture medium). The infected cells were then incubated at 8% CO2 and 37 ⁰ C. After 18 hours, enhancer was added to enhance transduction was added, and the cells were further incubated for 6 days to produce the encoded proteins.

Protein Purification

[0246] To purify the produced proteins, the cells were first lysed and centrifuged to remove cell debris. The supernatant was loaded onto an affinity column (anti-His, anti-ALFA, or Protein A resins) to capture the tagged antibody proteins.

[0247] Briefly, the anti-ALFA nanobody open columns were prepared by washing the column using 20 mM Tris-HCl, 200 mM NaCl buffer at pH 8.0 after filling the resin, but before protein loading. The resin passed through the supernatant was washed using 10 column volumes (CV) of 20 mM Tris-HCl, 200 mM NaCl buffer at pH 8.0. An amount of thrombin corresponding to 1% of a CV and 1 CV 20 mM Tris 200 mM NaCl pH 8.0 solution were reacted together at 4° C for 16 hours to achieve cleavage of the ALFA tag. The tag and the antibody protein were then eluted from the column. The eluted protein was concentrated using an Amicon Ultra- 15 centrifugal filter, 100 K (Millipore), and then reacted with a PreScission protease (2% of the protein amount) at 4⁰ C for 16 hours to cleave the mCherry and GFP fluorescent proteins to obtain the pure protein. [0248] For purification using the anti-His columns, the supernatant was loaded onto the column. The resin passed through the supernatant was washed with a solution of 10 CV of 20 mM Tris-HCl, 200 mM NaCl, pH 8.0, 10 mM Imidazole solution column. The concentration of imidazole was increased by 100, 300, 500 mM step by step using 20 mM Tris-HCl, 200 mM NaCl, pH 8.0, and the protein was eluted by flowing the solution twice the amount of the column, and the eluted protein was concentrated using the Amicon Ultra- 15 centrifugal filter, 100K (Millipore). [0249] For purification using the Protein A columns, the resin passed through the supernatant was washed with 20 mM Tris-HCl, 200 mM NaCl, pH 8.0 solution 10 CV. Antibody proteins attached to resin were eluted into a 100 mM Glycine, pH 2.5 solution and collected by neutralizing them in tubes containing 2 M Tris-HCl, pH 8.0 buffers. Example 2: Confirmation of Trispecific Heterodimer Antibody Formation

[0250] Trispecific heterodimer antibody fused to fluorescent proteins (GFP or mCherry) was expressed in in Expi293 cells as described above in Example E The heterodimer antibody bound to the ALFA nanobodies, allowing for the purification of only trispecific heterodimers. Because chain A and chain B were fused to different fluorescent proteins (GFP and mCherry, respectively), two distinct bands were observed in the SDS-PAGE gel under reducing conditions (see FIG. 4A). This confirmed that the purified protein was present as a heterodimer. Next, the purified protein was concentrated, and then the fluorescent proteins were cleaved using PreScission protease. The successful cleavage of the fluorescent proteins was confirmed via SDS-PAGE analysis (see FIG. 4B).

[0251] To further confirm the heterodimer nature of the produced trispecific antibodies, the fluorescent proteins (GFP and mCherry) were identified in SDS-PAGE gel under non-reducing and reducing conditions (see FIG. 5). Chain A (B7H3-CHA) and chain B (CD3a-CD27-CHn) appeared as two distinct bands under the reducing condition (see bands at 44.6 kDa and 72.1 kDa, respectively), but only a single band was observed under the non-reducing condition.

[0252] To assess the purity of the tri specific heterodimer antibody produced and described above, a Chemi-doc analysis was performed using the heterodimeric single band observed in the SDS-PAGE under the non-reducing condition. Purity of the trispecific heterodimer antibody was about 75% (see FIGs. 6A and 6B).

Example 3: Analysis of Baculovirus Infection Ratio on Trispecific Heterodimer Antibody Formation

[0253] The culture media used in the baculovirus infection of Expi293 human cells with chain A (B7H3-CHA) and chain B (CD3a-CD27-CHn) (as described in Example 1) performed at a 1 : 1 virus:human cell ratio were loaded onto an anti-ALFA and anti-GFP nanobody resins, respectively, in order to measure the yield of the heterodimer. The columns were eluted as described in Example 1, and the eluent was analyzed by SDS-PAGE. As shown in FIG. 7, excessive chain A was detected in the eluent for anti-ALFA nanobody resin, whereas only a small amount of the protein was observed in anti-GFP nanobody resin.

[0254] Therefore, in order to increase the productivity of the heterodimers, several baculovirus virus:human cell infection ratios (z.e., 4: 1, 8: 1, 10: 1) were tested, and analyzed via SDS-PAGE under non-reducing conditions. The expressed protein was purified using ALFA nanobody resins as described earlier (see FIG. 8), and the fluorescent protein was cleaved using PreScission protease. Productivity of the protein was assessed with SDS-PAGE analysis.

[0255] As shown in FIG. 9, there was much more heterodimer formation at infection ratios of 4: 1 and 8: 1, with greatest formation observed when the cells were infected at a ratio of 8: 1. The heterodimeric antibody resulting from these experiments was then analyzed via mass photometry, which showed that the peak molecular weight was similar to the expected molecular weight (115.5 kDa) of the fluorescence-deactivated heterodimer (see FIG. 10).

[0256] The above results further confirm that polypeptide chains A and B described herein were useful in producing heterodimeric proteins, such as the trispecific heterodimer antibody.

Example 4: Analysis of Baculovirus Infection Ratio on Trispecific Heterodimer Antibody Formation

[0257] To additionally confirm the results described in the above examples, Construct #3 (encoding chain B without a fluorescent protein) and Construct #4 (encoding chain A without a florescent protein) were used in producing the trispecific heterodimer antibodies. Briefly, as described in Example 1, cells were infected with recombinant baculovirus particle comprising Constructs #3 and #4 at a 1 : 1 ratio, and then the encoded proteins expressed. To purify the produced proteins, culture medium from the infected cells was loaded onto anti-His resin column, and then the captured proteins were eluted using successive imidazole treatment (with increasing concentrations - ie., 100, 300 and 500 mM). The eluted protein was analyzed by SDS-PAGE analysis (see FIG. 11).

[0258] The eluted proteins were further purified using a Protein A column, which captured only the antibody proteins. The captured proteins were eluted into a 100 mM glycine solution at pH 2.5 and collected by neutralizing them in tubes containing a buffer solution of 2M Tris-HCl at pH 8.0. The eluted proteins were analyzed by SDS-PAGE, which showed that a total of 8.8 mg (0.018 g/L) was obtained from a 500 mL prep of the infected cells with a purity of about 85% (see FIG. 12).

[0259] The above results demonstrate that the removal of the fluorescent tags (e.g., GFP and mCherry) not only simplified the purification process but also helped increase the purity of the produced trispecific heterodimer antibody.

Example 5: Additional Confirmation of Trispecific Heterodimer Antibody Formation [0260] To confirm that the trispecific antibody purified in Example 4 is indeed a heterodimer, the eluted protein (from Example 4) was assessed using SDS-PAGE analysis under both reducing and non-reducing conditions. As shown in FIG. 13, under reducing conditions, two distinct bands corresponding to chain A (B7H3-CHA; 43.6 kDa) and chain B (CD3a-CD27-CHn; 72 kDa) were observed. However, under non-reducing conditions, only a single band at roughly 115.5 kDa was observed. ChemiDoc analysis confirmed the results shown in FIG. 13, and further provided that the purified trispecific heterodimer antibody had a purity of about 83% (see FIG. 14). [0261] Next, capillary electrophoresis in sodium dodecyl sulfate (CE-SDS) analysis was performed to confirm the accuracy of the results described above for the SDS-PAGE. The CE-SDS analysis is an experiment that can automatically quantify and purify both heterodimer triple antibodies and reduced monoclonal antibodies, making it an ideal analytical method for analyzing the heterodimer formation of samples. As shown in FIG. 15, under reducing conditions, there were two peaks observed, corresponding to chain A (B7H3-CHA) and chain B (CD3a-CD27-CHn) subunits. However, under non-reducing conditions, a single, stronger peak corresponding to the dimer was observed, with a purity of about 90%. As shown in FIG. 16, the trispecific heterodimer formation was also confirmed with Western Blot analysis.

[0262] Collectively, the above results confirm that the heterodimeric proteins provided herein (e.g., comprising chain A and chain B described in FIG. 3) can be used in producing multispecific heterodimer antibodies.

Example 6: Modifications for Promoting Heterodimer Formation

[0263] As described throughout the present disclosure, Applicant has identified that certain amino acid modifications within an immunoglobulin CH3 domain can promote heterodimer formation. To better understand such amino acid modifications, polypeptide chains A and B described in Example 1 will be modified to comprise one or more additional heterodimerization modifications (e.g., negative and positive charge amino acid modifications described herein) within the CH3 domain. Exemplary polypeptide chains A and/or B will comprise the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 16, or SEQ ID NO: 20 and one or more of the following positive charge 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 negative charge 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 sequence comprising such positive charge modifications include: SEQ ID NO: 27 (z.e., D197R; "Polypeptide Chain A #2"). Exemplary chain B polypeptide amino acid sequence comprising such negative charge modifications include: SEQ ID NO: 29 (z.e., R207D; "Polypeptide Chain B #2") and SEQ ID NO: 32 (z.e., R207E; "Polypeptide Chain B #3"). Exemplary combinations of chain A and chain B polypeptides to form the heterodimers will 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, control sequences will include knob-into-hole (KiH) modifications to the CH3 domain (e.g., chain A comprising the amino acid sequence set forth in SEQ ID NO: 34 with chain B comprising the amino acid sequence set forth in SEQ ID NO: 38).

[0264] To produce the above-described heterodimers, materials and methods (such as those described in Example 1) will be used. Then, properties such as heterodimer formation, purity, and stability will be assessed.

Table 3. Exemplary Sequences

Example 7: Nano Differential Scanning Fluorimetry (Nano DSF) [0265] Further 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 (z.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). Both NIT-Fc-04 and NIT-Fc-05 do not contain the specific CH3 domain modifications shown in Table 4 and were used as control. To further characterize these heterodimers, nano differential scanning fluorimetry (nano DSF) measurements were performed using a Prometheus NT.48 instrument (NanoTemper Technologies GmbH, Miinchen, Germany) to evaluate the thermal stability of the different heterodimers. Briefly, the heterodimers were prepared at a concentration of 2.0 mg/ml in lx PBS (pH 7.4). Each experimental condition was set up and measured in standard capillaries (NanoTemper Technologies GmbH). The experiments were conducted at 40% sensitivity, with a temperature range from 20 to 90°C (FIG. 21) and a heating rate of l°C/min. The melting temperature (Tm) of the antibodies 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 were determined by identifying the peak of the first derivative of the fluorescence ratios (F350/330) (FIG.22). As summarized in Table 4, each of the heterodimers comprising the CH3 domain modifications described herein (i.e., NIT-Fc-01, NIT-Fc-02, and NIT- Fc-03) had comparable thermal stability compared to the control heterodimers (i.e., NIT-Fc-04, NIT-Fc-05).

Table 4. Temperatures from Nano differential scanning fluorimetry assays comparing heterodimers NIT-Fc-01 to NIT-Fc-05.

Example 8: Dynamic Light Scattering (DLS)

[0266] To assess the tendencies of the heterodimers described in Example 7 to form aggregates, DLS measurements were conducted employing a WYATT-493 -WPR3 instrument, with the detector oriented at a 150° angle relative to the incident light. Utilizing a monochromatic laser set at a wavelength of 825.1 nm (with auto-attenuation activated), the heterodimer sample housed within a 96-well plate was subjected to irradiation. The experimental procedure involved incrementally increasing the temperature from 25°C to 85°C to observe temperature-dependent changes in the size of antibody sample (FIG. 23). From the data, it can be shown that the heterodimers of NIT -Fc-01, NIT -Fc-02, and NIT-Fc-03 have comparable thermal stability to the control heterodimers.

Example 9: Re-heterodimerization

[0267] As described in at least Example 6, the specific CH3 modifications described herein were designed to promote heterodimerization. Accordingly, the ability of the heterodimers described in Example 7 to re-heterodimerize when denatured was assessed. Briefly, the heterodimers, initially prepared at a concentration of 2.0 mg/ml, were reduced by the addition of 10 mM P -mercaptoethanol (BME) for a duration of 1 hour at room temperature. This reduction step aimed to break any disulfide bonds present within the antibody molecules. Following reduction, the denatured polypeptide chains were subjected to the heterodimerization process through dialysis in PBS buffer for 18 hours. This prolonged dialysis duration ensured thorough equilibration of the reduced antibody molecules, promoting the formation of heterodimers. Finally, the re-heterodimerization was confirmed by analyzing the samples using SDS-PAGE gel.

[0268] As shown in Table 5, the heterodimers comprising the CH3 modifications described herein (z.e., NIT -Fc-01, NIT -Fc-02, and NIT-Fc-03) were much better at re-heterodimerizing compared to the control (z.e., NIT -Fc-04 and NIT -Fc-05). Table 5. Comparison of re-heterodimerization on heterodimers

Table 6. Information on expression yield and purity of heterodimers

Example 10: Thermal Stability Assessment and Further Characterization

[0269] To further characterize the heterodimers described in Example 6, stability at 37°C was next assessed. Briefly, the heterodimer samples were prepared at a concentration of 2.0 mg/ml in phosphate-buffered saline (PBS). The prepared heterodimer samples were subjected to incubation at 37°C for 7 days. The alterations in concentration were monitored by measuring the absorbance at 280 nm (A280). As shown in FIG. 24, the heterodimers all appeared to have similar stability. Table 6 provides data showing the expression yield and purity of the heterodimers when expressed in vitro. The heterodimers comprising the CH3 modifications described herein (z.e., NIT-Fc-01, NIT -Fc-02, and NIT-Fc-03) had improved purity and/or improved total yield as compared to each of the controls. Not to be bound by any one theory, such data highlight the improved manufacturability of the heterodimers comprising the CH3 modifications described herein. .

[0270] Collectively, the data provided herein highlight the usefulness of the CH3 domain modifications described herein to produce multispecific heterodimeric proteins (e.g., antibodies).

[0271] It is to be appreciated that the Detailed Description section, and not the Summary and Abstract sections, is intended to be used to interpret the claims. The Summary and Abstract sections can set forth one or more but not all exemplary aspects of the present disclosure as contemplated by the inventor(s), and thus, are not intended to limit the present disclosure and the appended claims in any way.

[0272] The present disclosure has been described above with the aid of functional building blocks illustrating the implementation of specified functions and relationships thereof. The boundaries of these functional building blocks have been arbitrarily defined herein for the convenience of the description. Alternate boundaries can be defined so long as the specified functions and relationships thereof are appropriately performed.

[0273] The foregoing description of the specific aspects will so fully reveal the general nature of the disclosure that others can, by applying knowledge within the skill of the art, readily modify and/or adapt for various applications such specific aspects, without undue experimentation, without departing from the general concept of the present disclosure. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed aspects, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance.

[0274] The breadth and scope of the present disclosure should not be limited by any of the above-described exemplary aspects, but should be defined only in accordance with the following claims and their equivalents.

[0275] All publications, patents, patent applications, internet sites, and accession numbers/database sequences (including both polynucleotide and polypeptide sequences) cited herein are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, internet site, or accession number/database sequence were specifically and individually indicated to be so incorporated by reference.

Claims

WHAT IS CLAIMED IS:

1. 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: 32 or SEQ ID NO: 29.

2. The heterodimeric protein of claim 1, wherein the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 32.

3. The heterodimer protein of claim 1, wherein the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 29.

4. 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: 16, and (b) the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 20.

5. A heterodimeric protein comprising a first polypeptide and a second polypeptide, wherein the first polypeptide and second polypeptide are not the same, 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: 24, and (b) the second CH3 domain comprises the amino acid sequence set forth in SEQ ID NO: 26, wherein:

(1) amino acid residue N50, S60, or both of the first CH3 domain are not modified or modified with a positive charge modification, and amino acid residue N50, S60, or both of the second CH3 domain are not modified 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 not modified or modified with a negative charge modification, and amino acid residue N50, S60, or both of the second CH3 domain are not modified 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 not modified or modified with a positive charge modification, and amino acid residue D59, R69, or both of the second CH3 domain are not modified 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 not modified or modified with a negative charge modification, and amino acid residue D59, R69, or both of the second CH3 domain are not modified 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 not modified or modified with a positive charge modification, and amino acid residue F65 of the second CH3 domain is not modified 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 not modified or modified with a negative charge modification, and amino acid residue F65 of the second CH3 domain is not modified or modified with a positive charge modification, such 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 comain 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 of any one of claims 5 to 7, wherein:

(a) the amino acid residue N50 of the first CH3 domain is modified to N50K or N50R and the amino acid residue S60 of the second CH3 domain is modified to S60D or S60E,

(b) the amino acid residue S60 of the first CH3 domain is modified to S60D or S60E and the amino acid residue N50 of the second CH3 domain is modified to N50K or N50R,

(c) the amino acid residue K52 of the first CH3 domain is not modified or modified to K52R and the amino acid residue F65 of the second CH3 domain is modified to F65D or F65E,

(d) the amino acid residue D59 of the first CH3 domain is modified to D59K or D59R and the amino acid residue R69 of the second CH3 domain is modified to R69D or R69E,

(e) the amino acid residue R69 of the first CH3 domain is modified R69D or R69E and the amino acid residue D59 of the second CH3 domain is modified D59K or D59R, or

(f) any combination of (a) to (e).

9. The heterodimeric protein of any one of claims 5 to 8, wherein 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.

10. The heterodimeric protein of any one of claims 5 to 8, wherein 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.

11. The heterodimeric protein of 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 an IgGl hinge, an IgG2 hinge, an IgG3 hinge, an IgG4 hinge, an IgD hinge, or a synthetic linker.

13. The heterodimeric protein of claim 12, wherein the synthetic linker comprises a glycineserine linker, a glycine-alanine linker, an alanine-serine linker, or combinations 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 of any one of claims 11 to 14, wherein: (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 forth 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 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).

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

19. The heterodimeric protein of any one of claim 18, wherein the first biologically active 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 claim 19, wherein the first biologically active molecule is attached directly or via a linker.

21. The heterodimeric protein of claim 20, wherein the linker is selected from an IgGl hinge, an IgG2 hinge, an IgG3 hinge, an IgG4 hinge, an IgD hinge, or a synthetic linker.

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

23. The heterodimeric protein of claim 22, wherein the second biologically active 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 biologically active molecule, or any combination thereof.

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

25. The heterodimeric protein of claim 24, wherein the linker is selected from an IgGl hinge, an IgG2 hinge, an IgG3 hinge, an IgG4 hinge, an IgD hinge, or a synthetic linker.

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

27. The heterodimeric protein of claim 26, wherein the one or more additional biologically active 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 biologically active molecule, the second biologically active molecule, or any combination thereof.

28. The heterodimeric protein of any one of claims 18 to 27, wherein 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.

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 an Ig NAR, 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 combinations thereof.

31. The heterodimeric protein of claim 29, wherein the antibody comprises a T cell engager (e.g., bispecific T-cell engager (BiTE) antibody), dual-affinity retargeting molecule (DART), CrossMAb antibody, DutaMab™ antibody, DuoBody antibody, Triomab, TandAb, bispecific NanoBody, Tandem scFv, diabody, single chain diabody, HSA body, (scFv)2 HSA Antibody, scFv-IgG antibody, Dock and Lock bispecific antibody, DVD-IgG antibody, TBTI DVD-IgG, IgG- fynomer, Tetravalent bispecific tandem IgG antibody, dual targeting domain antibody, chemically linked bispecific (Fab')2 molecule, crosslinked mAb, Dual-action Fab IgG (DAF-IgG), orthoFab- IgG, bispecific CovX-Body, bispecific hexavalent trimerbody, 2 scFv linked to diphtheria toxin, ART-Ig, IgM T-cell engager, Humabody™ human heavy-chain only antibody (HCAb), Uni Ab™ HCAb, shark heavy chain-only antibody (VNAR), or combinations thereof.

32. The heterodimeric protein of any one of claims 28 to 31, wherein the ligand-binding protein binds to a 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 a 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 (F0LR1), CD4, CD 19, 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, MRP3cadherin 19 (CDH19), epidermal growth factor 2 (FIER2), 5T4, 8H9, anbo 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, Mucl6, NCAM, NKG2D ligands, NY-ESO-1, PRAME, PSC1, PSCA, PSMA, R0R1, R0R2, SP17, survivin, TAG72, TEMs, 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 an immune cell comprises a CD3, CD27, B7H3, CD2, CD4, CD5, CD8, CDl lb, 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, 0X40, CD40L, ICOS, LIGHT, or combinations thereof.

36. The heterodimeric protein of any one of claims 29 to 35, wherein the cytokine comprises an IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21, IL-23, IFNa, IFNp. IFNy, TNF, combinations thereof an IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21, IL-23, IFNa, IFN . IFNy, TNF, combinations thereof.

37. A fusion protein comprising the heterodimeric protein of any one of claims 1 to 36.

38. A bispecific antibody comprising (a) a first antigen-binding domain, (b) a second antigenbinding domain, and (c) the heterodimeric protein of 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 of claim 39, wherein: (a) the first antigen-binding domain is conjugated directly, (b) the first antigen-binding domain is conjugated via a first linker, (c) the second antigen binding domain is conjugated directly, (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 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.

42. The bispecific antibody of 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 of any one of claims 42 to 44, wherein the tumor antigen comprises a 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 (F0LR1), CD4, CD 19, 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, MRP3cadherin 19 (CDH19), epidermal growth factor 2 (FIER2), 5T4, 8H9, anbo 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, Mucl6, NCAM, NKG2D ligands, NY-ESO-1, PRAME, PSC1, PSCA, PSMA, R0R1, R0R2, SP17, survivin, TAG72, TEMs, carcinoembryonic antigen, HMW-MAA, VEGF, CLDN18.2, programmed death-ligand 1 (PDL-

1), or combinations thereof.

46. The bispecific antibody of any one of claims 43 to 45, wherein the antigen expressed on an immune cell comprises a CD3, CD27, B7H3, CD2, CD4, CD5, CD8, CD1 lb, 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, 0X40, CD40L, ICOS, LIGHT, or combinations thereof.

47. The bispecific antibody of any one of claims 38 to 46, which comprises an Ig NAR, 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, 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) the heterodimeric protein of 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 combinations thereof are conjugated to the heterodimeric protein.

50. The multispecific antibody of claim 49, wherein: (a) the first antigen-binding domain is conjugated directly, (b) the first antigen-binding domain is conjugated via a first linker, (c) the second antigen-binding domain is conjugated directly, (d) the second antigen-binding domain is conjugated via a second linker, (e) the third antigen-binding domain is conjugated directly, (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 linker, the second linker, the third linker, or combinations thereof are selected from an IgGl hinge, an IgG2 hinge, an IgG3 hinge, an IgG4 hinge, an IgD hinge, or a synthetic linker.

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 linker, the second antigenbinding domain is attached to the N terminus of the 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, second linker, and third linker are selected from IgGl hinge, IgG2 hinge, IgG3 hinge, IgG4 hinge, IgD hinge, or synthetic linker.

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 linker, the second antigenbinding 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 the second polypeptide via a third linker, and wherein the first linker, second linker, and third linker are selected from IgGl hinge, IgG2 hinge, IgG3 hinge, IgG4 hinge, IgD hinge, or synthetic linker.

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 combinations thereof bind 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 combinations thereof bind to an antigen expressed on an immune cell.

56. The multispecific antibody of any one of claims 48 to 55, which comprises 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 of any one of claims 54 to 57, wherein the tumor antigen comprises a 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 (F0LR1), CD4, CD 19, 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, LI CAM, AXL, Tissue Factor (TF), CD74, EpCAM, EphA2, MRP3cadherin 19 (CDH19), epidermal growth factor 2 (FIER2), 5T4, 8H9, anbo 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, Mucl6, NCAM, NKG2D ligands, NY-ESO-1, PRAME, PSC1, PSCA, PSMA, R0R1, R0R2, SP17, survivin, TAG72, TEMs, carcinoembryonic antigen, HMW-MAA, VEGF, CLDN18.2, programmed death-ligand 1 (PDL- 1), or combinations thereof.

59. The multispecific antibody of any one of claims 55 to 58, wherein the antigen expressed on an immune cell comprises a CD3, CD27, B7H3, CD2, CD4, CD5, CD8, CDl lb, 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, 0X40, CD40L, ICOS, LIGHT, or combinations thereof.

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

61. The conjugate of claim 60, wherein the one or more conjugate moieties comprise a clearance-modifying agent, a chemotherapeutic agent, a toxin, a radioactive isotope, a lanthanide, a luminescent label, a fluorescent label, an enzyme-substrate label, a DNA-alkylators, a topoisomerase inhibitor, a tubulin-binders, an anticancer drug, or combinations thereof.

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

63. A vector comprising the isolated nucleic acid of claim 62.

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

65. A vector comprising the isolated nucleic acid of claim 64.

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

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

68. A method of preparing the heterodimeric protein of any one of claims 1 to 36, comprising culturing a cell comprising the isolated nucleic acid of claim 62 or the vector of claim 63, and optionally, recovering the heterodimeric protein that is produced.

69. A method of producing a fusion protein, comprising conjugating one or more biologically active molecules to the heterodimeric protein of any one of claims 1 to 36.

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

71. The method of claim 70, wherein the linker is selected from an IgGl hinge, an IgG2 hinge, an IgG3 hinge, an IgG4 hinge, an IgD hinge, or a synthetic linker.

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

73. The method of claim 72, wherein the disease or condition comprises a cancer, an infectious disease, or both.

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

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

76. The method of any one of claims 72 to 75, wherein the heterodimeric protein, the fusion protein, the bispecific antibody, the multispecific antibody, the conjugate, the isolated nucleic acid, the vector, or the pharmaceutical composition is administered to the subject intramuscularly, parenthetically, subcutaneously, ophthalmic, intravenously, intraperitoneally, intradermally, intraorbitally, intracerebrally, intracranially, intraspinally, intraventricular, intrathecally, intraci stemally, intracapsularly, or intratumorally.

77. The method of any one of claims 72 to 76, which comprises 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, parenthetically, subcutaneously, ophthalmic, intravenously, intraperitoneally, intradermally, intraorbitally, intracerebrally, intracranially, intraspinally, intraventricular, intrathecally, intraci stemally, intracapsularly, or intratumorally.

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

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

81. The method of claim 79 or 80, wherein the immune checkpoint activator comprises an 0X40 agonist (e.g., anti-OX40 antibody), a LAG-3 agonist (e.g, anti-LAG3 antibody), a 4- IBB (CD137) (e.g., anti-CD137 antibody), a GITR agonist (e.g., anti-GITR antibody), or combinations thereof.

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

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