Heterodimeric proteins with fc mutations

Abstract

To provide multispecific antibodies that heterodimerize in a specific and stable manner and can target multiple antigens correctly.SOLUTION: Provided is a heterodimeric protein comprising a first polypeptide comprising a first immunoglobulin heavy chain constant domain 3 (CH3 domain) and a second polypeptide comprising a second CH3 domain, as well as comprising polypeptides having CH3 domains with engineered residues that form disulfide bonds and / or salt bridges.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to International Application No. PCT / CN2020 / 073960, filed January 23, 2020, which is incorporated herein by reference in its entirety.

[0002] FIELD OF THE INVENTION This application relates to heterodimeric proteins (e.g., bispecific antibodies) and activatable antibodies, methods for their preparation, and methods of use.

[0003] Sequence Listing Reference The contents of the following submission in an ASCII text file are incorporated herein by reference in their entirety: Sequence Listing in Computer Readable Form (CRF) (Filename: 695402001041SEQLIST.txt, Recorded: January 21, 2020, Size: 656KB). [Background technology]

[0004] Multispecific antibodies can simultaneously bind to multiple different antigens. This property allows for the development of therapeutic strategies that are not possible with conventional monoclonal antibodies. One format of multispecific antibodies is a heterodimeric protein, e.g., an antibody composed of separate chains that bind to different antigens. Such heterodimeric multispecific antibodies can precisely target multiple antigens only when assembled with the appropriate complement of monomer components. Therefore, there is a need in the art for multispecific antibodies that heterodimerize in a specific and stable manner.

[0005] Activatable antibodies exhibit an "activatable" conformation in which the antigen-binding moiety contained therein is less likely to bind to its target when uncleaved than after cleavage in the presence of one or more specific proteases. Thus, activatable antibodies provide antigen-specific binding proteins that can bind to their target only under certain circumstances (e.g., a protease-rich tumor microenvironment). Bispecific T cell engagers are bispecific antibodies (BiTEs) that can bind to both T cells and target cells, such as tumor cells. Due to their on-target and off-tumor effects, BiTE molecules are associated with high cytotoxicity, including central nervous system (CNS) toxicity and cytokine storm. Activatable BiTE molecules with improved specificity and reduced side effects are needed.

[0006] All references cited herein, including patent applications, patent publications, non-patent literature, and UniProtKB / Swiss-Prot / GenBank accession numbers, are hereby incorporated by reference in their entirety, as if each individual reference was specifically and individually indicated to be incorporated by reference. Summary of the Invention

[0007] The present application provides heterodimeric proteins comprising a CH3 domain with engineered residues that form disulfide bonds and / or salt bridges. Activatable antibodies targeting CD3 and / or HER2 are also provided.

[0008] Accordingly, one aspect of the present application provides a heterodimeric protein comprising a first polypeptide comprising a first immunoglobulin heavy chain constant domain 3 (CH3 domain) and a second polypeptide comprising a second CH3 domain, wherein i) the first CH3 domain comprises a cysteine ​​(C) residue at position 390 and the second CH3 domain comprises a cysteine ​​residue at position 400, or the first CH3 domain comprises a cysteine ​​residue at position 400 and the second CH3 domain comprises a cysteine ​​residue at position 390; or ii) the first CH3 domain comprises a cysteine ​​residue at position 392. residue and the second CH3 domain comprises a cysteine ​​residue at position 397, or the first CH3 domain comprises a cysteine ​​residue at position 397 and the second CH3 domain comprises a cysteine ​​residue at position 392; or iii) the first CH3 domain comprises a cysteine ​​residue at position 392 and the second CH3 domain comprises a cysteine ​​residue at position 400, or the first CH3 domain comprises a cysteine ​​residue at position 400 and the second CH3 domain comprises a cysteine ​​residue at position 392; amino acid residue numbering is based on EU numbering. In some embodiments, i) the first CH3 domain comprises an N390C substitution and the second CH3 domain comprises an S400C substitution, or the first CH3 domain comprises an S400C substitution and the second CH3 domain comprises an N390C substitution; or ii) the first CH3 domain comprises a K392C substitution and the second CH3 domain comprises a V397C substitution, or the first CH3 domain comprises a V397C substitution and the second CH3 domain comprises a K392C substitution; or iii) the first CH3 domain comprises a K392C substitution and the second CH3 domain comprises an S400C substitution, or the first CH3 domain comprises an S400C substitution and the second CH3 domain comprises a K392C substitution.

[0009] In some embodiments of any one of the above heterodimeric proteins, i) the first CH3 domain further comprises a positively charged residue at position 357 and the second CH3 domain further comprises a negatively charged residue at position 351, or the first CH3 domain further comprises a negatively charged residue at position 351 and the second CH3 domain further comprises a positively charged residue at position 357; or ii) the first CH3 domain further comprises a positively charged residue at position 411 and the second CH3 domain further comprises a negatively charged residue at position 370, or the first CH3 domain or iii) the first CH3 domain further comprises a positively charged residue at position 364 and the second CH3 domain further comprises a negatively charged residue at position 370, or the first CH3 domain further comprises a negatively charged residue at position 370 and the second CH3 domain further comprises a positively charged residue at position 364; or a combination of i) and ii) or a combination of i) and iii), wherein the amino acid residue numbering is based on EU numbering. In some embodiments, the first CH3 domain further comprises a positively charged residue at position 356 and the second CH3 domain further comprises a negatively charged residue at position 439, or the first CH3 domain further comprises a negatively charged residue at position 439 and the second CH3 domain further comprises a positively charged residue at position 356; amino acid residue numbering is based on EU numbering. In some embodiments, i) the positively charged residue is a lysine (K) residue and the negatively charged residue is an aspartic acid (D) residue; or ii) the positively charged residue is a lysine (K) residue and the negatively charged residue is a glutamic acid (E) residue; or iii) the positively charged residue is an arginine (R) residue and the negatively charged residue is an aspartic acid (D) residue; or iv) the positively charged residue is an arginine (R) residue and the negatively charged residue is a glutamic acid (E) residue.In some embodiments, i) the first CH3 domain comprises E357K and T411K substitutions and the second CH3 domain comprises L351D and K370D substitutions, or the first CH3 domain comprises L351D and K370D substitutions and the second CH3 domain comprises E357K and T411K substitutions; or ii) the first CH3 domain comprises E357K and S364K substitutions and the second CH3 domain comprises L351D and K370D substitutions, or the H3 domain comprises L351D and K370D substitutions and the second CH3 domain comprises E357K and S364K substitutions; or iii) the first CH3 domain comprises D356K, E357K, and S364K substitutions and the second CH3 domain comprises L351D, K370D, and K439D substitutions, or the first CH3 domain comprises L351D, K370D, and K439D substitutions and the second CH3 domain comprises D356K, E357K, and S364K substitutions.

[0010] In some embodiments of any one of the above heterodimeric proteins, i) the first CH3 domain further comprises K392D and K409D substitutions and the second CH3 domain further comprises D356K and D399K substitutions, or the first CH3 domain further comprises D356K and D399K substitutions and the second CH3 domain further comprises K392D and K409D substitutions; or ii) the first CH3 domain further comprises , L368D and K370S substitutions and the second CH3 domain further comprises E357Q and S364K substitutions, or the first CH3 domain further comprises E357Q and S364K substitutions and the second CH3 domain further comprises L368D and K370S substitutions; or iii) the first CH3 domain further comprises L351K and T366K substitutions and the second CH3 domain further comprises L351D and L368E substitutions. or (iv) the first CH3 domain further comprises L351D and L368E substitutions and the second CH3 domain further comprises L351K and T366K substitutions; or (iv) the first CH3 domain further comprises P395K, P396K, and V397K substitutions and the second CH3 domain further comprises T394D, P395D, and P396D substitutions, or the first CH3 domain further comprises T394D, P395D, and P396D substitutions. and the second CH3 domain further comprises P395K, P396K, and V397K substitutions; or (v) the first CH3 domain further comprises F405E, Y407E, and K409E substitutions and the second CH3 domain further comprises F405K and Y407K substitutions; or the first CH3 domain further comprises F405K and Y407K substitutions and the second CH3 domain further comprises F405E, Y407E, and K409E substitutions.

[0011] In some embodiments of any one of the above heterodimeric proteins, i) the first CH3 domain comprises E357K, S364K, and N390C substitutions and the second CH3 domain comprises L351D, K370D, and S400C substitutions; or i) the first CH3 domain comprises L351D, K370D, and S400C substitutions and the second CH3 domain comprises E357K, S364K, and or ii) the first CH3 domain comprises E357K, S364K, and S400C substitutions and the second CH3 domain comprises L351D, K370D, and N390C substitutions, or the first CH3 domain comprises L351D, K370D, and N390C substitutions and the second CH3 domain comprises E357K, S364K, and S400C substitutions; or iii) the first CH3 domain comprises D356K, E357K, S364K, and S400C substitutions and the second CH3 domain comprises L351D, K370D, N390C, and K439D substitutions, or the first CH3 domain comprises L351D, K370D, N390C, and K439D substitutions and the second CH3 domain comprises D356K, E357K, S364K, and S400C substitutions; or iv) the first CH3 domain comprises D356K, E357K, S364K, and N390C substitutions and the second CH3 domain comprises L351D, K370D, K439D, and S400C substitutions, or the first CH3 domain comprises L351D, K370D, K439D, and S400C substitutions and the second CH3 domain comprises D356K, E357K, S364K, and N390C substitutions.

[0012] In some embodiments of any one of the above heterodimeric proteins, the first CH3 domain and the second CH3 domain further comprise knob-into-hole residues. In some embodiments, i) the first CH3 domain comprises T336S, L368A, and Y407V substitutions and the second CH3 domain comprises T366W substitution, or the first CH3 domain comprises T366W substitution and the second CH3 domain comprises T336S, L368A, and Y407V substitutions; or ii) the first CH3 domain comprises L368V and Y407V substitutions and the second CH3 domain comprises T366W substitution, or the first CH3 domain comprises T366W substitution and the second CH3 domain comprises L368V and Y407V substitutions.

[0013] Another aspect of the present application provides a heterodimeric protein comprising a first polypeptide comprising a first CH3 domain and a second polypeptide comprising a second CH3 domain, wherein i) the first CH3 domain comprises a positively charged residue at position 357 and the second CH3 domain comprises a negatively charged residue at position 351, or the first CH3 domain comprises a negatively charged residue at position 351 and the second CH3 domain comprises a positively charged residue at position 357; or ii) the first CH3 domain comprises a positively charged residue at position 411 and the second CH3 domain comprises a negatively charged residue at position 412. or iii) the first CH3 domain comprises a negatively charged residue at position 364 and the second CH3 domain comprises a negatively charged residue at position 370, or the first CH3 domain comprises a negatively charged residue at position 370 and the second CH3 domain comprises a positively charged residue at position 411; or iii) the first CH3 domain comprises a positively charged residue at position 364 and the second CH3 domain comprises a negatively charged residue at position 370, or the first CH3 domain comprises a negatively charged residue at position 370 and the second CH3 domain comprises a positively charged residue at position 364, where amino acid residue numbering is based on EU numbering. In some embodiments, the first CH3 domain comprises a positively charged residue at position 356 and the second CH3 domain comprises a negatively charged residue at position 439, or the first CH3 domain comprises a negatively charged residue at position 439 and the second CH3 domain comprises a positively charged residue at position 356; where amino acid residue numbering is based on EU numbering. In some embodiments, i) the positively charged residue is a lysine (K) residue and the negatively charged residue is an aspartic acid (D) residue; or ii) the positively charged residue is a lysine (K) residue and the negatively charged residue is a glutamic acid (E) residue; or iii) the positively charged residue is an arginine (R) residue and the negatively charged residue is an aspartic acid (D) residue; or iv) the positively charged residue is an arginine (R) residue and the negatively charged residue is a glutamic acid (E) residue.In some embodiments, i) the first CH3 domain comprises E357K and T411K substitutions and the second CH3 domain comprises L351D and K370D substitutions, or the first CH3 domain comprises L351D and K370D substitutions and the second CH3 domain comprises E357K and T411K substitutions; or ii) the first CH3 domain comprises E357K and S364K substitutions and the second CH3 domain comprises L351D and K370D substitutions, or the H3 domain comprises L351D and K370D substitutions and the second CH3 domain comprises E357K and S364K substitutions; or iii) the first CH3 domain comprises D356K, E357K, and S364K substitutions and the second CH3 domain comprises L351D, K370D, and K439D substitutions, or the first CH3 domain comprises L351D, K370D, and K439D substitutions and the second CH3 domain comprises D356K, E357K, and S364K substitutions.

[0014] In some embodiments of any one of the above heterodimeric proteins, i) the first CH3 domain further comprises a K392C substitution and the second CH3 domain further comprises a D399C substitution, or the first CH3 domain further comprises a D399C substitution and the second CH3 domain further comprises a K392C substitution; or ii) the first CH3 domain further comprises a Y394C substitution and the second CH3 domain further comprises a Y394C substitution. , and further comprises a S354C substitution, or wherein the first CH3 domain further comprises a S354C substitution and the second CH3 domain further comprises a Y394C substitution; or iii) wherein the first CH3 domain further comprises a D356C substitution and the second CH3 domain further comprises a Y349C substitution, or wherein the first CH3 domain further comprises a Y349C substitution and the second CH3 domain further comprises a D356C substitution.

[0015] In some embodiments of any one of the above heterodimeric proteins, the first CH3 domain and the second CH3 domain are human CH3 domains.

[0016] In some embodiments of any one of the above heterodimeric proteins, the first polypeptide and the second polypeptide each comprise, from N-terminus to C-terminus, at least a portion of an immunoglobulin hinge region, an immunoglobulin heavy chain constant domain 2 (CH2 domain), and a CH3 domain. In some embodiments, the CH2 domain and the CH3 domain form an IgG Fc region. In some embodiments, the Fc region is of the human IgG1 subclass. In some embodiments, the Fc region is of the human IgG4 subclass. In some embodiments, the Fc region further comprises a S228P substitution. In some embodiments, the Fc region further comprises a N297A substitution.

[0017] In some embodiments of any one of the above heterodimeric proteins, the first polypeptide and the second polypeptide are antibody heavy chains. In some embodiments, the heterodimeric protein further comprises one or more antibody light chains. In some embodiments, the heterodimeric protein is a multispecific antibody.

[0018] In some embodiments of any one of the above heterodimeric proteins, the heterodimeric protein further comprises a third polypeptide and a fourth polypeptide; (i) the first polypeptide comprises a structure represented by the following formula: VH1-CH1-hinge-CH2-1st CH3-L1-scFv1(Ia), (ii) the second polypeptide comprises a structure represented by the formula: VH2-CH1-hinge-CH2-2nd CH3-L2-scFv2(IIa), (iii) the third polypeptide comprises a structure represented by the following formula: VL1-CL(Ib), (iv) the fourth polypeptide comprises a structure represented by the following formula: VL2-CL(IIb), VL1 is a first immunoglobulin light chain variable domain; VH1 is a first immunoglobulin heavy chain variable domain; VL2 is a second immunoglobulin light chain variable domain; VH2 is a second immunoglobulin heavy chain variable domain; scFv1 is a first single chain variable fragment; scFv2 is a second single chain variable fragment; CL is an immunoglobulin light chain constant domain; CH1 is immunoglobulin heavy chain constant domain 1; CH2 is immunoglobulin heavy chain constant domain 2; wherein VL1 and VH1 are a first Fv that specifically binds to a first target; VL2 and VH2 are a second Fv that specifically binds to a second target; scFv1 specifically binds to a third target; and scFv2 specifically binds to a fourth target. In some embodiments, scFv1 and scFv2 are identical. In some embodiments, the first Fv and the second Fv are identical. In some embodiments, the first Fv and the second Fv are different. In some embodiments, the first Fv specifically binds to PDL1, the second Fv specifically binds to CD137, and scFv1 and scFv2 specifically bind to CTLA-4. In some embodiments, scFv1 and / or scFv2 comprise, from N-terminus to C-terminus: VH-L-VL, where L is a peptide linker. In some embodiments, scFv1 and / or scFv2 comprise a first cysteine ​​residue at position 44 of VH and a second cysteine ​​residue at position 100 of VL, where the first and second cysteine ​​residues form a disulfide bond. In some embodiments, L1 and / or L2 are peptide linkers comprising the amino acid sequence of SEQ ID NO: 80 or SEQ ID NO: 81. In some embodiments, VL1 and VL2 are identical. In some embodiments, VL1 and VL2 are different.

[0019] In some embodiments of any one of the above heterodimeric proteins, the heterodimeric protein comprises a first polypeptide, a second polypeptide, and a third polypeptide; (i) the first polypeptide comprises a structure represented by the following formula: VH-CH1-hinge-CH2-1st CH3(IIIa), (ii) the second polypeptide comprises a structure represented by the formula: scFv-hinge-CH2-second CH3(IVa), (iii) the third polypeptide comprises a structure represented by the following formula: VL-CL(IIIb), VL is an immunoglobulin light chain variable domain; VH is an immunoglobulin heavy chain variable domain; scFv is a single-chain variable fragment; CL is an immunoglobulin light chain constant domain; CH1 is immunoglobulin heavy chain constant domain 1; CH2 is immunoglobulin heavy chain constant domain 2; hinge is an immunoglobulin hinge region connecting the CH1 and CH2 domains; VL and VH associate to form an Fv that specifically binds to a first target; and the scFv specifically binds to a second target. In some embodiments, the first target is a tumor antigen and the second target is CD3. In some embodiments, the first target is HER2. In some embodiments, the first target is a first immune checkpoint molecule and the second target is a second immune checkpoint molecule. In some embodiments, the first target is PDL1 and the second target is CD137. In some embodiments, the first target is CD137 and the second target is PDL1. In some embodiments, the scFv comprises, from N- to C-terminus: VH-L-VL, where L is a peptide linker. In some embodiments, the scFv comprises a first cysteine ​​residue at position 44 of VH and a second cysteine ​​residue at position 100 of VL, wherein the first and second cysteine ​​residues form a disulfide bond. In some embodiments, the scFv is fused to the hinge of a second polypeptide via a peptide linker comprising the amino acid sequence of SEQ ID NO:80 or SEQ ID NO:81.

[0020] In some embodiments of any one of the above heterodimeric proteins, the heterodimeric protein is an activatable antibody, and the heterodimeric protein comprises a first polypeptide, a second polypeptide, and a third polypeptide; (i) the first polypeptide comprises a structure represented by the following formula: VH-CH1-hinge-CH2-1st CH3 (Va), (ii) the second polypeptide comprises a structure represented by the formula: MM1-CM1-scFv-hinge-CH2-second CH3 (VIa), (iii) the third polypeptide comprises a structure represented by the following formula: MM2-CM2-VL-CL(IVb), VL is an immunoglobulin light chain variable domain; VH is an immunoglobulin heavy chain variable domain; scFv is a single-chain variable fragment; CL is an immunoglobulin light chain constant domain; CH1 is immunoglobulin heavy chain constant domain 1; CH2 is immunoglobulin heavy chain constant domain 2; hinge is an immunoglobulin hinge region connecting the CH1 and CH2 domains; MM1 is a first masking peptide; MM2 is a second masking peptide; CM1 is a first cleavable peptide; CM2 is a second cleavable peptide; VL and VH associate to form a first Fv that specifically binds to a first target; the scFv specifically binds to a second target; if CM1 is not cleaved, MM1 inhibits binding of the first Fv to the first target; if CM2 is not cleaved, MM2 inhibits binding of the scFv to the second target. In some embodiments, the first target is a tumor antigen and the second target is CD3. In some embodiments, MM1 comprises the amino acid sequence of SEQ ID NO: 35. In some embodiments, the first Fv comprises a VH comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 61, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 62, and / or a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 63. In some embodiments, the TBM comprises a VL comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 64, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 65, and / or a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 66. In some embodiments, the first target is HER2. In some embodiments, MM2 comprises the amino acid sequence of SEQ ID NO: 36. In some embodiments, the scFv comprises a VH comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 69, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 70, and / or a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 71. In some embodiments, the TBM comprises a VL comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 72, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 73, and / or a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 74.

[0021] One aspect of the present application provides an activatable antibody comprising a first polypeptide comprising, from N-terminus to C-terminus, a masking moiety (MM), a cleavable moiety (CM), and a target binding moiety (TBM), wherein the MM comprises the amino acid sequence of SEQ ID NO: 35; when the CM is not cleaved, the MM inhibits binding of the activatable antibody to human CD3; the CM comprises at least a first cleavage site; a) the TBM comprises a VL, and the activatable antibody further comprises a second polypeptide comprising a VH; b) the TBM comprises a VH, and the activatable antibody further comprises a second polypeptide comprising a VL; c) the TBM comprises, from N-terminus to C-terminus, a VL and a VH; or d) the TBM comprises, from N-terminus to C-terminus, a VH and a VL; when the CM is cleaved, the activatable antibody binds to human CD3 via the VH and VL. In some embodiments, the TBM comprises a VH comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 61, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 62, and / or a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 63. In some embodiments, the TBM comprises a VL comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 64, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 65, and / or a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 66.

[0022] One aspect of the present application provides an activatable antibody comprising a first polypeptide comprising, from N-terminus to C-terminus, a masking moiety (MM), a cleavable moiety (CM), and a target binding moiety (TBM), wherein the MM comprises the amino acid sequence of SEQ ID NO: 36; when the CM is not cleaved, the MM inhibits binding of the activatable antibody to human HER2; the CM comprises at least a first cleavage site; a) the TBM comprises a VL, and the activatable antibody further comprises a second polypeptide comprising a VH; b) the TBM comprises a VH, and the activatable antibody further comprises a second polypeptide comprising a VL; c) the TBM comprises, from N-terminus to C-terminus, a VL and a VH; or d) the TBM comprises, from N-terminus to C-terminus, a VH and a VL; when the CM is cleaved, the activatable antibody binds to human HER2 via the VH and VL. In some embodiments, the TBM comprises a VH comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 69, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 70, and / or a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 71. In some embodiments, the TBM comprises a VL comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 72, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 73, and / or a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 74.

[0023] In some embodiments of any one of the above activatable antibodies, the activatable antibody comprises a first polypeptide, a second polypeptide, and a third polypeptide: (i) the first polypeptide comprises a structure represented by the following formula: VH-CH1-hinge-CH2-1st CH3 (Va), (ii) the second polypeptide comprises a structure represented by the formula: MM1-CM1-scFv-hinge-CH2-second CH3 (VIa), (iii) the third polypeptide comprises a structure represented by the following formula: MM2-CM2-VL-CL(IVb), VL is an immunoglobulin light chain variable domain; VH is an immunoglobulin heavy chain variable domain; scFv is a single chain variable fragment; CL is an immunoglobulin light chain constant domain; CH1 is immunoglobulin heavy chain constant domain 1; CH2 is immunoglobulin heavy chain constant domain 2; The hinge is an immunoglobulin hinge region that connects the CH1 and CH2 domains, MM1 is the first masking peptide, MM2 is a second masking peptide, CM1 is the first cleavable peptide, CM2 is a second cleavable peptide, The VL and VH associate to form a first Fv that specifically binds to a first target; the scFv specifically binds to a second target; and the MM is MM1 or MM2.

[0024] In some embodiments of any one of the above activatable antibodies, the activatable antibody comprises an Fc region comprising a first CH3 domain and a second CH3 domain, wherein the first CH3 domain comprises D356K, E357K, S364K, and S400C substitutions and the second CH3 domain comprises L351D, K370D, N390C, and K439D substitutions; or wherein the first CH3 domain comprises L351D, K370D, N390C, and K439D substitutions and the second CH3 domain comprises D356K, E357K, S364K, and S400C substitutions.

[0025] One aspect of the present application provides one or more nucleic acids encoding a heterodimeric protein according to any one of the above heterodimeric proteins, or an activatable antibody according to any one of the above activatable antibodies, a vector(s) comprising the one or more nucleic acids, and a host cell comprising the one or more nucleic acids or the vector. In some embodiments, a method for preparing the heterodimeric protein or activatable antibody is provided, the method comprising: (a) culturing a host cell according to any one of the above host cells under conditions allowing expression of the one or more nucleic acids or vectors; and (b) recovering the heterodimeric protein or activatable antibody from the host cell culture.

[0026] One aspect of the present application provides a pharmaceutical composition comprising a heterodimeric protein according to any one of the above heterodimeric proteins, or an activatable antibody according to any one of the above activatable antibodies, and a pharmaceutically acceptable carrier.

[0027] One aspect of the present application provides a method for treating a disease or condition in a subject in need thereof, the method comprising administering to the subject an effective amount of any one of the pharmaceutical compositions described above. In some embodiments, the disease or condition is cancer. In some embodiments, the cancer is lung cancer. In some embodiments, the cancer is HER-2 positive cancer. In some embodiments, the cancer is ovarian cancer. In some embodiments, the cancer is prostate cancer or melanoma. In some embodiments, the cancer is advanced cancer.

[0028] Compositions, uses, kits, and articles of manufacture comprising any one of the above heterodimeric proteins are also provided. [Brief explanation of the drawings]

[0029] [Figure 1]

[0023] Figure 1 provides schematic diagrams of exemplary antibody designs of the present application. A shows a schematic diagram of the Fab-Fc / Fc1 arm scaffold. B shows a schematic diagram of a general light chain scaffold. [Figure 2] Figure 1 provides a schematic representation of exemplary antibody designs of the present application. A schematic representation of a bispecific scaffold in Morrison format is shown. Shown on the right are PD-L1 x CD137 and CD137 x PD-L1 bispecific antibodies in Morrison format. [Figure 3] Schematics of exemplary antibody designs of the present application are provided. On the right, a schematic of a trispecific scaffold comprising a trispecific antibody against PD-L1, CD137, and CTLA4 is shown. [Figure 4] Schematics of exemplary antibody designs of the present application are provided. On the right, a schematic of an ScFv bispecific scaffold is shown, including a schematic of a HER2 and CD3 bispecific antibody. [Figure 5] Schematics of exemplary antibody designs of the present application are provided. On the right, a schematic of an activatable scaffold is shown, including a schematic of an activatable antibody against HER2 and CD3. A masking peptide (represented as a ball) can be fused to the antigen-binding domain via a cleavable linker. [Figure 6] A 10% SDS-PAGE gel showing the yield of heterodimeric proteins is provided. Three bands correspond to light chain-heavy chain homodimer, light chain-heavy chain-Fc heterodimer, and light chain-heavy chain half-body, as indicated. [Figure 7] Figure 1 shows size-exclusion high-performance liquid chromatography data for the heterodimeric protein. Time is shown on the x-axis and relative protein abundance on the y-axis. Stars indicate peaks corresponding to the heterodimeric protein. [Figure 8] 1 provides size-exclusion high performance liquid chromatography analytical data showing the heterodimeric protein after 1 hour of incubation at the indicated temperatures. The x-axis indicates temperature (from left to right: control, 40°C, 50°C, 60°C, 65°C, or 67.5°C), and the y-axis indicates the peak area corresponding to the heterodimeric protein after incubation compared to the control (no incubation). [Figure 9]Figure 1 provides size-exclusion high-performance liquid chromatography spectra of the heterodimeric protein after storage for 7, 14, 21, or 28 days at 4°C or 37°C, as indicated. Time is on the x-axis and relative protein abundance is on the y-axis. [Figure 10] 1 shows the effect of bispecific antibodies in an NFκB-activated luciferase reporter assay. The x-axis shows the log-transformed antibody concentration in nM, and the y-axis shows the relative light units ("RLU") of the luciferase reporter. [Figure 11A] Figure 1 provides an assessment of the quality of purified anti-PDL1 and CD137 bispecific antibodies in different formats. Protein quality assessed by analytical size exclusion chromatography after 3 and 6 freeze-thaw cycles is shown. Time is shown on the x-axis, relative protein abundance on the y-axis, and the bispecific antibody format is indicated. [Figure 11B] Figure 1 provides an assessment of the quality of purified anti-PDL1 and CD137 bispecific antibodies in different formats. Protein quality assessed by analytical size exclusion chromatography after 28 days of incubation at 40°C is shown. Time is shown on the x-axis, relative protein abundance on the y-axis, and the bispecific antibody format is indicated. [Figure 11C] This figure provides an evaluation of the quality of purified anti-PDL1 and CD137 bispecific antibodies in different formats. This figure also provides an analysis of the thermal stability of purified anti-PDL1 and CD137 bispecific antibodies in different formats. The antibodies were incubated for 1 hour at the temperatures indicated on the x-axis (from left to right: control, 50°C, 60°C, 65°C, and 70°C), and the y-axis represents the percentage of the major peak area. PDL1xCD137 TYF01 is indicated by squares, CD137xPDL1 TYF01 is indicated by circles, PDL1xCD137 TYF02 is indicated by triangles pointing up, and CD137xPDL1 TYF02 is indicated by triangles pointing down. [Figure 12] 1 provides flow cytometry analysis of anti-PDL1xCD137 bispecific antibody binding to PDL1 and CD137. The x-axis of each plot represents antigen presentation in the APC channel, and the y-axis represents ligand binding in the PE channel. [Figure 13] Figure 1 provides flow cytometry analysis of binding and cross-reactivity of anti-PDL1 and CD137 bispecific antibodies. The TYF01 antibody is shown in the top set of plots, and the TYF02 antibody is shown in the bottom set of plots. The x-axis of each plot represents antigen display in the FITC channel, and the y-axis represents antibody binding in the APC channel. The ability of bispecific antibodies in different formats to bind to PDL1 or CD137 of mouse, monkey, or human origin was tested, as indicated. [Figure 14]

[0033] Figure 1 provides the effect of PDL1xCD137 and CD137xPDL1 bispecific antibodies on PDL1 and CD137 reporter gene assays. The top plot shows the PDL1 reporter gene assay, with log-transformed antibody concentration in ng / ml on the x-axis and relative luminescence units ("RLU") on the y-axis. In the PDL1 reporter gene assay plot, squares represent the PDL1xCTLA4 bispecific antibody, downward-pointing triangles represent PDL1 monomer, circles represent the PDL1xCD137 bispecific antibody, and upward-pointing triangles represent the CD137xPDL1 bispecific antibody. The bottom plot shows the CD137-NFκB reporter gene assay, with antibody concentration in nM on the x-axis and relative luminescence units ("RLU") on the y-axis. In the CD137-NFκB reporter assay plot, squares indicate PDL1xCD137 bispecific antibody, upward pointing triangles indicate CD137xPDL1 bispecific antibody, diamonds indicate CD137xCTLA4 bispecific antibody, downward pointing triangles indicate CD137 monomer, and circles indicate negative control. [Figure 15A] Figure 1 shows the effect of anti-PDL1 and / or anti-CD137 antibodies on in vivo tumor growth in a 3LL syngeneic mouse model. The x-axis indicates days after treatment initiation, and the y-axis indicates tumor volume (mm). Mono-IgG, bispecific, or trispecific antibodies were tested alone or in combination at the concentrations indicated. [Figure 15B]Figure 1 shows the effect of anti-PDL1 and / or anti-CD137 antibodies on in vivo tumor growth in a 3LL syngeneic mouse model. The x-axis indicates days after treatment initiation, and the y-axis indicates tumor volume (mm). Mono-IgG, bispecific, or trispecific antibodies were tested alone or in combination at the concentrations indicated. [Figure 15C] Figure 1 shows the effect of anti-PDL1 and / or anti-CD137 antibodies on in vivo tumor growth in a 3LL syngeneic mouse model. The x-axis indicates days after treatment initiation, and the y-axis indicates tumor volume (mm). Mono-IgG, bispecific, or trispecific antibodies were tested alone or in combination at the concentrations indicated. [Figure 16] Characterization of bispecific antibodies by SDS-PAGE electrophoresis is shown. The gel on the left is a 12% SDS-PAGE gel under reducing conditions, and the gel on the right is a 4-15% SDS-PAGE gel under non-reducing conditions. The MW lanes indicate molecular weight markers, which are labeled in kilodaltons on the left side of each gel. In both gels, lane 1 indicates antibody TY24051, lane 2 indicates antibody TY24052, and lane 3 indicates antibody TY24053. [Figure 17] Figure 1 shows size-exclusion high-performance liquid chromatography analysis of bispecific antibodies. The top plot shows the TY24051 antibody, the middle plot shows the TY24105 antibody, and the bottom plot shows the TY24106 antibody. For each plot, time is shown on the x-axis and relative protein abundance on the y-axis. Peaks corresponding to heterodimeric proteins and aggregates are indicated. [Figure 18] Figure 1 provides enzyme-linked immunosorbent assay (ELISA) analysis of antibodies TY24051 and TY24052. A shows HER2 binding by TY24051 (squares), TY24052 (triangles pointing up), and TY24052 after activation (triangles pointing down). B shows CD3 binding by TY24051 (squares), TY24052 (triangles pointing up), and TY24052 after activation (triangles pointing down). A and B show antibody concentration in M ​​on the x-axis and absorbance at 450 nm on the y-axis. [Figure 19]Assay of T cell-mediated cytotoxic killing upon treatment with bispecific antibodies. Antibody concentration (ng / ml) is shown on the x-axis, and percentage of cell lysis is shown on the y-axis. Target cells were incubated with T cells for 24 hours with TY24051 (circles), TY24052 (squares), isotype control (upward triangles), or no antibody (downward triangles). DETAILED DESCRIPTION OF THE INVENTION

[0030] The present application provides heterodimeric proteins comprising a CH3 domain with engineered disulfide bond(s) and / or salt bridge(s), including multispecific antibodies, e.g., bispecific antibodies, comprising an Fc region with engineered disulfide bond(s) and / or salt bridge(s). In some embodiments, the heterodimeric protein comprises an engineered disulfide bond between C390 of the first CH3 domain and C400 of the second CH3 domain, between C392 of the first CH3 domain and C397 of the second CH3 domain, or between C392 of the first CH3 domain and C400 of the second CH3 domain. In some embodiments, the heterodimeric protein comprises a rearranged salt bridge network compared to the wild-type CH3 domain, for example, between positions 357 and 411 of the first CH3 domain and positions 351 and 370 of the second CH3 domain (e.g., E357K:T411K-L351'D:K370'D), or between positions 357 and 364 of the first CH3 domain and positions 351 and 370 of the second CH3 domain (e.g., E357K:S364K-L351'D:K370'D). In some embodiments, the heterodimeric protein comprises an inverted salt bridge between position 356 of the first CH3 domain and position 439 of the second CH3 domain (e.g., D356-K439') compared to the wild-type CH3 domain. The heterodimeric proteins described herein provide a platform for preparing multispecific proteins and antibodies in a variety of formats with high yields, excellent stability (e.g., resistance to aggregation and precipitation due to high temperature or freeze-thaw cycles), and potent activity.

[0031] I. Definition Unless otherwise defined below, terms are used herein in the sense commonly used in the art.

[0032] The term "antibody" as used herein is used in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, provided they exhibit the desired antigen-binding activity.

[0033] The term "antibody" includes, but is not limited to, fragments capable of binding to antigen, such as Fv, Fab, Fab', and (Fab')2. Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, the name reflecting their ability to crystallize readily. Pepsin treatment yields an F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen. The term antibody also includes, but is not limited to, chimeric antibodies, humanized antibodies, and antibodies of various species, such as mouse, human, and cynomolgus monkey.

[0034] The term "antigen-binding fragment" refers to one or more portions of an antibody that retain the ability of the antibody to bind to an antigen. Examples of "antigen-binding fragments" of antibodies include, but are not limited to, (i) a Fab fragment (a monovalent fragment consisting of the VL, VH, CL, and CH1 domains); (ii) a F(ab')2 fragment (a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region); (iii) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a single-chain Fv fragment comprising the VH and VL domains of an antibody (in which the VH and VL domains are fused together); and (vi) a single-chain Fab fragment comprising a single polypeptide comprising the VL, VH, CL, and CH1 domains.

[0035] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical and / or bind to the same epitope. This excludes possible variant antibodies that contain, for example, naturally occurring mutations or that arise during production of the monoclonal antibody preparation, although such variants are generally present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention may be produced by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci; such methods and other exemplary methods for producing monoclonal antibodies are described herein.

[0036] As used herein, the term "hypervariable region" or "HVR" refers to each of the regions of an antibody variable domain that are hypervariable in sequence. HVRs may form structurally defined loops ("hypervariable loops"). Generally, naturally occurring four-chain antibodies contain six HVRs: three in the VH (H1, H2, and H3) and three in the VL (L1, L2, and L3). HVRs generally contain amino acid residues from the hypervariable loops and / or "complementarity-determining regions" (CDRs), which are those with the highest sequence variability and / or are involved in antigen recognition. Exemplary hypervariable loops are located at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3). (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). Exemplary CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) are located at amino acid residues 24-34 of L1, 50-56 of L2, 89-97 of L3, 31-35B of H1, 50-65 of H2, and 95-102 of H3 (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)). With the exception of CDR1 of VH, CDRs generally comprise amino acid residues that form hypervariable loops. CDRs also contain "specificity-determining residues" or "SDRs," which are residues that contact the antigen. SDRs are contained within regions of CDRs called truncated CDRs or a-CDRs. Exemplary a-CDRs (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2, and a-CDR-H3) are located at amino acid residues 31-34 of L1, 50-55 of L2, 89-96 of L3, 31-35B of H1, 50-58 of H2, and 95-102 of H3. (See Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)).Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al., supra.

[0037] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The heavy and light chain variable domains of natural antibodies (VH and VL, respectively) generally have a similar structure, with each domain containing four framework regions (FR) and three hypervariable regions (HVR), arranged from the amino terminus to the carboxy terminus in the following order: FR1, HVR1, FR2, HVR2, FR3, HVR3, FR4. (See, for example, Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007)). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a specific antigen may be isolated by using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0038] The terms "EU numbering" or "amino acid position numbering based on EU numbering" and variations thereof refer to the numbering system used for the heavy chain constant domains of antibodies in Edelman, GM et al., Proc. Natl. Acad. USA, 63, 78-85 (1969). The EU numbering of residues can be determined for a given antibody by aligning the "standard" EU numbered sequence with the homologous regions of the antibody's sequence.

[0039] The Kabat numbering system is commonly used to refer to residues within the variable domain (roughly corresponding to residues 1-107 in the light chain and residues 1-113 in the heavy chain) (e.g., Kabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to shortening or insertion of the FRs or HVRs of the variable domain. For example, a heavy chain variable domain may contain a single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat), inserted residues after residue 82 of the heavy chain FR (e.g., residues 82a, 82b, and 82c according to Kabat, etc.). The Kabat numbering of residues can be determined for a given antibody by aligning the "standard" Kabat numbered sequence with the homologous regions of the antibody's sequence.

[0040] For heterodimeric proteins having two CH3 domains, a given amino acid position in the first CH3 domain is designated X, and the corresponding amino acid position in the second CH3 domain is designated X'. For example, N390C-S400'C refers to a heterodimeric protein having a first CH3 domain with an N390C mutation and a second CH3 domain with an S400C mutation. All mutations or substitutions in the heterodimeric proteins described herein are referred to herein with respect to the wild-type, naturally occurring CH3 domain.

[0041] Unless otherwise indicated, all formulas of polypeptide chains described herein list the components of the polypeptide in N- to C-terminal order. For example, the formula VH1-CH1-hinge-CH2-first CH3-L1-scFv1 indicates that the polypeptide comprises, from N- to C-terminus, the following structural components: VH1, CH1, hinge, CH2, first CH3, L1, and scFv1.

[0042] As used herein, the term "heavy chain constant region" refers to a region comprising at least three heavy chain constant domains, CH1, CH2, and CH3, as well as the hinge region between CH1 and CH2. Non-limiting examples of heavy chain constant regions include gamma, delta, and alpha. Non-limiting examples of heavy chain constant regions also include epsilon and mu. Each heavy chain constant region corresponds to an antibody isotype. For example, an antibody comprising a gamma constant region is an IgG antibody, an antibody comprising a delta constant region is an IgD antibody, and an antibody comprising an alpha constant region is an IgA antibody. Furthermore, an antibody comprising a mu constant region is an IgM antibody, and an antibody comprising an epsilon constant region is an IgE antibody. A particular isotype can be further subdivided into subclasses. For example, IgG antibodies include, but are not limited to, IgG1 (containing a γ1 constant region), IgG2 (containing a γ2 constant region), IgG3 (containing a γ3 constant region), and IgG4 (containing a γ4 constant region) antibodies; IgA antibodies include, but are not limited to, IgA1 (containing an α1 constant region) and IgA2 (containing an α2 constant region) antibodies; and IgM antibodies include, but are not limited to, IgM1 and IgM2.

[0043] The term "CH2 domain" of the human IgG Fc region typically spans approximately residues 231 to 340 of IgG in the EU numbering system. The CH2 domain is unique in that it is not tightly paired with another domain. Rather, two N-linked branched carbohydrate chains are inserted between the two CH2 domains in intact native IgG molecules. It has been speculated that carbohydrates may provide a surrogate for domain-domain pairing and help stabilize the CH2 domain. Burton, Molec. Immunol. 22:161-206 (1985).

[0044] The term "CH3 domain" includes the stretch of residues from the C-terminus of the Fc region to the CH2 domain (ie, from about amino acid residue 341 to about amino acid residue 447 of IgG according to the EU numbering system).

[0045] As used herein, the term "heavy chain" refers to a polypeptide comprising at least a heavy chain variable region, with or without a leader sequence. In some embodiments, a heavy chain also comprises at least a portion of a heavy chain constant region. As used herein, the term "full-length heavy chain" refers to a polypeptide comprising a heavy chain variable region and a heavy chain constant region, with or without a leader sequence.

[0046] As used herein, the term "light chain constant region" refers to a region comprising a light chain constant domain, CL. Non-limiting exemplary light chain constant regions include λ and κ.

[0047] As used herein, the term "light chain" refers to a polypeptide comprising at least a light chain variable region, with or without a leader sequence. In some embodiments, a light chain also comprises at least a portion of a light chain constant region. As used herein, the term "full-length light chain" refers to a polypeptide comprising a light chain variable region and a light chain constant region, with or without a leader sequence.

[0048] "Affinity" refers to the strength of the sum of non-covalent interactions between the binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). The affinity of molecule X for partner Y is generally determined by the dissociation constant (K d ) Affinity can be measured by common methods known in the art, including those described herein. In the context of multispecific antibodies (e.g., bispecific or trispecific antibodies), the affinity of the antibody to each binding specificity (i.e., target) can be measured.

[0049] The terms "bind," "specifically bind," or "specific for" refer to a measurable, reproducible interaction, such as binding between a target and an antibody, which determines the presence of a target in the presence of a heterogeneous population of molecules, including biomolecules. For example, an antibody that binds to or specifically binds to a target (which may be an epitope) is an antibody that binds to this target with greater affinity, avidity, more readily, and / or longer duration than it binds to other targets. In some embodiments, the extent of binding of an antibody to an unrelated target is less than about 10% of the binding of the antibody to the target (e.g., as measured by radioimmunoassay (RIA)). In some embodiments, an antibody that specifically binds to a target has a dissociation constant (Kd) of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, or ≦0.1 nM. In some embodiments, an antibody specifically binds to an epitope on a protein that is conserved among proteins from different species. In some embodiments, specific binding can include, but does not require, exclusive binding.

[0050] The term "multispecific" when used in connection with an antibody refers to an antibody that has polyepitopic specificity (i.e., capable of specifically binding to two, three, or more different epitopes on one biomolecule, or capable of specifically binding to epitopes on two, three, or more different biomolecules).

[0051] An "affinity matured" antibody refers to an antibody with one or more modifications in one or more hypervariable regions (HVRs), compared to a parent antibody (not possessing such modifications), which modifications result in an improvement in the affinity of the antibody for antigen. In some examples, an affinity matured antibody refers to an antibody with one or more modifications in one or more complementarity determining regions (CDRs), compared to a parent antibody (not possessing such modifications), which modifications result in an improvement in the affinity of the antibody for antigen.

[0052] As used herein, a "chimeric antibody" refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species. In some embodiments, a chimeric antibody refers to an antibody that comprises at least one variable region derived from a first species (e.g., mouse, rat, cynomolgus monkey, etc.) and at least one constant region derived from a second species (e.g., human, cynomolgus monkey, etc.). In some embodiments, a chimeric antibody comprises at least one mouse variable region and at least one human constant region. In some embodiments, a chimeric antibody comprises at least one cynomolgus monkey variable region and at least one human constant region. In some embodiments, all of the variable regions of a chimeric antibody are derived from a first species, and all of the constant regions of a chimeric antibody are derived from a second species.

[0053] As used herein, "humanized antibody" refers to an antibody in which at least one amino acid in the framework region of a non-human variable region has been replaced with the corresponding amino acid from a human variable region. In some embodiments, a humanized antibody comprises at least one human constant region or fragment thereof. In some embodiments, a humanized antibody is Fab, (Fab'), etc.

[0054] As used herein, "HVR-grafted antibody" refers to a humanized antibody in which one or more hypervariable regions (HVRs) of a first (non-human) species have been grafted onto framework regions (FRs) of a second (human) species. In some examples, "CDR-grafted antibody" as used herein refers to a humanized antibody in which one or more complementarity-determining regions (CDRs) of a first (non-human) species have been grafted onto framework regions (FRs) of a second (human) species.

[0055] As used herein, "human antibody" refers to antibodies produced in humans, antibodies produced in non-human animals that contain human immunoglobulin genes, e.g., XENOMOUSE®, and antibodies selected using in vitro methods such as phage display, where the antibody repertoire is based on human immunoglobulin sequences.

[0056] The terms "nucleic acid molecule," "nucleic acid," and "polynucleotide" may be used interchangeably and refer to a polymer of nucleotides. Such polymers of nucleotides may contain natural and / or non-natural nucleotides and include, but are not limited to, DNA, RNA, and PNA. A "nucleic acid sequence" refers to the linear sequence of nucleotides that comprises a nucleic acid molecule or polynucleotide.

[0057] The terms "polypeptide" and "peptide" are used interchangeably to refer to a polymer of amino acid residues and are not limited to a minimum length. Such a polymer of amino acid residues may contain natural or unnatural amino acid residues. Both full-length proteins and fragments thereof are encompassed by the definition. The term also includes post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, and the like. Furthermore, "polypeptide" includes modifications to the native sequence, such as deletions, additions, and substitutions (generally conservative in nature), so long as the polypeptide maintains the desired activity. These modifications may be deliberate (e.g., via site-directed mutagenesis) or accidental (e.g., via host mutation), which may result in a protein or errors due to PCR amplification.

[0058] A polypeptide "variant" refers to a biologically active polypeptide having at least about 80% amino acid sequence identity with a native sequence polypeptide, after aligning the sequences to achieve the maximum percent sequence identity (without considering any conservative substitutions as part of the sequence identity) and introducing gaps, if necessary. Such variants include, for example, polypeptides in which one or more amino acid residues are added or deleted at the N- or C-terminus of the polypeptide. In some embodiments, a variant has at least about 80% amino acid sequence identity. In some embodiments, a variant has at least about 90% amino acid sequence identity. In some embodiments, a variant has at least about 95% amino acid sequence identity with a native sequence polypeptide.

[0059] As used herein, "percent (%) amino acid sequence identity" with respect to a peptide, polypeptide, or antibody sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues of a particular peptide or polypeptide sequence after aligning the sequences to achieve the maximum percent sequence identity (not considering any conservative substitutions as part of the sequence identity) and introducing gaps as necessary. Alignment to determine percent amino acid sequence identity can be achieved in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN™ (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared.

[0060] Amino acid substitutions may include, but are not limited to, replacing one amino acid in a polypeptide with another. Exemplary substitutions are shown in Table A. Amino acid substitutions may be introduced into an antibody of interest, and the products may be screened for a desired activity, such as retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC. [Table 1]

[0061] Amino acids may be grouped according to common side chain properties: (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile, (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln, (3) Acidic: Asp, Glu, (4) Basic: His, Lys, Arg, (5) Residues that influence chain orientation: Gly, Pro, (6) Aromatic: Trp, Tyr, Phe. Non-conservative substitutions will involve exchanging a member of one of these classes for another class.

[0062] The term "vector" is used to describe a polynucleotide that can be engineered to contain cloned polynucleotide(s) that can be propagated in a host cell. A vector may contain one or more of the following elements: an origin of replication, one or more regulatory sequences (e.g., promoters and / or enhancers) that control the expression of a polypeptide of interest, and / or one or more selectable marker genes (e.g., antibiotic resistance genes and genes that can be used in colorimetric assays, e.g., β-galactosidase). The term "expression vector" refers to a vector used to express a polypeptide of interest in a host cell.

[0063] A "host cell" refers to a cell that can be or is the recipient of a vector or isolated polynucleotide. A host cell can be a prokaryotic or eukaryotic cell. Exemplary eukaryotic cells include mammalian cells, e.g., primate or non-primate cells; fungal cells, e.g., yeast; plant cells; and insect cells. Non-limiting examples of mammalian cells include, but are not limited to, NSO cells, PER.C6® cells (Crucell), and 293 and CHO cells, and their derivatives, e.g., 293-6E and DG44 cells, respectively. The term "cell" includes the primary subject cell and its progeny.

[0064] As used herein, the term "isolated" refers to a molecule that is separated from at least some of the components with which it is normally found or produced in nature. For example, a polypeptide is referred to as "isolated" if it is separated from at least some of the components of the cell in which it is produced. If a polypeptide is secreted by a cell after expression, physically separating the supernatant containing it from the cell that produced it is considered to be "isolating" the polypeptide. Similarly, a polynucleotide is referred to as "isolated" if it is not part of a larger polynucleotide with which it is normally found in nature (e.g., genomic DNA or mitochondrial DNA in the case of a DNA polynucleotide), or if it is separated from at least some of the components of the cell in which it is produced, for example, in the case of an RNA polynucleotide. Thus, a DNA polynucleotide contained in a vector within a host cell can be referred to as "isolated."

[0065] The terms "individual" or "subject" are used interchangeably herein to refer to a mammal. In some embodiments, methods are provided for treating mammals, including but not limited to humans, rodents, monkeys, cats, dogs, horses, cows, pigs, sheep, goats, mammalian laboratory animals, mammalian farm animals, mammalian sport animals, and mammalian pets. In some examples, "individual" or "subject" refers to an individual or subject in need of treatment for a disease or disorder.

[0066] As used herein, "treatment" or "treating" is an approach to obtaining beneficial or desired results, including clinical results. For purposes of the present invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: reducing one or more symptoms attributable to the disease, alleviating the extent of the disease, stabilizing the disease (e.g., preventing or slowing the worsening of the disease), preventing or slowing the spread of the disease (e.g., metastasis), preventing or slowing the recurrence of the disease, slowing or slowing the progression of the disease, ameliorating the condition of the disease, providing remission (partial or total) of the disease, reducing the dosage of one or more other therapeutic agents required to treat the disease, slowing the progression of the disease, improving quality of life, and / or prolonging survival. Reduction of the pathological consequences of cancer is also encompassed by "treatment." The methods of the present invention contemplate any one or more of these aspects of treatment.

[0067] The term "prevent" and similar terms such as "prevented," "preventing," etc. refer to an approach for preventing, inhibiting, or reducing the likelihood of recurrence of a disease or condition, e.g., cancer. It also refers to delaying the recurrence of the disease or condition, or delaying the recurrence of symptoms of the disease or condition. As used herein, "prevention" and similar terms also include reducing the extent, impact, symptoms, and / or burden of a disease or condition prior to the recurrence of the disease or condition.

[0068] As used herein, "delaying" the onset of cancer means extending, preventing, slowing, inhibiting, stabilizing, and / or postponing the onset of the disease. This delay can be of varying duration, depending on the history of the disease and / or the individual being treated. A method of "delaying" the onset of cancer is one that reduces the likelihood of disease onset in a given timeframe and / or reduces the extent of disease in a given timeframe compared to not using the method. Such comparisons are usually based on clinical studies using a statistically significant number of individuals. Cancer onset can be detected using standard methods, including, but not limited to, computed tomography (CAT scan), magnetic resonance imaging (MRI), abdominal ultrasound, coagulation tests, arteriography, or biopsy. Onset also refers to the progression of cancer that was initially undetectable, including onset, recurrence, and development.

[0069] As used herein, the term "effective amount" refers to an amount of an agent or combination of agents sufficient to treat a particular disorder, condition, or disease, for example, to ameliorate, alleviate, relieve, and / or delay one or more of the symptoms. With respect to cancer, an effective amount includes an amount sufficient to shrink a tumor and / or reduce the rate of tumor growth (e.g., inhibit tumor growth), or prevent or delay other undesirable cell proliferation. In some embodiments, an effective amount is an amount sufficient to delay the onset of the disease. In some embodiments, an effective amount is an amount sufficient to prevent or delay recurrence. An effective amount can be administered in one or more administrations. An effective amount of a drug or composition can (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) inhibit, suppress, slow to some extent, and preferably stop cancer cell invasion into peripheral organs; (iv) inhibit (i.e., slow to some extent, and preferably stop) tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay tumor onset and / or recurrence; and / or (vii) alleviate to some extent one or more symptoms associated with cancer.

[0070] It is understood that embodiments of the invention described herein include "consisting of" and / or "consisting essentially of" embodiments.

[0071] Reference herein to "about" a value or parameter includes (and accounts for) variations on that value or parameter itself. For example, a statement referring to "about X" includes the statement "X."

[0072] As used herein, a reference to a value or parameter "not" generally means and describes a value or parameter "other than." For example, a method is not used to treat cancer type X means that the method is used to treat cancers other than type X.

[0073] As used herein, the term "about XY" has the same meaning as "about X to about Y."

[0074] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0075] As used herein, the term "and / or," phrases such as "A and / or B," are intended to include both A and B; A or B; A (alone); and B (alone). Similarly, as used herein, the term "and / or," phrases such as "A, B, and / or C," are intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0076] It is understood that certain features of the invention that are described for clarity in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, for brevity, various features of the invention that are described in the context of a single embodiment may also be provided separately or in any suitable subcombination. All combinations of embodiments relating to heterodimeric proteins are specifically embraced by the present invention and are disclosed herein as if each and every combination were individually and expressly disclosed. In addition, all subcombinations of heterodimeric proteins listed in embodiments describing such variations are also specifically embraced by the present invention and are disclosed herein as if each and every such subcombination of heterodimeric proteins were individually and expressly disclosed herein.

[0077] II. Heterodimeric Proteins The present application provides heterodimeric proteins comprising a CH3 domain with any one or combination of engineered residues that promote heterodimer formation, as described in the subsection "CH3 Domain Mutations." Heterodimers comprising multiple heterodimers formed by a first polypeptide comprising a first engineered CH3 domain and a second polypeptide comprising a second engineered CH3 domain are also contemplated herein.

[0078] In some embodiments, a heterodimeric protein (e.g., a multispecific antibody) is provided comprising a first polypeptide comprising a first CH3 domain and a second polypeptide comprising a second CH3 domain, wherein i) the first CH3 domain comprises a cysteine ​​(C) residue at position 390 and the second CH3 domain comprises a cysteine ​​residue at position 400, or the first CH3 domain comprises a cysteine ​​residue at position 400 and the second CH3 domain comprises a cysteine ​​residue at position 390; or ii) the first CH3 domain comprises a cysteine ​​residue at position 392. and the second CH3 domain comprises a cysteine ​​residue at position 397, or the first CH3 domain comprises a cysteine ​​residue at position 397 and the second CH3 domain comprises a cysteine ​​residue at position 392; or iii) the first CH3 domain comprises a cysteine ​​residue at position 392 and the second CH3 domain comprises a cysteine ​​residue at position 400, or the first CH3 domain comprises a cysteine ​​residue at position 400 and the second CH3 domain comprises a cysteine ​​residue at position 392; amino acid residue numbering is based on EU numbering.

[0079] In some embodiments, a heterodimeric protein (e.g., a multispecific antibody) is provided comprising a first polypeptide comprising a first CH3 domain and a second polypeptide comprising a second CH3 domain, wherein i) the first CH3 domain further comprises a positively charged residue at position 357 and the second CH3 domain further comprises a negatively charged residue at position 351, or the first CH3 domain further comprises a negatively charged residue at position 351 and the second CH3 domain further comprises a positively charged residue at position 357; or ii) the first CH3 domain further comprises a positively charged residue at position 411 and the second CH3 domain further comprises a positively charged residue at position 370. or wherein the first CH3 domain further comprises a negatively charged residue at position 370 and the second CH3 domain further comprises a positively charged residue at position 411; or iii) the first CH3 domain further comprises a positively charged residue at position 364 and the second CH3 domain further comprises a negatively charged residue at position 370, or wherein the first CH3 domain further comprises a negatively charged residue at position 370 and the second CH3 domain further comprises a positively charged residue at position 364; or a combination of i) and ii) or a combination of i) and iii), wherein the amino acid residue numbering is based on EU numbering. In some embodiments, the first CH3 domain further comprises a positively charged residue at position 356 and the second CH3 domain further comprises a negatively charged residue at position 439, or the first CH3 domain further comprises a negatively charged residue at position 439 and the second CH3 domain further comprises a positively charged residue at position 356, where amino acid residue numbering is based on EU numbering.

[0080] In some embodiments, a heterodimeric protein (e.g., a multispecific antibody) is provided comprising a first polypeptide comprising a first CH3 domain and a second polypeptide comprising a second CH3 domain, wherein i) the first CH3 domain comprises a cysteine ​​(C) residue at position 390 and the second CH3 domain comprises a cysteine ​​residue at position 400, or the first CH3 domain comprises a cysteine ​​residue at position 400 and the second CH3 domain comprises a cysteine ​​residue at position 390; or ii) the first CH3 domain comprises a cysteine ​​residue at position 400 and the second CH3 domain comprises a cysteine ​​residue at position 390. or iii) the first CH3 domain comprises a cysteine ​​residue at position 392 and the second CH3 domain comprises a cysteine ​​residue at position 397, or the first CH3 domain comprises a cysteine ​​residue at position 397 and the second CH3 domain comprises a cysteine ​​residue at position 392; or iv) the first CH3 domain comprises a cysteine ​​residue at position 392 and the second CH3 domain comprises a cysteine ​​residue at position 400, or the first CH3 domain comprises a cysteine ​​residue at position 400 and the second CH3 domain comprises a cysteine ​​residue at position 392. a) the first CH3 domain further comprises a positively charged residue at position 357 and the second CH3 domain further comprises a negatively charged residue at position 351, or the first CH3 domain further comprises a negatively charged residue at position 351 and the second CH3 domain further comprises a positively charged residue at position 357; or b) the first CH3 domain further comprises a positively charged residue at position 411 and the second CH3 domain further comprises a negatively charged residue at position 370, or the first CH3 domain further comprises a negatively charged residue at position 370. and the second CH3 domain further comprises a positively charged residue at position 411; or c) the first CH3 domain further comprises a positively charged residue at position 364 and the second CH3 domain further comprises a negatively charged residue at position 370, or the first CH3 domain further comprises a negatively charged residue at position 370 and the second CH3 domain further comprises a positively charged residue at position 364; or a combination of a) and b) or a combination of a) and c), wherein the amino acid residue numbering is based on EU numbering.In some embodiments, the first CH3 domain further comprises a positively charged residue at position 356 and the second CH3 domain further comprises a negatively charged residue at position 439, or the first CH3 domain further comprises a negatively charged residue at position 439 and the second CH3 domain further comprises a positively charged residue at position 356, where amino acid residue numbering is based on EU numbering.

[0081] The CH3 domain may be derived from any naturally occurring immunoglobulin molecule. In some embodiments, the CH3 domain is derived from an IgG1 molecule, an IgG2 molecule, an IgG3 molecule, or an IgG4 molecule. In some embodiments, the CH3 domain is a human CH3 domain. In some embodiments, the CH3 domain is derived from a human IgG1 molecule.

[0082] In some embodiments, a heterodimeric protein (e.g., a multispecific antibody) is provided that comprises a first polypeptide comprising a first CH3 domain and a second polypeptide comprising a second CH3 domain, wherein i) the first CH3 domain comprises an N390C substitution and the second CH3 domain comprises an S400C substitution, or the first CH3 domain comprises an S400C substitution and the second CH3 domain comprises an N390C substitution; or ii) the first wherein the CH3 domain of said polypeptide comprises a K392C substitution and the second CH3 domain comprises a V397C substitution, or wherein the first CH3 domain comprises a V397C substitution and the second CH3 domain comprises a K392C substitution; or wherein iii) the first CH3 domain comprises a K392C substitution and the second CH3 domain comprises an S400C substitution, or wherein the first CH3 domain comprises an S400C substitution and the second CH3 domain comprises a K392C substitution.

[0083] In some embodiments, a heterodimeric protein (e.g., a multispecific antibody) is provided that comprises a first polypeptide comprising a first CH3 domain and a second polypeptide comprising a second CH3 domain, wherein i) the first CH3 domain comprises E357K and T411K substitutions and the second CH3 domain comprises L351D and K370D substitutions, or the first CH3 domain comprises L351D and K370D substitutions and the second CH3 domain comprises E357K and T411K substitutions; or ii) the first CH3 domain comprises E357K and S364K substitutions, the second CH3 domain comprises L351D and K370D substitutions, or the first CH3 domain comprises L351D and K370D substitutions and the second CH3 domain comprises E357K and S364K substitutions; or iii) the first CH3 domain comprises D356K, E357K, and S364K substitutions and the second CH3 domain comprises L351D, K370D, and K439D substitutions, or the first CH3 domain comprises L351D, K370D, and K439D substitutions and the second CH3 domain comprises D356K, E357K, and S364K substitutions.

[0084] In some embodiments, a heterodimeric protein (e.g., a multispecific antibody) is provided that comprises a first polypeptide comprising a first CH3 domain and a second polypeptide comprising a second CH3 domain, wherein the first CH3 domain comprises E357K, S364K, and N390C substitutions and the second CH3 domain comprises L351D, K370D, and S400C substitutions, or wherein the first CH3 domain comprises L351D, K370D, and S400C substitutions and the second CH3 domain comprises E357K, S364K, and N390C substitutions.

[0085] In some embodiments, a heterodimeric protein (e.g., a multispecific antibody) is provided that comprises a first polypeptide comprising a first CH3 domain and a second polypeptide comprising a second CH3 domain, wherein the first CH3 domain comprises E357K, S364K, and S400C substitutions and the second CH3 domain comprises L351D, K370D, and N390C substitutions, or wherein the first CH3 domain comprises L351D, K370D, and N390C substitutions and the second CH3 domain comprises E357K, S364K, and S400C substitutions.

[0086] In some embodiments, a heterodimeric protein (e.g., a multispecific antibody) is provided that comprises a first polypeptide comprising a first CH3 domain and a second polypeptide comprising a second CH3 domain, wherein the first CH3 domain comprises D356K, E357K, S364K, and S400C substitutions and the second CH3 domain comprises L351D, K370D, N390C, and K439D substitutions; or wherein the first CH3 domain comprises L351D, K370D, N390C, and K439D substitutions and the second CH3 domain comprises D356K, E357K, S364K, and S400C substitutions.

[0087] In some embodiments, a heterodimeric protein (e.g., a multispecific antibody) is provided that comprises a first polypeptide comprising a first CH3 domain and a second polypeptide comprising a second CH3 domain, wherein the first CH3 domain comprises D356K, E357K, S364K, and N390C substitutions and the second CH3 domain comprises L351D, K370D, K439D, and S400C substitutions, or wherein the first CH3 domain comprises L351D, K370D, K439D, and S400C substitutions and the second CH3 domain comprises D356K, E357K, S364K, and N390C substitutions.

[0088] In some embodiments, the heterodimeric protein comprises an IgG Fc region comprising an engineered CH3 domain. The Fc region may be derived from any suitable Fc subclass, including but not limited to, IgG1, IgG2, IgG3, and IgG4 subclasses.

[0089] Tables 1A-1B in the Examples section list exemplary polypeptide sequences (SEQ ID NOS: 1-28) of CH3 domains (or Fc regions) comprising engineered disulfide bond(s) and / or salt bridge(s) described herein. Other polypeptide sequences of engineered CH3 domains or Fc region polypeptides include SEQ ID NOS: 138-365. Polypeptides comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 1-28 and 138-365 are also provided.

[0090] In some embodiments, a heterodimeric protein (e.g., a multispecific antibody) is provided comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 1 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 2. In some embodiments, a heterodimeric protein (e.g., a multispecific antibody) is provided comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 3 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 4. In some embodiments, a heterodimeric protein (e.g., a multispecific antibody) is provided comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 5 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 6. In some embodiments, a heterodimeric protein (e.g., a multispecific antibody) is provided comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 7 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 8. In some embodiments, a heterodimeric protein (e.g., a multispecific antibody) is provided comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 9 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 10. In some embodiments, a heterodimeric protein (e.g., a multispecific antibody) is provided comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 11 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments, a heterodimeric protein (e.g., a multispecific antibody) is provided comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 13 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, a heterodimeric protein (e.g., a multispecific antibody) is provided comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 15 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 16. In some embodiments, a heterodimeric protein (e.g., a multispecific antibody) is provided comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 17 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 18. In some embodiments, a heterodimeric protein (e.g., a multispecific antibody) is provided comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 19 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 20.In some embodiments, a heterodimeric protein (e.g., a multispecific antibody) is provided that includes a first polypeptide comprising the amino acid sequence of SEQ ID NO: 21 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 22. In some embodiments, a heterodimeric protein (e.g., a multispecific antibody) is provided that includes a first polypeptide comprising the amino acid sequence of SEQ ID NO: 23 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 24.

[0091] CH3 domain mutations The heterodimeric proteins described herein may have one or more engineered disulfide bonds, one or more engineered (e.g., rearranged or inverted) salt bridges, or a combination thereof. Unless otherwise specified, all amino acid residue numbering herein is based on EU numbering, and amino acid substitutions are relative to the wild-type (or naturally occurring) sequence at the corresponding amino acid position in the wild-type (or naturally occurring) CH3 domain sequence. It is understood that the mutations or substitutions described herein are applicable to all IgG subclasses and allotypes. IgG allotypes are described, for example, in Jefferis R. and Lefranc M. mAbs 1:4, 1-7 (2009), which is incorporated herein by reference in its entirety. In some embodiments, the amino acid mutations or substitutions described herein are relative to the wild-type CH3 domain sequence of IgG1, e.g., IgG1 allotype G1m, 1(a), 2(x), 3(f), or 17(z). In some embodiments, the amino acid mutations or substitutions described herein are relative to the wild-type CH3 domain sequence of IgG4. For example, a D356K substitution in the wild-type CH3 domain of one human IgG1 allotype (Uniprot ID P01857; SEQ ID NO: 29) is equivalent to an E356K substitution in the wild-type CH3 domain of a second human IgG1 allotype (SEQ ID NO: 30), or in the wild-type CH3 domain of human IgG4 (SEQ ID NO: 31). Exemplary CH3 domain mutations are shown in Tables 1A-1B. In some embodiments, the amino acid mutations or substitutions described herein are relative to a wild-type Fc region sequence, e.g., an IgG1 Fc region (SEQ ID NO: 32 or 33) or an IgG4 Fc region (SEQ ID NO: 34).

[0092] Novel cysteine ​​mutations In some embodiments, the heterodimeric proteins described herein comprise a first polypeptide comprising a first CH3 domain and a second polypeptide comprising a second CH3 domain, wherein the first CH3 domain comprises a first engineered cysteine ​​residue and the second CH3 domain comprises a second engineered cysteine ​​residue, and wherein the first engineered cysteine ​​residue and the second cysteine ​​residue form a disulfide bond.

[0093] In some embodiments, the first CH3 domain comprises a C at position 390 and the second CH3 domain comprises a C at position 400, or the first CH3 domain comprises a C at position 400 and the second CH3 domain comprises a C at position 390. In some embodiments, the first CH3 domain comprises an N390C substitution and the second CH3 domain comprises an S400C substitution, or the first CH3 domain comprises an S400C substitution and the second CH3 domain comprises an N390C substitution.

[0094] In some embodiments, the first CH3 domain comprises a C at position 392 and the second CH3 domain comprises a C at position 397, or the first CH3 domain comprises a C at position 397 and the second CH3 domain comprises a C at position 392. In some embodiments, the first CH3 domain comprises a K392C substitution and the second CH3 domain comprises a V397C substitution, or the first CH3 domain comprises a V397C substitution and the second CH3 domain comprises a K392C substitution.

[0095] In some embodiments, the first CH3 domain comprises a C at position 392 and the second CH3 domain comprises a C at position 400, or the first CH3 domain comprises a C at position 400 and the second CH3 domain comprises a C at position 392. In some embodiments, the first CH3 domain comprises a K392C substitution and the second CH3 domain comprises an S400C substitution, or the first CH3 domain comprises an S400C substitution and the second CH3 domain comprises a K392C substitution.

[0096] Novel salt bridge mutation In some embodiments, the heterodimeric proteins described herein comprise a first polypeptide comprising a first CH3 domain and a second polypeptide comprising a second CH3 domain, wherein the first CH3 domain comprises an engineered positively charged residue and the second CH3 domain comprises an engineered negatively charged residue, and wherein the engineered positively charged residue and the engineered negatively charged residue form a salt bridge. The engineered salt bridge may introduce a new salt bridge between the CH3 domains, rearrange a salt bridge network between two or more amino acid residues, or reverse the charge of the amino acid residues forming the salt bridge relative to the wild-type CH3 domain (i.e., "flip" the salt bridge). In some embodiments, the engineered positively charged residue replaces a negatively charged residue in the wild-type CH3 domain with a positively charged residue. In some embodiments, the engineered negatively charged residue replaces a positively charged residue in the wild-type CH3 domain with a negatively charged residue. The rearranged and flipped salt bridges may result in a change in the isoelectric point (PI) of heterodimers and homodimers containing the engineered CH3 domain, thereby allowing for better separation of heterodimers from homodimers during the purification process.

[0097] In some embodiments, the first CH3 domain comprises a positively charged residue at position 357 and the second CH3 domain comprises a negatively charged residue at position 351, or the first CH3 domain comprises a negatively charged residue at position 351 and the second CH3 domain comprises a positively charged residue at position 357. In some embodiments, the first CH3 domain comprises a K at position 357 and the second CH3 domain comprises a D at position 351, or the first CH3 domain comprises a D at position 351 and the second CH3 domain comprises a K at position 357. In some embodiments, the first CH3 domain comprises a K at position 357 and the second CH3 domain comprises an E at position 351, or the first CH3 domain comprises an E at position 351 and the second CH3 domain comprises a K at position 357. In some embodiments, the first CH3 domain comprises an R at position 357 and the second CH3 domain comprises a D at position 351, or the first CH3 domain comprises a D at position 351 and the second CH3 domain comprises an R at position 357. In some embodiments, the first CH3 domain comprises an R at position 357 and the second CH3 domain comprises an E at position 351, or the first CH3 domain comprises an E at position 351 and the second CH3 domain comprises an R at position 357. In some embodiments, the first CH3 domain comprises an E357K substitution and the second CH3 domain comprises an L351D substitution, or the first CH3 domain comprises an L351D substitution and the second CH3 domain comprises an E357K substitution.

[0098] In some embodiments, the first CH3 domain comprises a positively charged residue at position 411 and the second CH3 domain comprises a negatively charged residue at position 370, or the first CH3 domain comprises a negatively charged residue at position 370 and the second CH3 domain comprises a positively charged residue at position 411. In some embodiments, the first CH3 domain comprises a K at position 411 and the second CH3 domain comprises a D at position 370, or the first CH3 domain comprises a D at position 370 and the second CH3 domain comprises a K at position 411. In some embodiments, the first CH3 domain comprises a K at position 411 and the second CH3 domain comprises an E at position 370, or the first CH3 domain comprises an E at position 370 and the second CH3 domain comprises a K at position 411. In some embodiments, the first CH3 domain comprises an R at position 411 and the second CH3 domain comprises a D at position 370, or the first CH3 domain comprises a D at position 370 and the second CH3 domain comprises an R at position 411. In some embodiments, the first CH3 domain comprises an R at position 411 and the second CH3 domain comprises an E at position 370, or the first CH3 domain comprises an E at position 370 and the second CH3 domain comprises an R at position 411. In some embodiments, the first CH3 domain comprises a T411K substitution and the second CH3 domain comprises a K370D substitution, or the first CH3 domain comprises a K370D substitution and the second CH3 domain comprises a T411K substitution.

[0099] In some embodiments, the first CH3 domain comprises a positively charged residue at position 364 and the second CH3 domain comprises a negatively charged residue at position 370, or the first CH3 domain comprises a negatively charged residue at position 370 and the second CH3 domain comprises a positively charged residue at position 364. In some embodiments, the first CH3 domain comprises a K at position 364 and the second CH3 domain comprises a D at position 370, or the first CH3 domain comprises a D at position 370 and the second CH3 domain comprises a K at position 364. In some embodiments, the first CH3 domain comprises a K at position 364 and the second CH3 domain comprises an E at position 370, or the first CH3 domain comprises an E at position 370 and the second CH3 domain comprises a K at position 364. In some embodiments, the first CH3 domain comprises an R at position 364 and the second CH3 domain comprises a D at position 370, or the first CH3 domain comprises a D at position 370 and the second CH3 domain comprises an R at position 364. In some embodiments, the first CH3 domain comprises an R at position 364 and the second CH3 domain comprises an E at position 370, or the first CH3 domain comprises an E at position 370 and the second CH3 domain comprises an R at position 364. In some embodiments, the first CH3 domain comprises an S364K substitution and the second CH3 domain comprises a K370D substitution, or the first CH3 domain comprises a K370D substitution and the second CH3 domain comprises an S364K substitution.

[0100] In some embodiments, the first CH3 domain comprises a positively charged residue at position 356 and the second CH3 domain comprises a negatively charged residue at position 439, or the first CH3 domain comprises a negatively charged residue at position 439 and the second CH3 domain comprises a positively charged residue at position 356. In some embodiments, the first CH3 domain comprises a K at position 356 and the second CH3 domain comprises a D at position 439, or the first CH3 domain comprises a D at position 439 and the second CH3 domain comprises a K at position 356. In some embodiments, the first CH3 domain comprises a K at position 356 and the second CH3 domain comprises an E at position 439, or the first CH3 domain comprises an E at position 439 and the second CH3 domain comprises a K at position 356. In some embodiments, the first CH3 domain comprises an R at position 356 and the second CH3 domain comprises a D at position 439, or the first CH3 domain comprises a D at position 439 and the second CH3 domain comprises an R at position 356. In some embodiments, the first CH3 domain comprises an R at position 356 and the second CH3 domain comprises an E at position 439, or the first CH3 domain comprises an E at position 439 and the second CH3 domain comprises an R at position 356. In some embodiments, the first CH3 domain comprises a D356K substitution and the second CH3 domain comprises a K439D substitution, or the first CH3 domain comprises a K439D substitution and the second CH3 domain comprises a D356K substitution.

[0101] Any of the engineered salt bridges described herein may be combined with one another. In some embodiments, a first CH3 domain comprises a positively charged residue at position 357 and a positively charged residue at position 411, and a second CH3 domain comprises a negatively charged residue at position 351 and a negatively charged residue at position 370; or, a first CH3 domain comprises a negatively charged residue at position 351 and a negatively charged residue at position 370, and a second CH3 domain comprises a positively charged residue at position 357 and a positively charged residue at position 411. In some embodiments, a first CH3 domain comprises E357K and T411K substitutions, and a second CH3 domain comprises L351D and K370D substitutions, or a first CH3 domain comprises L351D and K370D substitutions, and a second CH3 domain comprises E357K and T411K substitutions.

[0102] In some embodiments, the first CH3 domain comprises a positively charged residue at position 357 and a positively charged residue at position 364, and the second CH3 domain comprises a negatively charged residue at position 351 and a negatively charged residue at position 370, or the first CH3 domain comprises a negatively charged residue at position 351 and a negatively charged residue at position 370, and the second CH3 domain comprises a positively charged residue at position 357 and a positively charged residue at position 364. In some embodiments, the first CH3 domain comprises E357K and S364K substitutions and the second CH3 domain comprises L351D and K370D substitutions, or the first CH3 domain comprises L351D and K370D substitutions and the second CH3 domain comprises E357K and S364K substitutions.

[0103] In some embodiments, the first CH3 domain comprises a positively charged residue at position 356, a positively charged residue at position 357, and a positively charged residue at position 364; the second CH3 domain comprises a negatively charged residue at position 351, a negatively charged residue at position 370, and a negatively charged residue at position 439; or the first CH3 domain comprises a negatively charged residue at position 351, a negatively charged residue at position 370, and a negatively charged residue at position 439; and the second CH3 domain comprises a positively charged residue at position 356, a positively charged residue at position 357, and a positively charged residue at position 364. In some embodiments, the first CH3 domain comprises D356K, E357K, and S364K substitutions and the second CH3 domain comprises L351D, K370D, and K439D substitutions, or the first CH3 domain comprises L351D, K370D, and K439D substitutions and the second CH3 domain comprises D356K, E357K, and S364K substitutions.

[0104] Other mutations The CH3 domains or Fc regions described herein may further comprise engineered disulfide bonds and / or salt bridges as listed in Table B below. [Table 2-1] [Table 2-2]

[0105] In some embodiments, the first CH3 domain further comprises a C at position 392 and the second CH3 domain further comprises a C at position 399, or the first CH3 domain further comprises a C at position 399 and the second CH3 domain further comprises a C at position 392. In some embodiments, the first CH3 domain further comprises a K392C substitution and the second CH3 domain further comprises a D399C substitution, or the first CH3 domain further comprises a D399C substitution and the second CH3 domain further comprises a K392C substitution.

[0106] In some embodiments, the first CH3 domain further comprises a C at position 394 and the second CH3 domain further comprises a C at position 354, or the first CH3 domain further comprises a C at position 354 and the second CH3 domain further comprises a C at position 394. In some embodiments, the first CH3 domain further comprises a Y394C substitution and the second CH3 domain further comprises a S354C substitution, or the first CH3 domain further comprises a S354C substitution and the second CH3 domain further comprises a Y394C substitution.

[0107] In some embodiments, the first CH3 domain further comprises a C at position 356 and the second CH3 domain further comprises a C at position 349, or the first CH3 domain further comprises a C at position 349 and the second CH3 domain further comprises a C at position 356. In some embodiments, the first CH3 domain further comprises a D356C substitution and the second CH3 domain further comprises a Y349C substitution, or the first CH3 domain further comprises a Y349C substitution and the second CH3 domain further comprises a D356C substitution.

[0108] In some embodiments, the first CH3 domain further comprises K392D and K409D substitutions and the second CH3 domain further comprises D356K and D399K substitutions, or the first CH3 domain further comprises D356K and D399K substitutions and the second CH3 domain further comprises K392D and K409D substitutions.

[0109] In some embodiments, the first CH3 domain further comprises L368D and K370S substitutions and the second CH3 domain further comprises E357Q and S364K substitutions; or the first CH3 domain further comprises E357Q and S364K substitutions and the second CH3 domain further comprises L368D and K370S substitutions.

[0110] In some embodiments, the first CH3 domain further comprises L351K and T366K substitutions and the second CH3 domain further comprises L351D and L368E substitutions, or the first CH3 domain further comprises L351D and L368E substitutions and the second CH3 domain further comprises L351K and T366K substitutions.

[0111] In some embodiments, the first CH3 domain further comprises P395K, P396K, and V397K substitutions and the second CH3 domain further comprises T394D, P395D, and P396D substitutions; or the first CH3 domain further comprises T394D, P395D, and P396D substitutions and the second CH3 domain further comprises P395K, P396K, and V397K substitutions.

[0112] In some embodiments, the first CH3 domain further comprises F405E, Y407E, and K409E substitutions and the second CH3 domain further comprises F405K and Y407K substitutions; or the first CH3 domain further comprises F405K and Y407K substitutions and the second CH3 domain further comprises F405E, Y407E, and K409E substitutions.

[0113] Heterodimeric proteins containing engineered CH3 domain disulfide bonds and / or salt bridges described herein may further comprise one or more knob-into-hole residues. "Knobs-into-hole" or "KIH" refers to an art-known approach for generating bispecific antibodies, also known as the "protuberance-into-cavity" approach (see, e.g., U.S. Pat. No. 5,731,168). In this approach, two immunoglobulin polypeptides (e.g., heavy chain polypeptides) each constitute an interface. The interface of one immunoglobulin polypeptide interacts with a corresponding interface on the other immunoglobulin polypeptide, thereby allowing the two immunoglobulin polypeptides to associate. These interfaces may be engineered so that a "knob" or "protuberance" (these terms may be used interchangeably herein) located at the interface of one immunoglobulin polypeptide corresponds to a "hole" or "cavity" (these terms may be used interchangeably herein) located at the interface of the other immunoglobulin polypeptide. In some embodiments, the holes are the same size as or similar to the knobs and are appropriately positioned so that when the two interfaces interact, the knobs on one interface can be positioned into the corresponding holes on the other interface. Without wishing to be bound by theory, this is believed to stabilize the heteromultimer and promote the formation of heteromultimers over other species, e.g., homomultimers. In some embodiments, the KIH approach is used in combination with the engineered disulfide bonds and / or salt bridges described herein to promote heteromultimerization of two different immunoglobulin polypeptides and generate bispecific antibodies comprising two immunoglobulin polypeptides with binding specificities for different epitopes. In some embodiments, the CH3 domains of the heterodimeric proteins described herein do not contain KIH residues.

[0114] In some embodiments, the first CH3 domain further comprises a T336S, L368A, and Y407V substitution and the second CH3 domain further comprises a T366W substitution, or the first CH3 domain further comprises a T366W substitution and the second CH3 domain further comprises a T336S, L368A, and Y407V substitution.

[0115] In some embodiments, the first CH3 domain comprises an L368V and a Y407V substitution and the second CH3 domain comprises a T366W substitution, or the first CH3 domain comprises a T366W substitution and the second CH3 domain comprises an L368V and a Y407V substitution.

[0116] III. Multispecific antibodies In some embodiments, the heterodimeric proteins described herein are multispecific antibodies, e.g., bispecific or trispecific antibodies.

[0117] In some embodiments, a multispecific antibody is provided comprising a first polypeptide comprising a first CH3 domain and a first target-binding moiety (TBM), and a second polypeptide comprising a second CH3 domain and a second TBM, wherein the first CH3 domain and the second CH3 domain comprise any one or combination of engineered disulfide bonds or salt bridges described herein, and the first TBM specifically binds to a first target and the second TBM specifically binds to a second target different from the first target. In some embodiments, the TBM is an antigen-binding domain. In some embodiments, the TBM is an scFv or VHH. In some embodiments, the first CH3 domain comprises an N390C substitution and the second CH3 domain comprises an S400C substitution, or the first CH3 domain comprises an S400C substitution and the second CH3 domain comprises an N390C substitution. In some embodiments, the first CH3 domain comprises E357K, S364K, and S400C substitutions and the second CH3 domain comprises L351D, K370D, and N390C substitutions, or the first CH3 domain comprises L351D, K370D, and N390C substitutions and the second CH3 domain comprises E357K, S364K, and S400C substitutions. In some embodiments, the first CH3 domain comprises D356K, E357K, S364K, and N390C substitutions and the second CH3 domain comprises L351D, K370D, K439D, and S400C substitutions; or the first CH3 domain comprises L351D, K370D, K439D, and S400C substitutions and the second CH3 domain comprises D356K, E357K, S364K, and N390C substitutions. In some embodiments, the first CH3 domain comprises D356K, E357K, S364K, and S400C substitutions and the second CH3 domain comprises L351D, K370D, N390C, and K439D substitutions, or the first CH3 domain comprises L351D, K370D, N390C, and K439D substitutions and the second CH3 domain comprises D356K, E357K, S364K, and S400C substitutions. In some embodiments, the multispecific antibody comprises an IgG1 Fc region, e.g., an IgG1 Fc with an N297A substitution.In some embodiments, the multispecific antibody comprises an IgG4 Fc region, for example, an IgG4 with an S228P substitution.

[0118] In some embodiments, a multispecific antibody is provided comprising a first CH3 domain, a second polypeptide comprising a second CH3 domain, a third polypeptide, and a fourth polypeptide, wherein the first CH3 domain and the second CH3 domain comprise any one or combination of engineered disulfide bonds or salt bridges described herein, wherein the first polypeptide is a first antibody heavy chain, the second polypeptide is a second antibody heavy chain, the third polypeptide is a first antibody light chain, and the fourth polypeptide is a second antibody light chain, wherein the first polypeptide and the third polypeptide associate to form a first antigen-binding site that specifically binds to a first target, and the second polypeptide and the fourth polypeptide associate to form a second antigen-binding site that specifically binds to a second target, different from the first target. In some embodiments, the first CH3 domain comprises an N390C substitution and the second CH3 domain comprises an S400C substitution, or the first CH3 domain comprises an S400C substitution and the second CH3 domain comprises an N390C substitution, hi some embodiments, the first CH3 domain comprises an E357K, S364K, and S400C substitution and the second CH3 domain comprises an L351D, K370D, and N390C substitution, or the first CH3 domain comprises an L351D, K370D, and N390C substitution and the second CH3 domain comprises an E357K, S364K, and S400C substitution. In some embodiments, the first CH3 domain comprises D356K, E357K, S364K, and N390C substitutions and the second CH3 domain comprises L351D, K370D, K439D, and S400C substitutions; or the first CH3 domain comprises L351D, K370D, K439D, and S400C substitutions and the second CH3 domain comprises D356K, E357K, S364K, and N390C substitutions.In some embodiments, the first CH3 domain comprises D356K, E357K, S364K, and S400C substitutions and the second CH3 domain comprises L351D, K370D, N390C, and K439D substitutions, or the first CH3 domain comprises L351D, K370D, N390C, and K439D substitutions and the second CH3 domain comprises D356K, E357K, S364K, and S400C substitutions. In some embodiments, the multispecific antibody comprises an IgG1 Fc region, e.g., an IgG1 Fc with an N297A substitution. In some embodiments, the multispecific antibody comprises an IgG4 Fc region, e.g., an IgG4 with an S228P substitution.

[0119] In some embodiments, a multispecific antibody is provided, comprising a first polypeptide comprising a first CH3 domain, a second polypeptide comprising a second CH3 domain, a third polypeptide, and a fourth polypeptide, wherein the first CH3 domain and the second CH3 domain comprise any one or combination of engineered disulfide bonds or salt bridges as described herein; (i) the first polypeptide comprises a structure represented by the following formula: VH1-CH1-hinge-CH2-1st CH3, (ii) the second polypeptide comprises a structure represented by the formula: VH2-CH1-hinge-CH2-2nd CH3, (iii) the third polypeptide comprises a structure represented by the following formula: VL1-CL, (iv) the fourth polypeptide comprises a structure represented by the following formula: VL2-CL, VL1 is the first immunoglobulin light chain variable domain, VH1 is the first immunoglobulin heavy chain variable domain, VL2 is the second immunoglobulin light chain variable domain, VH2 is the second immunoglobulin heavy chain variable domain, CL is an immunoglobulin light chain constant domain; CH1 is immunoglobulin heavy chain constant domain 1; CH2 is immunoglobulin heavy chain constant domain 2; The hinge is an immunoglobulin hinge region that connects the CH1 and CH2 domains, VL1 and VH1 associate to form a first Fv that specifically binds to a first target; VL2 and VH2 associate to form a second Fv that specifically binds to a second target. In some embodiments, VL1 is identical to VL2 (e.g., a multispecific antibody is a common light chain antibody). In some embodiments, VL1 is different from VL2. In some embodiments, the first CH3 domain comprises an N390C substitution and the second CH3 domain comprises an S400C substitution, or the first CH3 domain comprises an S400C substitution and the second CH3 domain comprises an N390C substitution. In some embodiments, the first CH3 domain comprises E357K, S364K, and S400C substitutions and the second CH3 domain comprises L351D, K370D, and N390C substitutions, or the first CH3 domain comprises L351D, K370D, and N390C substitutions and the second CH3 domain comprises E357K, S364K, and S400C substitutions. In some embodiments, the first CH3 domain comprises D356K, E357K, S364K, and N390C substitutions and the second CH3 domain comprises L351D, K370D, K439D, and S400C substitutions; or the first CH3 domain comprises L351D, K370D, K439D, and S400C substitutions and the second CH3 domain comprises D356K, E357K, S364K, and N390C substitutions. In some embodiments, the first CH3 domain comprises D356K, E357K, S364K, and S400C substitutions and the second CH3 domain comprises L351D, K370D, N390C, and K439D substitutions, or the first CH3 domain comprises L351D, K370D, N390C, and K439D substitutions and the second CH3 domain comprises D356K, E357K, S364K, and S400C substitutions. In some embodiments, the multispecific antibody comprises an IgG1 Fc region, e.g., an IgG1 Fc with an N297A substitution. In some embodiments, the multispecific antibody comprises an IgG4 Fc region, e.g., an IgG4 with an S228P substitution. In some embodiments, the first target is PDL1 and the second target is CD137, or the first target is CD137 and the second target is PDL1.

[0120] In some embodiments, a multispecific antibody is provided, comprising a first polypeptide comprising a first CH3 domain, a second polypeptide comprising a second CH3 domain, a third polypeptide, and a fourth polypeptide, wherein the first CH3 domain and the second CH3 domain comprise any one or combination of engineered disulfide bonds or salt bridges as described herein; (i) the first polypeptide comprises a structure represented by the following formula: VH1-CH1-hinge-CH2-1st CH3-L1-scFv1, (ii) the second polypeptide comprises a structure represented by the formula: VH2-CH1-hinge-CH2-2nd CH3-L2-scFv2, (iii) the third polypeptide comprises a structure represented by the following formula: VL1-CL, (iv) the fourth polypeptide comprises a structure represented by the following formula: VL2-CL, VL1 is the first immunoglobulin light chain variable domain, VH1 is the first immunoglobulin heavy chain variable domain, VL2 is the second immunoglobulin light chain variable domain, VH2 is the second immunoglobulin heavy chain variable domain, scFv1 is the first single chain variable fragment, scFv2 is a second single chain variable fragment, CL is an immunoglobulin light chain constant domain; CH1 is immunoglobulin heavy chain constant domain 1; CH2 is immunoglobulin heavy chain constant domain 2; hinge is an immunoglobulin hinge region connecting the CH1 and CH2 domains; L1 and L2 are each independently a bond or a peptide linker; VL1 and VH1 associate to form a first Fv that specifically binds to a first target; VL2 and VH2 associate to form a second Fv that specifically binds to a second target; scFv1 specifically binds to a third target; and scFv2 specifically binds to a fourth target. In some embodiments, the heterodimeric protein is a bispecific antibody, where the first target and the second target are the same, and the third target and the fourth target are the same. In some embodiments, the heterodimeric protein is a trispecific antibody, where the third target and the fourth target are the same, or the first target and the second target are the same. In some embodiments, the heterodimeric protein is a tetraspecific antibody. In some embodiments, VL1 is identical to VL2 (e.g., a multispecific antibody is an antibody with a common light chain). In some embodiments, VL1 is different from VL2. In some embodiments, the first CH3 domain comprises an N390C substitution and the second CH3 domain comprises an S400C substitution, or the first CH3 domain comprises an S400C substitution and the second CH3 domain comprises an N390C substitution, hi some embodiments, the first CH3 domain comprises an E357K, S364K, and S400C substitution and the second CH3 domain comprises an L351D, K370D, and N390C substitution, or the first CH3 domain comprises an L351D, K370D, and N390C substitution and the second CH3 domain comprises an E357K, S364K, and S400C substitution. In some embodiments, the first CH3 domain comprises D356K, E357K, S364K, and N390C substitutions and the second CH3 domain comprises L351D, K370D, K439D, and S400C substitutions; or the first CH3 domain comprises L351D, K370D, K439D, and S400C substitutions and the second CH3 domain comprises D356K, E357K, S364K, and N390C substitutions.In some embodiments, the first CH3 domain comprises D356K, E357K, S364K, and S400C substitutions and the second CH3 domain comprises L351D, K370D, N390C, and K439D substitutions, or the first CH3 domain comprises L351D, K370D, N390C, and K439D substitutions and the second CH3 domain comprises D356K, E357K, S364K, and S400C substitutions. In some embodiments, the multispecific antibody comprises an IgG1 Fc region, e.g., an IgG1 Fc with an N297A substitution. In some embodiments, the multispecific antibody comprises an IgG4 Fc region, e.g., an IgG4 with an S228P substitution.

[0121] In some embodiments, a multispecific antibody is provided, comprising: a first polypeptide comprising a first CH3 domain; a second polypeptide comprising a second CH3 domain and a first target-binding moiety (TBM) that specifically binds to a first target; and a third polypeptide, wherein the first CH3 domain and the second CH3 domain comprise any one or combination of engineered disulfide bonds or salt bridges described herein, and the first polypeptide and the third polypeptide associate to form a second antigen-binding site that specifically binds to a second target. In some embodiments, the first target-binding moiety (TBM) is an scFv or VHH. In some embodiments, the first CH3 domain comprises an N390C substitution and the second CH3 domain comprises an S400C substitution, or the first CH3 domain comprises an S400C substitution and the second CH3 domain comprises an N390C substitution. In some embodiments, the first CH3 domain comprises E357K, S364K, and S400C substitutions and the second CH3 domain comprises L351D, K370D, and N390C substitutions, or the first CH3 domain comprises L351D, K370D, and N390C substitutions and the second CH3 domain comprises E357K, S364K, and S400C substitutions. In some embodiments, the first CH3 domain comprises D356K, E357K, S364K, and N390C substitutions and the second CH3 domain comprises L351D, K370D, K439D, and S400C substitutions; or the first CH3 domain comprises L351D, K370D, K439D, and S400C substitutions and the second CH3 domain comprises D356K, E357K, S364K, and N390C substitutions. In some embodiments, the first CH3 domain comprises D356K, E357K, S364K, and S400C substitutions and the second CH3 domain comprises L351D, K370D, N390C, and K439D substitutions, or the first CH3 domain comprises L351D, K370D, N390C, and K439D substitutions and the second CH3 domain comprises D356K, E357K, S364K, and S400C substitutions. In some embodiments, the multispecific antibody comprises an IgG1 Fc region, e.g., an IgG1 Fc with an N297A substitution.In some embodiments, the multispecific antibody comprises an IgG4 Fc region, e.g., an IgG4 with an S228P substitution. In some embodiments, the first target is PDL1 and the second target is CD137. In some embodiments, the first target is CD137 and the second target is PDL1. In some embodiments, the first target is CD137 and the second target is CTLA4. In some embodiments, the first target is CTLA4 and the second target is PDL1.

[0122] In some embodiments, a multispecific antibody is provided, comprising a first polypeptide comprising a first CH3 domain, a second polypeptide, and a third polypeptide, wherein the first CH3 domain and the second CH3 domain comprise any one or combination of engineered disulfide bonds or salt bridges as described herein; (i) the first polypeptide comprises a structure represented by the following formula: VH-CH1-hinge-CH2-1st CH3, (ii) the second polypeptide comprises a structure represented by the formula: scFv-hinge-CH2-2nd CH3, (iii) the third polypeptide comprises a structure represented by the following formula: VL-CL, VL is an immunoglobulin light chain variable domain; VH is an immunoglobulin heavy chain variable domain; scFv is a single chain variable fragment; CL is an immunoglobulin light chain constant domain; CH1 is immunoglobulin heavy chain constant domain 1; CH2 is immunoglobulin heavy chain constant domain 2; The hinge is an immunoglobulin hinge region that connects the CH1 and CH2 domains, the VL and VH associate to form an Fv that specifically binds to a first target; The scFv specifically binds to a second target. In some embodiments, the first CH3 domain comprises an N390C substitution and the second CH3 domain comprises an S400C substitution, or the first CH3 domain comprises an S400C substitution and the second CH3 domain comprises an N390C substitution. In some embodiments, the first CH3 domain comprises an E357K, S364K, and S400C substitution and the second CH3 domain comprises an L351D, K370D, and N390C substitution, or the first CH3 domain comprises an L351D, K370D, and N390C substitution and the second CH3 domain comprises an E357K, S364K, and S400C substitution. In some embodiments, the first CH3 domain comprises D356K, E357K, S364K, and N390C substitutions and the second CH3 domain comprises L351D, K370D, K439D, and S400C substitutions; or the first CH3 domain comprises L351D, K370D, K439D, and S400C substitutions and the second CH3 domain comprises D356K, E357K, S364K, and N390C substitutions. In some embodiments, the first CH3 domain comprises D356K, E357K, S364K, and S400C substitutions and the second CH3 domain comprises L351D, K370D, N390C, and K439D substitutions, or the first CH3 domain comprises L351D, K370D, N390C, and K439D substitutions and the second CH3 domain comprises D356K, E357K, S364K, and S400C substitutions. In some embodiments, the multispecific antibody comprises an IgG1 Fc region, e.g., an IgG1 Fc with an N297A substitution. In some embodiments, the multispecific antibody comprises an IgG4 Fc region, e.g., an IgG4 with an S228P substitution. In some embodiments, the scFv is linked to the hinge of the second polypeptide via a linker, such as a peptide linker comprising the amino acid sequence of SEQ ID NO: 80 or 81. In some embodiments, the first target is CD3 and the second target is a tumor antigen (e.g., HER2). In some embodiments, the first target is a tumor antigen (e.g., HER2) and the second target is CD3.

[0123] In some embodiments, the multispecific antibody comprises one or more antibody constant regions. In some embodiments, the human heavy chain constant region is of an isotype selected from IgA, IgG, and IgD. In some embodiments, the human light chain constant region is of an isotype selected from Kappa and Lambda. In some embodiments, the multispecific antibody comprises a human IgG constant region. In some embodiments, the multispecific antibody comprises a human IgG4 heavy chain constant region. In some embodiments, the multispecific antibody comprises a human IgG1 heavy chain constant region. In some such embodiments, the multispecific antibody comprises a S228P mutation in the human IgG4 constant region. In some embodiments, the first polypeptide and the second polypeptide further comprise a S228P substitution.

[0124] Whether effector function is desirable may depend on the particular treatment method desired for the multispecific antibody. In some embodiments, if effector function is desirable, a multispecific antibody comprising a human IgG1 heavy chain constant region or a human IgG3 heavy chain constant region is selected. In some embodiments, if effector function is undesirable, a multispecific antibody comprising a human IgG4 or IgG2 heavy chain constant region is selected. In some embodiments, the multispecific antibody comprises a human IgG1 heavy chain constant region comprising one or more mutations that reduce effector function. In some embodiments, the multispecific antibody comprises an IgG1 heavy chain constant region comprising an N297A substitution. In some embodiments, the first polypeptide and the second polypeptide further comprise an N297A substitution.

[0125] Any of the multispecific antibodies described herein can specifically bind to at least two different targets or epitopes. The at least two different epitopes recognized can be located on the same antigen or on different antigens. In some embodiments, the antigen is a cell surface molecule. In some embodiments, the antigen is an extracellular molecule.

[0126] In some embodiments, the first target, second target, third target, and / or fourth target is a cell surface antigen. In some embodiments, the cell surface antigen is an antigen on an immune effector cell, such as, for example, a T cell (e.g., a helper T cell, a cytotoxic T cell, a memory T cell, etc.), a B cell, a macrophage, or a natural killer (NK) cell. In some embodiments, the cell surface antigen is a T cell surface antigen, such as CD3.

[0127] In some embodiments, the cell surface antigen is a tumor antigen. Tumor antigens are proteins produced by tumor cells that can induce an immune response, particularly a T cell-mediated immune response. In some embodiments, the tumor antigen is a tumor-specific antigen (TSA) or tumor-associated antigen (TAA). TSAs are unique to tumor cells and are not present on other cells in the body. TAA-associated antigens are not unique to tumor cells, but are instead expressed on normal cells under conditions that do not induce a state of immune tolerance to the antigen. Antigen expression on tumors can occur under conditions that allow the immune system to respond to the antigen. TAAs can be antigens expressed on normal cells during fetal development, when the immune system is immature and unable to respond, or they can be antigens that are normally present at very low levels on normal cells but expressed at much higher levels on tumor cells.

[0128] Non-limiting examples of TSA or TAA antigens include differentiation antigens such as MART-1 / MelanA (MART-1), gp100 (Pmel17), tyrosinase, TRP-1, TRP-2, and tumor-specific multilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor suppressor genes such as p53, Ras, HER2 / neu; unique tumor antigens resulting from chromosomal translocations such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens such as Epstein-Barr virus antigen EBVA and human papillomavirus (HPV) antigens E6 and E7. Other large protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, pl85erbB2, pl80erbB-3, c-met, nm-23HI, PSA, TAG-72, CA19-9, CA72-4, CAM17.1, NuMa, K-ras, beta-catenin, CDK4, Mum-1, p15, p16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BT These include AA, CA125, CA15-3, CA27.29, BCAA, CA195, CA242, CA-50, CAM43, CD68, P1, CO-029, FGF-5, G250, Ga733, EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90, Mac-2 binding protein, cyclophilin C-related protein, TAAL6, TAG72, TLP, and TPS.

[0129] In some embodiments, the first, second, third, and / or fourth target is an immune checkpoint molecule. In some embodiments, the immune checkpoint molecule is a stimulatory immune checkpoint molecule. Exemplary stimulatory immune checkpoint molecules include, but are not limited to, CD28, OX40, ICOS, GITR, 4-1BB, CD27, CD40, CD3, HVEM, and TCR (e.g., MHC class I or class II molecules). In some embodiments, the immune checkpoint molecule is an inhibitory immune checkpoint molecule. Exemplary inhibitory immune checkpoint molecules include, but are not limited to, CTLA-4, TIM-3, A2a receptor, LAG-3, BTLA, KIR, PD-1, IDO, CD47, and their ligands, e.g., B7.1, B7.2, PDL1, PD-L2, HVEM, B7-H4, NKTR-218, and SIRP-alpha receptor.

[0130] Target binding moiety (TBM) In some embodiments, the target binding moiety (TBM) comprises an antibody light chain variable region (VL) and / or an antibody heavy chain variable region (VH). In some embodiments, the TBM comprises a VL. In some embodiments, the TBM comprises a VH. In some embodiments, the TBM comprises VL and / or VH specificity for any target of interest, including, for example, CTLA4, CD137, PD1, PDL1, PDL2, LAG3, TIM3, B7-H3, OX40, CD3, CD19, CD20, CD40, CD95, CD120a, BTLA, VISTA, ICOS, BCMA, HER1, HER2, HER3, and / or B7-H4.

[0131] In some embodiments, the TBM may be any of a variety of antibody fragments, including, but not limited to, (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CHI domains; (ii) an F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment, which is a VH and CHI domain; (iv) an Fv fragment, which is a VL and VH domain of a single arm of an antibody; (v) a dAb fragment, which is a VH domain (Ward et al., (1989) Nature 341:544-546); (vi) isolated CDRs; and (vii) a single-chain antibody (scFv), which is a polypeptide comprising the VL region of an antibody linked to the VH region of an antibody (e.g., Bird et al. (1988) Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883).

[0132] In some embodiments, the TBM is an scFv comprising, from N- to C-terminus, VL-L1-VH, where L1 is a peptide linker. In some embodiments, the TBM is an scFv comprising, from N- to C-terminus, VH-L1-VL, where L1 is a peptide linker. In some embodiments, L1 comprises the amino acid sequence of SEQ ID NO: 82. In some embodiments, the TBM is an scFv comprising an engineered disulfide bond between VH and VL, such as between C44 of VH and C100 of VL, where numbering is based on Kabat numbering. In some embodiments, the scFv comprises a first cysteine ​​residue at position 44 of VH and a second cysteine ​​residue at position 100 of VL, where the first and second cysteine ​​residues form a disulfide bond, where numbering is based on Kabat numbering.

[0133] In some embodiments, the TBM comprises a full-length antibody light chain and / or a full-length antibody heavy chain. The antibody light chain may be a kappa or lambda light chain. The antibody heavy chain may be of any class, such as IgG, IgM, IgE, IgA, or IgD. In some embodiments, the antibody heavy chain is of the IgG class, such as the IgG1, IgG2, IgG3, or IgG4 subclass. The antibody heavy chains described herein may be converted from one class or subclass to another using methods known in the art.

[0134] The multispecific antibodies described herein may comprise a TBM derived from any suitable antibody targeting an antigen of interest. The TBM described herein may incorporate any of the CDR sequences (e.g., one, two, or three heavy chain variable region CDR sequences and / or one, two, or three light chain variable region CDR sequences), heavy chain variable region sequences, and / or light chain variable region sequences of any of the antibodies described in WO2019 / 036856, WO2019 / 036842, WO2019 / 036855, WO2019148444, WO2019185035, and WO2019036855 (which are incorporated herein by reference in their entireties). Table C below shows the antibody CDR, VH, VL, and scFv sequences of exemplary TBMs described herein. [Table 3-1] [Table 3-2] [Table 3-3]

[0135] In some embodiments, the TBM is an anti-PDL1 antibody or an antigen-binding domain thereof, including, for example, a VH, VL, scFv, light chain, or heavy chain (e.g., IgG1, IgG2, IgG4). Any of the known anti-PD-L1 antibodies may be used in the present invention. See, for example, U.S. Patent Nos. US7943743, US7722868, US8217149, US8383796, US8552154, and US9102725; and U.S. Patent Application Publication Nos. US20140341917 and US20150203580; and International Patent Application No. PCT / US2001 / 020964. Exemplary anti-PD-L1 antibodies include, but are not limited to, BMS935559 (also known as MDX-1105), MPDL3280A, MEDI4736, avelumab (also known as MSB0010718C), KY-1003, MCLA-145, RG7446 (also known as atezolizumab), SHR-1316, STI-3031, ZKAB001, TQB2450, LY3300054, and STI-A1010.

[0136] In some embodiments, the TBM comprises a VH comprising an antibody heavy chain complementarity determining region (CDR-H)1 comprising the amino acid sequence of SEQ ID NO: 37, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 38, and / or a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 39. In some embodiments, the TBM comprises an antibody light chain complementarity determining region (CDR-L)1 comprising the amino acid sequence of SEQ ID NO: 40, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 41, and / or a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 42. In some embodiments, the TBM comprises a VH comprising the amino acid sequence of SEQ ID NO: 43. In some embodiments, the TBM comprises a VL comprising the amino acid sequence of SEQ ID NO: 44. In some embodiments, the TBM comprises an scFv comprising the amino acid sequence of SEQ ID NO: 77.

[0137] In some embodiments, the TBM is an anti-CD137 antibody or an antigen-binding domain thereof, including, for example, a VH, VL, scFv, light chain, or heavy chain (e.g., IgG1, IgG2, IgG4). Any known anti-CD137 antibody can be used in the present invention. See, for example, WO2016 / 134358. Exemplary anti-CD137 antibodies include, but are not limited to, urelumab (also known as BMS-663513), utomilumab (also known as PF-05082566), CTX-471, ATOR-1017, and AGEN2373.

[0138] In some embodiments, the TBM comprises a VH comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 45, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 46, and / or a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 47. In some embodiments, the TBM comprises a VL comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 48, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 49, and / or a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 50. In some embodiments, the TBM comprises a VH comprising the amino acid sequence of SEQ ID NO: 51. In some embodiments, the TBM comprises a VL comprising the amino acid sequence of SEQ ID NO: 52. In some embodiments, the TBM comprises an scFv comprising the amino acid sequence of SEQ ID NO: 78.

[0139] In some embodiments, the TBM is an anti-CTLA4 antibody or an antigen-binding domain thereof, including, for example, a VH, VL, scFv, light chain, or heavy chain (eg, IgG1, IgG2, IgG4). Other anti-CTLA-4 antibodies, such as, but not limited to, ipilimumab (see U.S. Patent Nos. 6,984,720, 7,452,535, 7,605,238, 8,017,114, and 8,142,778), tremilimumab (see U.S. Patent Nos. 6,68,736, 7,109,003, 7,132,281, 7,411,057, 7,807,797, 7,824,679, and 8,143,379), and single chain antibodies (e.g., U.S. Patent No. 5,811,097 Any of the known anti-CTLA4 antibodies may be used in the present invention, including U.S. Patent Publication Nos. 6,051,227, and 7,229,628, U.S. Patent Publication Nos. US20110044953, US Patent Publication No. US2018037654, US Patent Publication No. US2009025274, US Patent Publication No. US2019127468, International Patent Publication Nos. WO2019 / 152413, WO2018209701, WO2018 / 202649, and WO2019 / 152423. Other exemplary anti-CTLA-4 antibodies include RG2077, ONC-392, CS1002, BCD-145, IBI310, AGEN1884, AGEN1181, and AGEN2041.

[0140] In some embodiments, the TBM comprises a VH comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 53, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 54, and / or a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 55. In some embodiments, the TBM comprises a VL comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 56, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 57, and / or a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 58. In some embodiments, the TBM comprises a VH comprising the amino acid sequence of SEQ ID NO: 59. In some embodiments, the TBM comprises a VL comprising the amino acid sequence of SEQ ID NO: 60. In some embodiments, the TBM comprises an scFv comprising the amino acid sequence of SEQ ID NO: 83.

[0141] In some embodiments, the TBM is an anti-CD3 antibody or an antigen-binding domain thereof, including, for example, a VH, VL, scFv, light chain, or heavy chain (eg, IgG1, IgG2, IgG4). Non-limiting examples of such antibodies include Cris-7 monoclonal antibody (Reinherz, EL et al. (eds.), Leukocyte typing II, Springer Verlag, New York, (1986)), BC3 monoclonal antibody (Anasetti et al. (1990) J. Exp. Med. 172:1691), OKT3 (Ortho multicenter Transplant Study Group (1985) N. Engl. J. Med. 313:337), and derivatives thereof, such as OKT3 ala-ala (Herold et al. (2003) J. Clin. Invest. 11:409), visilizumab (Carpenter et al. (2002) Blood 99:2712), and 145-2C11 monoclonal antibody (Hirsch et al. (2003) J. Clin. Invest. 11:409). Any of the known anti-CD3 antibodies, including otelixizumab and foralarumab, may be used in the present invention. Additional CD3-binding molecules contemplated herein include UCHT-1 (Beverley, PC and Callard, RE (1981) Eur. J. Immunol. 11:329-334), SP34 (Silvana et al. (1985) The EMBO Journal. 4:337-344), and the CD3-binding molecules described in WO2004 / 106380; WO2010 / 037838; WO2008 / 119567; WO2007 / 042261; WO2010 / 0150918; WO2018 / 052503; WO2016 / 204966.

[0142] In some embodiments, the TBM comprises a VH comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 61, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 62, and / or a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 63. In some embodiments, the TBM comprises a VL comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 64, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 65, and / or a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 66. In some embodiments, the TBM comprises a VH comprising the amino acid sequence of SEQ ID NO: 67. In some embodiments, the TBM comprises a VL comprising the amino acid sequence of SEQ ID NO: 68. In some embodiments, the TBM comprises an scFv comprising the amino acid sequence of SEQ ID NO: 79.

[0143] In some embodiments, the TBM is an anti-HER2 antibody or its antigen-binding domain, including, for example, a VH, VL, scFv, light chain, or heavy chain (e.g., IgG1, IgG2, IgG4). Any known anti-HER2 antibody may be used in the present invention, including, but not limited to, Herceptin (1998, Cancer Res 58(13):2825-2831), MDXH210 (Schwaab et al., 2001, Journal of Immunotherapy, 24(1):79-87), dicitamab (Toxicol Lett. 2019.S0378-4274(19)30421-7), and pertuzumab (Agus DB, Gordon MS, Taylor C, et al. J Clin Oncol. 2005;23(11):2534-2543).

[0144] In some embodiments, the TBM comprises a VH comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 69, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 70, and / or a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 71. In some embodiments, the TBM comprises a VL comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 72, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 73, and / or a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 74. In some embodiments, the TBM comprises a VH comprising the amino acid sequence of SEQ ID NO: 75. In some embodiments, the TBM comprises a VL comprising the amino acid sequence of SEQ ID NO: 76.

[0145] The term "PDL1" as used in this application includes human PDL1 (e.g., UniProt accession number Q9NZQ7), as well as variants, isoforms, and species homologs thereof (e.g., mouse PDL1 (UniProt accession number Q9EP73), rat PDL1 (UniProt accession number P52944), canine PDL1 (UniProt accession number E2RKZ5), cynomolgus monkey PDL1, etc.).

[0146] The term "CTLA4" as used in this application includes human CTLA4 (e.g., UniProt accession number P16410), as well as variants, isoforms, and species homologs thereof (e.g., mouse CTLA4 (UniProt accession number P09793), rat CTLA4 (UniProt accession number Q9Z1A7), canine CTLA4 (UniProt accession number Q9XSI1), cynomolgus monkey CTLA4 (UniProt accession number G7PL88), etc.).

[0147] The term "CD137" as used in this application includes human CD137 (e.g., GenBank Accession Nos. NM_001561; NP_001552), as well as variants, isoforms, and species homologs thereof (e.g., mouse CD137 (GenBank Gene ID 21942), rat CD137 (GenBank Gene ID 500590), canine CD137 (GenBank Gene ID 608274), cynomolgus monkey CTLA4 (GenBank Gene ID 102127961), etc.).

[0148] The term "CD3" is known in the art as a six-chain multiprotein complex (see Abbas and Lichtman, 2003; Janeway et al., pp. 172 and 178, 1999). In mammals, the complex comprises a homodimer of the CD3 gamma chain, the CD3 delta chain, two CD3 epsilon chains, and the CD3 zeta chain. The CD3 gamma, CD3 delta, and CD3 epsilon chains are highly related cell surface proteins of the immunoglobulin superfamily that contain a single immunoglobulin domain. The transmembrane regions of the CD3 gamma, CD3 delta, and CD3 epsilon chains are negatively charged, a property that allows these chains to associate with positively charged T cell receptor chains. The intracellular tails of the CD3 gamma, CD3 delta, and CD3 epsilon chains each contain a single conserved motif known as an immunoreceptor tyrosine-based activation motif or ITAM, while the CD3 zeta chain has three. Without being bound by theory, ITAMs are believed to be important for the signal transduction ability of the TCR complex. CD3 as used herein may be derived from various animal species, including humans, primates, mice, rats, or other mammals. For example, CD3 as used herein includes human CD3e (i.e., CD3 epsilon; e.g., UniProt Accession No. P07766), as well as its variants, isoforms, and species homologs (e.g., mouse CD3e (UniProt Accession No. P22646), rat CD3e (UniProt Accession No. A0A0G2K986), dog CD3e (UniProt Accession No. P27597), and cynomolgus monkey CD3e (UniProt Accession No. Q95LI5)).

[0149] The term "HER2" as used in this application includes human HER2 (e.g., UniProt Accession No. P04626), as well as variants, isoforms, and species homologs thereof (e.g., mouse HER2 (UniProt Accession No. P70424), rat HER2 (UniProt Accession No. P06494), canine HER2, and cynomolgus monkey HER2). HER2 is also known as ERBB2.

[0150] The TBMs described herein can bind to human targets (e.g., PDL1, CTLA4, CD137, CD3, or HER2). In some cases, the TBMs may be completely specific for the human target and may not exhibit species or other types of cross-reactivity. In other cases, the TBMs also bind to targets of species other than human.

[0151] Linker The multispecific antibodies described herein may comprise one or more linkers (e.g., L1, L2, L3, etc.) positioned between various regions of the polypeptide.

[0152] For example, 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.); glycine-serine polymers (GS)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.), such as SGGGS (sequence Any suitable linker (e.g., flexible linker) known in the art may be used, including (G4S)4 (SEQ ID NO: 80), GGGSGGGGS (SEQ ID NO: 81), (G4S)4 (SEQ ID NO: 82), GGGGS (SEQ ID NO: 130), SGGS (SEQ ID NO: 131), GGSG (SEQ ID NO: 132), GGSGG (SEQ ID NO: 133), GSGSG (SEQ ID NO: 134), GSGGG (SEQ ID NO: 135), GGGSG (SEQ ID NO: 136), and / or GSSSG (SEQ ID NO: 137); glycine-alanine polymers; alanine-serine polymers; and the like. The linker sequence may be of any length, for example, from about 1 amino acid (e.g., glycine or serine) to about 20 amino acids (e.g., a 20-amino acid glycine polymer or glycine-serine polymer), from about 1 amino acid to about 15 amino acids, from about 3 amino acids to about 12 amino acids, from about 4 amino acids to about 10 amino acids, from about 5 amino acids to about 9 amino acids, from about 6 amino acids to about 8 amino acids, etc. In some embodiments, the linker is any of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length.

[0153] Exemplary Multispecific Antibodies Exemplary multispecific antibodies described herein include, but are not limited to, bispecific antibodies targeting PDL1 and CD137 (e.g., PDL1×CD137 and CD137×PDL1 antibodies), bispecific antibodies targeting CD137 and CTLA4 (e.g., CD137×CTLA4 antibodies), bispecific T cell engagers (BiTEs) targeting CD3 and a cell surface antigen, and trispecific antibodies targeting PDL1, CD137, and CTLA4 (also referred to herein as PDL1×CD137×CTLA4 antibodies). In some embodiments, a multispecific antibody comprises a CH3 domain or Fc region comprising any one or combination of engineered disulfide bonds and / or salt bridges described herein. In some embodiments, a multispecific antibody does not comprise a CH3 domain or Fc region comprising any one or combination of engineered disulfide bonds and / or salt bridges described herein.

[0154] In some embodiments, a bispecific antibody targeting PDL1 and CD137 is provided, comprising a first polypeptide and a second polypeptide, (i) the first polypeptide comprises a structure represented by the following formula: VH1-CH1-hinge-CH2-1st CH3-L1-scFv1, (ii) the second polypeptide comprises a structure represented by the formula: VL-CL, VL is an immunoglobulin light chain variable domain; VH is an immunoglobulin heavy chain variable domain; scFv is a single chain variable fragment; CL is an immunoglobulin light chain constant domain; CH1 is immunoglobulin heavy chain constant domain 1; CH2 is immunoglobulin heavy chain constant domain 2; The hinge is an immunoglobulin hinge region that connects the CH1 and CH2 domains, L1 is a bond or a peptide linker; The VL and VH associate to form an Fv that specifically binds to CD137, and the scFv specifically binds to PDL1. In some embodiments, the scFv comprises a VH that includes a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 37, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 38, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 39; and / or a VL that includes a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 40, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 41, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 42. In some embodiments, the scFv comprises a VH that includes the amino acid sequence of SEQ ID NO: 43 and / or a VL that includes the amino acid sequence of SEQ ID NO: 44. In some embodiments, the scFv comprises, from N- to C-terminus, VH-L1-VL, where L1 is a peptide linker. In some embodiments, L1 comprises the amino acid sequence of SEQ ID NO: 82. In some embodiments, the scFv comprises a first cysteine ​​residue at position 44 of the VH and a second cysteine ​​residue at position 100 of the VL, where the first and second cysteine ​​residues form a disulfide bond, and the numbering is based on Kabat numbering. In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 77. In some embodiments, the Fv comprises a VH comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 45, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 46, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 47; and / or a VL comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 48, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 49, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 50. In some embodiments, the Fv comprises a VH comprising the amino acid sequence of SEQ ID NO: 51 and / or a VL comprising the amino acid sequence of SEQ ID NO: 52. In some embodiments, the first CH3 domain comprises an N390C substitution and the second CH3 domain comprises an S400C substitution, or the first CH3 domain comprises an S400C substitution and the second CH3 domain comprises an N390C substitution. In some embodiments, the multispecific antibody comprises an IgG1 Fc region, e.g., an IgG1 Fc with an N297A substitution.In some embodiments, the multispecific antibody comprises an IgG4 Fc region, e.g., an IgG4 with an S228P substitution. In some embodiments, the scFv is linked to the hinge of a second polypeptide via a linker, such as a peptide linker comprising the amino acid sequence of SEQ ID NO: 80 or 81.

[0155] In some embodiments, a bispecific antibody is provided comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO:96 and a second polypeptide comprising the amino acid sequence of SEQ ID NO:97. In some embodiments, a bispecific antibody is provided comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO:98 and a second polypeptide comprising the amino acid sequence of SEQ ID NO:99. In some embodiments, a bispecific antibody is provided comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO:100 and a second polypeptide comprising the amino acid sequence of SEQ ID NO:101. In some embodiments, a bispecific antibody is provided comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO:102 and a second polypeptide comprising the amino acid sequence of SEQ ID NO:103. In some embodiments, a bispecific antibody is provided comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO:104 and a second polypeptide comprising the amino acid sequence of SEQ ID NO:105. In some embodiments, a bispecific antibody is provided comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO:106 and a second polypeptide comprising the amino acid sequence of SEQ ID NO:107. In some embodiments, a bispecific antibody is provided comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO:108 and a second polypeptide comprising the amino acid sequence of SEQ ID NO:109. In some embodiments, a bispecific antibody is provided comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO:110 and a second polypeptide comprising the amino acid sequence of SEQ ID NO:111.

[0156] In some embodiments, a bispecific antibody targeting PDL1 and CD137 is provided, comprising a first polypeptide and a second polypeptide, (i) the first polypeptide comprises a structure represented by the following formula: VH1-CH1-hinge-CH2-1st CH3-L1-scFv1, (ii) the second polypeptide comprises a structure represented by the formula: VL-CL, VL is an immunoglobulin light chain variable domain; VH is an immunoglobulin heavy chain variable domain; scFv is a single chain variable fragment; CL is an immunoglobulin light chain constant domain; CH1 is immunoglobulin heavy chain constant domain 1; CH2 is immunoglobulin heavy chain constant domain 2; The hinge is an immunoglobulin hinge region that connects the CH1 and CH2 domains, L1 is a bond or a peptide linker; VL and VH associate to form an Fv that specifically binds to PDL1, The scFv specifically binds to CD137. In some embodiments, the Fv comprises a VH comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 37, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 38, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 39; and / or a VL comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 40, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 41, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 42. In some embodiments, the Fv comprises a VH comprising the amino acid sequence of SEQ ID NO: 43 and / or a VL comprising the amino acid sequence of SEQ ID NO: 44. In some embodiments, the scFv comprises a VH comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 45, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 46, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 47; and / or a VL comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 48, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 49, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 50. In some embodiments, the scFv comprises a VH comprising the amino acid sequence of SEQ ID NO:51 and / or a VL comprising the amino acid sequence of SEQ ID NO:52. In some embodiments, the scFv comprises, from N- to C-terminus, VH-L1-VL, where L1 is a peptide linker. In some embodiments, L1 comprises the amino acid sequence of SEQ ID NO:82. In some embodiments, the scFv comprises a first cysteine ​​residue at position 44 of the VH and a second cysteine ​​residue at position 100 of the VL, the first and second cysteine ​​residues forming a disulfide bond, numbering according to Kabat numbering. In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO:78. In some embodiments, the first CH3 domain comprises an N390C substitution and the second CH3 domain comprises an S400C substitution, or the first CH3 domain comprises an S400C substitution and the second CH3 domain comprises an N390C substitution. In some embodiments, the multispecific antibody comprises an IgG1 Fc region, e.g., an IgG1 Fc with an N297A substitution. In some embodiments, the multispecific antibody comprises an IgG4 Fc region, e.g., an IgG4 with an S228P substitution.In some embodiments, the scFv is linked to the hinge of the second polypeptide via a linker, such as a peptide linker comprising the amino acid sequence of SEQ ID NO: 80 or 81.

[0157] In some embodiments, a bispecific antibody is provided comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 84 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 85. In some embodiments, a bispecific antibody is provided comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 86 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 87. In some embodiments, a bispecific antibody is provided comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 88 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 89. In some embodiments, a bispecific antibody is provided comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 90 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 91. In some embodiments, a bispecific antibody is provided comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 92 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 93. In some embodiments, a bispecific antibody is provided comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 94 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 95.

[0158] In some embodiments, a trispecific antibody targeting PDL1, CD137, and CTLA4 is provided, comprising a first polypeptide, a second polypeptide, and a third polypeptide; (i) the first polypeptide comprises a structure represented by the following formula: VH-CH1-hinge-CH2-1st CH3-L1-scFv1, (ii) the second polypeptide comprises a structure represented by the formula: VH-CH1-hinge-CH2-2nd CH3-L2-scFv2, (iii) the third polypeptide comprises a structure represented by the following formula: VL-CL, (iv) the fourth polypeptide comprises a structure represented by the following formula: VL-CL, VH is an immunoglobulin light chain variable domain; VL is an immunoglobulin light chain variable domain; scFv1 is the first single chain variable fragment, CL is an immunoglobulin light chain constant domain; CH1 is immunoglobulin heavy chain constant domain 1; CH2 is immunoglobulin heavy chain constant domain 2; The hinge is an immunoglobulin hinge region that connects the CH1 and CH2 domains, L1 and L2 are independently a bond or a peptide linker; The VL and VH associate to form an Fv that specifically binds to CD137, scFv1 specifically binds to PD-L1, and scFv2 specifically binds to CTLA4. In some embodiments, scFv1 comprises a VH that comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 37, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 38, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 39; and / or a VL that comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 40, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 41, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 42. In some embodiments, scFv1 comprises a VH that comprises the amino acid sequence of SEQ ID NO: 43 and / or a VL that comprises the amino acid sequence of SEQ ID NO: 44. In some embodiments, the Fv comprises a VH comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 45, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 46, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 47; and / or a VL comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 48, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 49, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 50. In some embodiments, the Fv comprises a VH comprising the amino acid sequence of SEQ ID NO: 51 and / or a VL comprising the amino acid sequence of SEQ ID NO: 52. In some embodiments, the scFv2 comprises a VH comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 53, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 54, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 55; and / or a VL comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 56, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 57, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 58. In some embodiments, the scFv2 comprises a VH comprising the amino acid sequence of SEQ ID NO: 59 and / or a VL comprising the amino acid sequence of SEQ ID NO: 60. In some embodiments, the first CH3 domain comprises E357K, S364K, and S400C substitutions and the second CH3 domain comprises L351D, K370D, and N390C substitutions, or the first CH3 domain comprises L351D, K370D, and N390C substitutions and the second CH3 domain comprises E357K, S364K, and S400C substitutions.In some embodiments, the multispecific antibody comprises an IgG1 Fc region, for example, an IgG1 Fc with an N297A substitution.

[0159] In some embodiments, a trispecific antibody is provided, comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 118, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 119, and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 120. In some embodiments, a trispecific antibody is provided, comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 121, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 122, and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 123. In some embodiments, a trispecific antibody is provided, comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 124, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 125, and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 126.

[0160] In some embodiments, a bispecific T cell engager (BiTE) molecule is provided that targets CD3 and a tumor antigen (e.g., HER2), comprising a first polypeptide, a second polypeptide, and a third polypeptide; (i) the first polypeptide comprises a structure represented by the following formula: VH-CH1-hinge-CH2-1st CH3, (ii) the second polypeptide comprises a structure represented by the formula: scFv-hinge-CH2-2nd CH3, (iii) the third polypeptide comprises a structure represented by the following formula: VL-CL, VL is an immunoglobulin light chain variable domain; VH is an immunoglobulin heavy chain variable domain; scFv is a single chain variable fragment; CL is an immunoglobulin light chain constant domain; CH1 is immunoglobulin heavy chain constant domain 1; CH2 is immunoglobulin heavy chain constant domain 2; The hinge is an immunoglobulin hinge region that connects the CH1 and CH2 domains, The VL and VH associate to form an Fv that specifically binds to a tumor antigen (e.g., HER2); the scFv specifically binds to CD3. In some embodiments, the scFv comprises a VH that comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 61, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 62, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 63; and / or a VL that comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 64, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 65, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 66. In some embodiments, the scFv comprises a VH that comprises the amino acid sequence of SEQ ID NO: 67 and / or a VL that comprises the amino acid sequence of SEQ ID NO: 68. In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 79. In some embodiments, the Fv specifically binds to HER2. In some embodiments, the Fv comprises a VH comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 69, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 70, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 71; and / or a VL comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 72, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 73, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 74. In some embodiments, the Fv comprises a VH comprising the amino acid sequence of SEQ ID NO: 75 and / or a VL comprising the amino acid sequence of SEQ ID NO: 76. In some embodiments, the first CH3 domain comprises D356K, E357K, S364K, and S400C substitutions and the second CH3 domain comprises L351D, K370D, N390C, and K439D substitutions, or the first CH3 domain comprises L351D, K370D, N390C, and K439D substitutions and the second CH3 domain comprises D356K, E357K, S364K, and S400C substitutions. In some embodiments, the multispecific antibody comprises an IgG1 Fc region, e.g., an IgG1 Fc with an N297A substitution. In some embodiments, a bispecific T cell engager molecule is provided, comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 112, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 113, and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 114.

[0161] IV. Activatable antibodies Certain embodiments of the present application relate to activatable antibodies (including activatable bispecific T cell engager molecules), activatable antigen-binding fragments thereof, or derivatives of activatable antibodies.

[0162] In some embodiments, the activatable antibody comprises a polypeptide comprising a target-binding moiety (TBM), a cleavable moiety (CM), and a masking moiety (MM). In some embodiments, the TBM comprises an amino acid sequence that binds to a target, such as CD3 or HER2. In some embodiments, the TBM comprises an antigen-binding domain (ABD) of an antibody or antibody fragment thereof. In some embodiments, the TBM comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH), where the VH and VL form a binding domain that binds to the target in the absence of MM. In some embodiments, the VH and VL are covalently linked, for example, in an scFv. In some embodiments, the VH and VL form an Fv fragment. In some embodiments, the VH is linked to an antibody heavy chain constant region and the VL is linked to an antibody light chain constant region. In some embodiments, the activatable antibody comprises an Fc region comprising any one or combination of engineered disulfide bonds or salt bridges described herein. In some embodiments, an activatable antibody comprises an Fc region that does not contain any one or combination of engineered disulfide bonds or salt bridges described herein.

[0163] In some embodiments, an activatable antibody comprises a polypeptide comprising, from its N-terminus to its C-terminus, the structure masking moiety (MM)-cleavable moiety (CM)-VL, and the activatable antibody further comprises a second polypeptide comprising a VH (e.g., a Fab fragment). In some embodiments, an activatable antibody comprises a polypeptide comprising, from its N-terminus to its C-terminus, the structure masking moiety (MM)-cleavable moiety (CM)-VL-VH (e.g., an scFv). In some embodiments, an activatable antibody comprises a polypeptide comprising, from its N-terminus to its C-terminus, the structure masking moiety (MM)-cleavable moiety (CM)-VH, and the activatable antibody further comprises a second polypeptide comprising a VL (e.g., a Fab fragment). In some embodiments, an activatable antibody comprises a polypeptide comprising, from its N-terminus to its C-terminus, the structure masking moiety (MM)-cleavable moiety (CM)-VH-VL (e.g., an scFv).

[0164] In some embodiments, an activatable antibody comprises a polypeptide comprising, from N- to C-terminus, the structure masking moiety (MM)-L1-cleavable moiety (CM)-L2-VL, and the activatable antibody further comprises a second polypeptide comprising a VH (e.g., a Fab fragment). In some embodiments, an activatable antibody comprises a polypeptide comprising, from N- to C-terminus, the structure masking moiety (MM)-L1-cleavable moiety (CM)-L2-VL-L3-VH (e.g., an scFv). In some embodiments, an activatable antibody comprises a polypeptide comprising, from N- to C-terminus, the structure masking moiety (MM)-cleavable moiety (CM)-L1-VH, and the activatable antibody further comprises a second polypeptide comprising a VL (e.g., a Fab fragment). In some embodiments, an activatable antibody comprises a polypeptide comprising, from N- to C-terminus, the structure masking moiety (MM)-L1-cleavable moiety (CM)-L2-VH-L3-VL (e.g., an scFv). In some embodiments, L1, L2, and / or L3 are linkers. In some embodiments, each of L1, L2, and L3 is independently a linker that can be either a bond or a peptide linker having a selected length of 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more amino acids.

[0165] In some embodiments, an activatable antibody is provided, comprising: a first polypeptide comprising a first CH3 domain, a second polypeptide comprising a second CH3 domain, and a third polypeptide; (i) the first polypeptide comprises a structure represented by the following formula: VH-CH1-hinge-CH2-1st CH3, (ii) the second polypeptide comprises a structure represented by the formula: MM1-CM1-scFv-hinge-CH2-2nd CH3, (iii) the third polypeptide comprises a structure represented by the following formula: MM2-CM2-VL-CL, VL is an immunoglobulin light chain variable domain; VH is an immunoglobulin heavy chain variable domain; scFv is a single chain variable fragment; CL is an immunoglobulin light chain constant domain; CH1 is immunoglobulin heavy chain constant domain 1; CH2 is immunoglobulin heavy chain constant domain 2; The hinge is an immunoglobulin hinge region that connects the CH1 and CH2 domains, MM1 is the first masking peptide, MM2 is a second masking peptide, CM1 is the first cleavable peptide, CM2 is a second cleavable peptide, The VL and VH associate to form a first Fv that specifically binds to a first target; the scFv specifically binds to a second target; if CM1 is not cleaved, MM1 inhibits binding of the scFv to the first target; and if CM2 is not cleaved, MM2 inhibits binding of the first Fv to the second target. In some embodiments, the first CH3 domain and the second CH3 domain do not comprise any one or combination of engineered disulfide bonds or salt bridges described herein. In some embodiments, the first CH3 domain and the second CH3 domain comprise any one or combination of engineered disulfide bonds or salt bridges described herein. In some embodiments, the first CH3 domain comprises an N390C substitution and the second CH3 domain comprises an S400C substitution, or the first CH3 domain comprises an S400C substitution and the second CH3 domain comprises an N390C substitution. In some embodiments, the first CH3 domain comprises E357K, S364K, and S400C substitutions and the second CH3 domain comprises L351D, K370D, and N390C substitutions, or the first CH3 domain comprises L351D, K370D, and N390C substitutions and the second CH3 domain comprises E357K, S364K, and S400C substitutions. In some embodiments, the first CH3 domain comprises D356K, E357K, S364K, and N390C substitutions and the second CH3 domain comprises L351D, K370D, K439D, and S400C substitutions; or the first CH3 domain comprises L351D, K370D, K439D, and S400C substitutions and the second CH3 domain comprises D356K, E357K, S364K, and N390C substitutions. In some embodiments, the first CH3 domain comprises D356K, E357K, S364K, and S400C substitutions and the second CH3 domain comprises L351D, K370D, N390C, and K439D substitutions; or the first CH3 domain comprises L351D, K370D, N390C, and K439D substitutions and the second CH3 domain comprises D356K, E357K, S364K, and S400C substitutions.In some embodiments, the activatable antibody comprises an IgG1 Fc region, such as an IgG1 Fc with an N297A substitution. In some embodiments, the first target is a tumor antigen (e.g., HER2) and the second target is CD3 (e.g., CD3e). In some embodiments, the first target is CD3 (e.g., CD3e) and the second target is a tumor antigen (e.g., HER2).

[0166] In some embodiments, an activatable antibody is provided, comprising: a first polypeptide comprising a first CH3 domain, a second polypeptide comprising a second CH3 domain, and a third polypeptide; (i) the first polypeptide comprises a structure represented by the following formula: mm1-cm1-vh-ch1-hinge-ch2-first ch3, (ii) the second polypeptide comprises a structure represented by the formula: MM2-CM2-scFv-hinge-CH2-2nd CH3, (iii) the third polypeptide comprises a structure represented by the following formula: VL-CL, VL is an immunoglobulin light chain variable domain; VH is an immunoglobulin heavy chain variable domain; scFv is a single chain variable fragment; CL is an immunoglobulin light chain constant domain; CH1 is immunoglobulin heavy chain constant domain 1; CH2 is immunoglobulin heavy chain constant domain 2; The hinge is an immunoglobulin hinge region that connects the CH1 and CH2 domains, MM1 is the first masking peptide, MM2 is a second masking peptide, CM1 is the first cleavable peptide, CM2 is a second cleavable peptide, The VL and VH associate to form a first Fv that specifically binds to a first target; the scFv specifically binds to a second target; if CM1 is not cleaved, MM1 inhibits binding of the first Fv to the first target; and if CM2 is not cleaved, MM2 inhibits binding of the scFv to the second target. In some embodiments, the first CH3 domain and the second CH3 domain do not comprise any one or combination of engineered disulfide bonds or salt bridges described herein. In some embodiments, the first CH3 domain and the second CH3 domain comprise any one or combination of engineered disulfide bonds or salt bridges described herein. In some embodiments, the first CH3 domain comprises an N390C substitution and the second CH3 domain comprises an S400C substitution, or the first CH3 domain comprises an S400C substitution and the second CH3 domain comprises an N390C substitution. In some embodiments, the first CH3 domain comprises E357K, S364K, and S400C substitutions and the second CH3 domain comprises L351D, K370D, and N390C substitutions, or the first CH3 domain comprises L351D, K370D, and N390C substitutions and the second CH3 domain comprises E357K, S364K, and S400C substitutions. In some embodiments, the first CH3 domain comprises D356K, E357K, S364K, and N390C substitutions and the second CH3 domain comprises L351D, K370D, K439D, and S400C substitutions; or the first CH3 domain comprises L351D, K370D, K439D, and S400C substitutions and the second CH3 domain comprises D356K, E357K, S364K, and N390C substitutions. In some embodiments, the first CH3 domain comprises D356K, E357K, S364K, and S400C substitutions and the second CH3 domain comprises L351D, K370D, N390C, and K439D substitutions; or the first CH3 domain comprises L351D, K370D, N390C, and K439D substitutions and the second CH3 domain comprises D356K, E357K, S364K, and S400C substitutions.In some embodiments, the activatable antibody comprises an IgG1 Fc region, such as an IgG1 Fc with an N297A substitution. In some embodiments, the first target is a tumor antigen (e.g., HER2) and the second target is CD3 (e.g., CD3e). In some embodiments, the first target is CD3 (e.g., CD3e) and the second target is a tumor antigen (e.g., HER2).

[0167] In some embodiments, an activatable antibody is provided that comprises a first polypeptide comprising a first CH3 domain, a second polypeptide comprising a second CH3 domain, a third polypeptide, and a fourth polypeptide, (i) the first polypeptide comprises a structure represented by the following formula: mm1-cm1-vh1-ch1-hinge-ch2-first ch3, (ii) the second polypeptide comprises a structure represented by the formula: MM2-CM2-VH2-CH1-hinge-CH2-2nd CH3, (iii) the third polypeptide comprises a structure represented by the following formula: VL1-CL, (iv) the fourth polypeptide comprises a structure represented by the following formula: VL2-CL, VL1 is the first immunoglobulin light chain variable domain, VH1 is the first immunoglobulin heavy chain variable domain, VL2 is the second immunoglobulin light chain variable domain, VH2 is the second immunoglobulin heavy chain variable domain, CL is an immunoglobulin light chain constant domain; CH1 is immunoglobulin heavy chain constant domain 1; CH2 is immunoglobulin heavy chain constant domain 2; The hinge is an immunoglobulin hinge region that connects the CH1 and CH2 domains, MM1 is the first masking peptide, MM2 is a second masking peptide, CM1 is the first cleavable peptide, CM2 is a second cleavable peptide, VL1 and VH1 associate to form a first Fv that specifically binds to a first target; VL2 and VH2 associate to form a second Fv that specifically binds to a second target; if CM1 is not cleaved, MM1 inhibits binding of the first Fv to the first target; if CM2 is not cleaved, MM2 inhibits binding of the second Fv to the second target. In some embodiments, the first CH3 domain and the second CH3 domain do not comprise any one or combination of engineered disulfide bonds or salt bridges described herein. In some embodiments, the first CH3 domain and the second CH3 domain comprise any one or combination of engineered disulfide bonds or salt bridges described herein. In some embodiments, the first CH3 domain comprises an N390C substitution and the second CH3 domain comprises an S400C substitution, or the first CH3 domain comprises an S400C substitution and the second CH3 domain comprises an N390C substitution. In some embodiments, the first CH3 domain comprises E357K, S364K, and S400C substitutions and the second CH3 domain comprises L351D, K370D, and N390C substitutions, or the first CH3 domain comprises L351D, K370D, and N390C substitutions and the second CH3 domain comprises E357K, S364K, and S400C substitutions. In some embodiments, the first CH3 domain comprises D356K, E357K, S364K, and N390C substitutions and the second CH3 domain comprises L351D, K370D, K439D, and S400C substitutions; or the first CH3 domain comprises L351D, K370D, K439D, and S400C substitutions and the second CH3 domain comprises D356K, E357K, S364K, and N390C substitutions. In some embodiments, the first CH3 domain comprises D356K, E357K, S364K, and S400C substitutions and the second CH3 domain comprises L351D, K370D, N390C, and K439D substitutions; or the first CH3 domain comprises L351D, K370D, N390C, and K439D substitutions and the second CH3 domain comprises D356K, E357K, S364K, and S400C substitutions.In some embodiments, the activatable antibody comprises an IgG1 Fc region, such as an IgG1 Fc with an N297A substitution. In some embodiments, the first target is a tumor antigen (e.g., HER2) and the second target is CD3 (e.g., CD3e). In some embodiments, the first target is CD3 (e.g., CD3e) and the second target is a tumor antigen (e.g., HER2).

[0168] In some embodiments, an activatable antibody is provided that comprises a first polypeptide comprising a first CH3 domain, a second polypeptide comprising a second CH3 domain, a third polypeptide, and a fourth polypeptide, (i) the first polypeptide comprises a structure represented by the following formula: mm1-cm1-vh1-ch1-hinge-ch2-first ch3, (ii) the second polypeptide comprises a structure represented by the formula: VH2-CH1-hinge-CH2-2nd CH3, (iii) the third polypeptide comprises a structure represented by the following formula: VL1-CL, (iv) the fourth polypeptide comprises a structure represented by the following formula: MM2-CM2-VL2-CL, VL1 is the first immunoglobulin light chain variable domain, VH1 is the first immunoglobulin heavy chain variable domain, VL2 is the second immunoglobulin light chain variable domain, VH2 is the second immunoglobulin heavy chain variable domain, CL is an immunoglobulin light chain constant domain; CH1 is immunoglobulin heavy chain constant domain 1; CH2 is immunoglobulin heavy chain constant domain 2; The hinge is an immunoglobulin hinge region that connects the CH1 and CH2 domains, MM1 is the first masking peptide, MM2 is a second masking peptide, CM1 is the first cleavable peptide, CM2 is a second cleavable peptide, VL1 and VH1 associate to form a first Fv that specifically binds to a first target; VL2 and VH2 associate to form a second Fv that specifically binds to a second target; if CM1 is not cleaved, MM1 inhibits binding of the first Fv to the first target; if CM2 is not cleaved, MM2 inhibits binding of the second Fv to the second target. In some embodiments, the first CH3 domain and the second CH3 domain do not comprise any one or combination of engineered disulfide bonds or salt bridges described herein. In some embodiments, the first CH3 domain and the second CH3 domain comprise any one or combination of engineered disulfide bonds or salt bridges described herein. In some embodiments, the first CH3 domain comprises an N390C substitution and the second CH3 domain comprises an S400C substitution, or the first CH3 domain comprises an S400C substitution and the second CH3 domain comprises an N390C substitution. In some embodiments, the first CH3 domain comprises E357K, S364K, and S400C substitutions and the second CH3 domain comprises L351D, K370D, and N390C substitutions, or the first CH3 domain comprises L351D, K370D, and N390C substitutions and the second CH3 domain comprises E357K, S364K, and S400C substitutions. In some embodiments, the first CH3 domain comprises D356K, E357K, S364K, and N390C substitutions and the second CH3 domain comprises L351D, K370D, K439D, and S400C substitutions; or the first CH3 domain comprises L351D, K370D, K439D, and S400C substitutions and the second CH3 domain comprises D356K, E357K, S364K, and N390C substitutions. In some embodiments, the first CH3 domain comprises D356K, E357K, S364K, and S400C substitutions and the second CH3 domain comprises L351D, K370D, N390C, and K439D substitutions; or the first CH3 domain comprises L351D, K370D, N390C, and K439D substitutions and the second CH3 domain comprises D356K, E357K, S364K, and S400C substitutions.In some embodiments, the activatable antibody comprises an IgG1 Fc region, such as an IgG1 Fc with an N297A substitution. In some embodiments, the first target is a tumor antigen (e.g., HER2) and the second target is CD3 (e.g., CD3e). In some embodiments, the first target is CD3 (e.g., CD3e) and the second target is a tumor antigen (e.g., HER2).

[0169] In some embodiments, an activatable antibody is provided that comprises a first polypeptide comprising a first CH3 domain, a second polypeptide comprising a second CH3 domain, a third polypeptide, and a fourth polypeptide, (i) the first polypeptide comprises a structure represented by the following formula: VH1-CH1-hinge-CH2-1st CH3, (ii) the second polypeptide comprises a structure represented by the formula: VH2-CH1-hinge-CH2-2nd CH3, (iii) the third polypeptide comprises a structure represented by the following formula: MM1-CM1-VL1-CL, (iv) the fourth polypeptide comprises a structure represented by the following formula: MM2-CM2-VL2-CL, VL1 is the first immunoglobulin light chain variable domain, VH1 is the first immunoglobulin heavy chain variable domain, VL2 is the second immunoglobulin light chain variable domain, VH2 is the second immunoglobulin heavy chain variable domain, CL is an immunoglobulin light chain constant domain; CH1 is immunoglobulin heavy chain constant domain 1; CH2 is immunoglobulin heavy chain constant domain 2; The hinge is an immunoglobulin hinge region that connects the CH1 and CH2 domains, MM1 is the first masking peptide, MM2 is a second masking peptide, CM1 is the first cleavable peptide, CM2 is a second cleavable peptide, VL1 and VH1 associate to form a first Fv that specifically binds to a first target; VL2 and VH2 associate to form a second Fv that specifically binds to a second target; if CM1 is not cleaved, MM1 inhibits binding of the first Fv to the first target; if CM2 is not cleaved, MM2 inhibits binding of the second Fv to the second target. In some embodiments, the first CH3 domain and the second CH3 domain do not comprise any one or combination of engineered disulfide bonds or salt bridges described herein. In some embodiments, the first CH3 domain and the second CH3 domain comprise any one or combination of engineered disulfide bonds or salt bridges described herein. In some embodiments, the first CH3 domain comprises an N390C substitution and the second CH3 domain comprises an S400C substitution, or the first CH3 domain comprises an S400C substitution and the second CH3 domain comprises an N390C substitution. In some embodiments, the first CH3 domain comprises E357K, S364K, and S400C substitutions and the second CH3 domain comprises L351D, K370D, and N390C substitutions, or the first CH3 domain comprises L351D, K370D, and N390C substitutions and the second CH3 domain comprises E357K, S364K, and S400C substitutions. In some embodiments, the first CH3 domain comprises D356K, E357K, S364K, and N390C substitutions and the second CH3 domain comprises L351D, K370D, K439D, and S400C substitutions; or the first CH3 domain comprises L351D, K370D, K439D, and S400C substitutions and the second CH3 domain comprises D356K, E357K, S364K, and N390C substitutions. In some embodiments, the first CH3 domain comprises D356K, E357K, S364K, and S400C substitutions and the second CH3 domain comprises L351D, K370D, N390C, and K439D substitutions; or the first CH3 domain comprises L351D, K370D, N390C, and K439D substitutions and the second CH3 domain comprises D356K, E357K, S364K, and S400C substitutions.In some embodiments, the activatable antibody comprises an IgG1 Fc region, such as an IgG1 Fc with an N297A substitution. In some embodiments, the first target is a tumor antigen (e.g., HER2) and the second target is CD3 (e.g., CD3e). In some embodiments, the first target is CD3 (e.g., CD3e) and the second target is a tumor antigen (e.g., HER2).

[0170] In some embodiments, an activatable antibody is designed based on any one of the multispecific antibodies described herein by fusing a masking moiety (MM) to the target binding moiety (TBM) of the multispecific antibody, e.g., via a cleavable moiety (CM), such that when the CM is not cleaved, the MM inhibits binding of the TBM to the target. Activatable antibodies are described, for example, in WO 2019 / 149282, the contents of which are incorporated herein by reference in their entirety. An activatable antibody may comprise any one of the TBMs described in Section III, "Multispecific Antibodies," subsection "Target Binding Moieties (TBM)." An activatable antibody described herein may comprise one or more linkers described in Section III, "Multispecific Antibodies," subsection "Linkers," e.g., positioned between the MM and CM of the Fc, between the CM and TBM, or between the TBM and hinge region.

[0171] MM refers to an amino acid sequence that interferes with or inhibits TBM binding to a target when the CM of an activatable antibody is intact (e.g., contains a cysteine-cysteine ​​disulfide bond that has not been cleaved and / or reduced by the corresponding enzyme). In some embodiments, the MM interferes with or inhibits TBM binding to a target so efficiently that binding of the TBM to the target is very low and / or below the limit of detection (e.g., binding is not detectable in an ELISA or flow cytometry assay). The amino acid sequence of the CM may overlap with or be contained within the MM. It should be noted that for convenience, "ABP" or "activatable antibody" is used herein to refer to both the uncleaved (or "native") and cleaved states of the ABP or activatable antibody. It will be apparent to one of skill in the art that in some embodiments, the cleaved ABP may lack the MM due to cleavage of the CM (e.g., by a protease), resulting in release of at least the MM (e.g., the MM is no longer covalently bound to the ABP (e.g., a disulfide bond between cysteine ​​residues)). Exemplary ABPs are described in more detail below.

[0172] A CM generally contains a cleavable amino acid sequence and serves as a substrate for enzymes and / or cysteine-cysteine ​​pairs capable of forming, for example, reducible disulfide bonds. Thus, when terms such as "cleavage," "cleavable," and "cleaved" are used in connection with a CM, the terms encompass enzymatic cleavage, e.g., by a protease, and cleavage of the disulfide bond between the cysteine-cysteine ​​pair via reduction of the disulfide bond, which may occur upon exposure to a reducing agent.

[0173] In some embodiments, the activatable antibody does not induce ADCC effect. Methods for measuring ADCC effect are known in the art. In some embodiments, the activatable antibody (in either active or inactive form) does not induce more than about 10% ADCC effect (does not induce more than about 10%, more than about 5%, more than about 1%, more than about 0.1%, or more than about 0.01%) compared to a control.

[0174] In some embodiments, the activatable antibody (e.g., BiTE molecule) is capable of inhibiting tumor cell growth and / or proliferation. In some embodiments, tumor cell growth and / or proliferation is inhibited by at least about 5% (e.g., at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 99%) when contacted with the activatable antibody and T cells, compared to corresponding tumor cells not contacted with the activatable antibody (or compared to corresponding tumor cells contacted with an isotype control antibody and T cells). In some embodiments, the activatable antibody is capable of reducing tumor volume in a subject when the subject is administered the activatable antibody. In some embodiments, an activatable antibody (e.g., a BiTE molecule) is capable of reducing a subject's tumor volume by at least about 5% (e.g., at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 99%) compared to the subject's initial tumor volume (e.g., before administration of the activatable antibody; compared to a corresponding tumor in a subject administered an isotype control antibody). Methods for measuring tumor cell growth / proliferation, tumor volume, and / or tumor inhibition are known in the art.

[0175] In some embodiments, the activatable antibody has a therapeutic effect against cancer. In some embodiments, the activatable antibody reduces one or more signs or symptoms of cancer. In some embodiments, a subject with cancer goes into partial or complete remission when administered the activatable antibody.

[0176] Masking part (MM) The activatable antibodies described herein comprise one, two, or more masking moieties. Exemplary masking moiety sequences are shown in Table D below. Masking moieties can be isolated from phage display libraries, for example, as described in WO2019 / 149282 (incorporated herein by reference in its entirety).

[0177] In some embodiments, the activatable antibody comprises a MM comprising the amino acid sequence of SEQ ID NO: 35. In some embodiments, the activatable antibody comprises a MM comprising the amino acid sequence of SEQ ID NO: 36. In some embodiments, the activatable antibody comprises a first MM comprising the amino acid sequence of SEQ ID NO: 35 and a second MM comprising the amino acid sequence of SEQ ID NO: 36. [Table 4]

[0178] In some embodiments, the masking peptide (MM) interferes with, blocks, reduces the ability of, inhibits, inhibits, or competes with the corresponding target-binding moiety for binding to the target (e.g., an "inactive activatable antibody"). In some embodiments, the masking peptide (MM) interferes with, blocks, reduces, inhibits, inhibits, or competes with the target-binding moiety for binding to the target only if the antibody has not been activated (e.g., activated by a change (increase or decrease) in pH, activated by a change (increase or decrease) in temperature, activated after contact with a second molecule (e.g., a small molecule or protein ligand, etc.)). In some embodiments, activation induces cleavage of the cleavable moiety. In some embodiments, activation induces a conformational change in the polypeptide(s) (e.g., displacement of the MM) such that the MM no longer inhibits binding of the activatable antibody to the target. In some embodiments, the MM interferes with, blocks, reduces the ability of, inhibits, inhibits, or competes with the target-binding moiety for binding to the target only if the cleavable moiety (CM) has not been cleaved by one or more proteases that cleave within the cleavable moiety (CM). In some embodiments, the MM has a masking efficiency, prior to activation, of at least about 2.0 (e.g., at least about 2.0, at least about 3.0, at least about 4.0, at least about 5.0, at least about 6.0, at least about 7.0, at least about 8.0, at least about 9.0, at least about 10, at least about 25, at least about 50, at least about 75, at least about 100, at least about 150, at least about 200, at least about 300, at least about 400, at least about 500, etc.). In some embodiments, the masking efficiency is measured as the difference in affinity for binding to a target of an activatable antibody comprising MM (prior to activation) compared to the affinity for binding to a target of a polypeptide lacking MM (e.g., the difference in affinity for a target antigen (e.g., CD3 or HER2) of an activatable antibody comprising MM (prior to activation) compared to the affinity for a target antigen of the activatable antibody after activation).In some embodiments, masking efficiency is measured by dividing the EC50 of binding of an activatable antibody comprising MM (before activation) by the EC50 of the parent antibody (e.g., measuring EC50 by ELISA). In some embodiments, masking efficiency is measured as the difference in affinity for binding to a target of an activatable antibody comprising MM before activation compared to the affinity for binding to the target of an activatable antibody comprising MM after activation (e.g., the difference in affinity for a target antigen (e.g., CD3 or HER2) of an activatable antibody before activation compared to the activatable antibody after activation). In some embodiments, the MM binds to the target-binding moiety (TBM) and inhibits the activatable antibody from binding to the target (e.g., an "inactive" activatable antibody). In some embodiments, the MM has a dissociation constant for binding to the target-binding moiety (TBM) that is greater than the dissociation constant of the target-binding moiety (TBM) for the target. The dissociation constant can be measured, for example, by techniques such as ELISA, surface plasmon resonance or biolayer interferometry (BLI), or flow cytometry.

[0179] In some embodiments, the MM does not interfere with, block, reduce, inhibit, inhibit, or compete with the target binding moiety (TBM) for binding to the target after the polypeptide has been activated (e.g., activated by treatment with one or more proteases that cleave within the cleavable moiety (CM), activated by a change (increase or decrease) in pH, activated by a change (increase or decrease) in temperature, activated after contact with a second molecule (e.g., an enzyme), etc.). In some embodiments, the MM does not interfere with, block, reduce, inhibit, inhibit, or compete with the target binding moiety (TBM) for binding to the target after the cleavable moiety (CM) has been cleaved with one or more proteases that cleave within the cleavable moiety (CM). In some embodiments, the MM has a masking efficiency (e.g., the relative affinity of the activatable antibody after activation compared to the affinity of the parent antibody) of at most about 1.75 (e.g., at most about 1.75, at most about 1.5, at most about 1.4, at most about 1.3, at most about 1.2, at most about 1.1, at most about 1.0, at most about 0.9, at most about 0.8, at most about 0.7, at most about 0.6, or at most about 0.5, etc.) after activation.

[0180] In some embodiments, any of the MMs described herein may further comprise one or more additional amino acid sequences (e.g., one or more polypeptide tags). Examples of suitable additional amino acid sequences may include, but are not limited to, purification tags (e.g., His tags, Flag tags, maltose binding protein, and glutathione-S-transferase tags), detection tags (e.g., tags that can be detected photometrically, such as red or green fluorescent protein), tags with detectable enzymatic activity (e.g., alkaline phosphatase), tags comprising secretion sequences, leader sequences, and / or stabilizing sequences, protease cleavage sites (e.g., furin cleavage sites, TEV cleavage sites, thrombin cleavage sites), and the like. In some embodiments, the one or more additional amino acid sequences are at the N-terminus of the MM.

[0181] Cuttable part (CM) In some embodiments, the activatable antibody comprises one or more CMs, each of which is located between the MM and the TBM.

[0182] In some embodiments, the CM comprises at least a first cleavage site (CS1) (e.g., a first protease cleavage site). In some embodiments, the first cleavage site is a first protease cleavage site. Any suitable protease cleavage site recognized and / or cleaved by any protease known in the art (e.g., a protease known to co-localize with a target of the polypeptide comprising the CM) may be used, including, for example, protease cleavage sites recognized and / or cleaved by urokinase-type plasminogen activator (uPA); matrix metalloproteinases (e.g., MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-16, MMP-17, MMP-19, MMP-20, MMP-23, MMP-24, MMP-26, and / or MMP-27; tobacco etch virus (TEV) protease; plasmin; thrombin; PSA; PSMA; ADAMS / ADAMTS (e.g., ADAM8, ADAM9, ADAMIO, ADAM12, ADAMIS, ADAM17 / TACE, ADAMDECI, ADAMTSI, ADAMTS4, and / or ADAMTS5); caspases (e.g., caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11, caspase-12, caspase-13, and / or caspase-14); aspartic acid proteases (e.g., RACE and / or renin); aspartic acid cathepsins (e.g., cathepsin D and / or cathepsin E); cysteine ​​cathepsins (e.g., cathepsin B, cathepsin C, cathepsin K, cathepsin L, cathepsin S, cathepsin V / L2, and / or cathepsin X / Z / P); cysteine ​​proteinases (e.g., cruzipain, legumain, and / or otubain-2); KLKs (e.g., KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, and / or KLK14);These include metalloproteinases (e.g., meprin, neprilysin, PSMA, and / or BMP-1); serine proteases (e.g., activated protein C, cathepsin A, cathepsin G, chymase, and / or coagulation factor proteases (e.g., FVIIa, FIXa, FXa, FX1a, FXIIa)); elastase; granzyme B; guanidinobenzoatase; HtrAl; human neutrophil elastase; lactoferrin; marapsin; NS3 / 4A; PACE4; tPA; tryptase; type II transmembrane serine proteases (TTSPs) (e.g., DESC1, DPP-4, FAP, hepsin, matriptase-2, MT-SP1 / matriptase, TMPRSS2, TMPRSS3, and / or TMPRSS4), and the like. In some embodiments, the first protease cleavage site is a cleavage site for a protease selected from uPA, MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, MMP-14, TEV protease, plasmin, thrombin, factor X, PSA, PSMA, cathepsin D, cathepsin K, cathepsin S, ADAM10, ADAM12, ADAMTS, caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase-8, caspase-9, caspase-10, caspase-11, caspase-12, caspase-13, caspase-14, and TACE. In some embodiments, the first protease cleavage site is a cleavage site for a protease selected from uPA, MMP-2, MMP-9, and / or TEV protease. In some embodiments, the protease cleavage comprises an amino acid sequence selected from SGRSA (SEQ ID NO: 127), PLGLAG (SEQ ID NO: 128), and ENLYFQG (SEQ ID NO: 129);

[0183] In some embodiments, the cleavable moiety (CM) further comprises at least a second cleavage site (e.g., at least a second, at least a third, at least a fourth, at least a fifth, etc.). In some embodiments, the cleavable moiety (CM) further comprises a second cleavage site (CS2). In some embodiments, the second cleavage site is a second protease cleavage site. The second protease cleavage site may be any suitable protease cleavage site recognized and / or cleaved by any of the proteases described above. In some embodiments, the first (CS1) and second (CS2) cleavage sites are protease cleavage sites recognized and / or cleaved by the same protease. In some embodiments, the first (CS1) and second (CS2) cleavage sites are protease cleavage sites recognized and / or cleaved by different proteases (e.g., the first protease cleavage site is recognized and / or cleaved by uPA and the second protease cleavage site is recognized and / or cleaved by MMP-2; the first protease cleavage site is recognized and / or cleaved by uPA and the second protease cleavage site is recognized and / or cleaved by MMP-9; the first protease cleavage site is recognized and / or cleaved by uPA and the second protease cleavage site is recognized and / or cleaved by TEV protease, etc.). In some embodiments, at least the second cleavage site (CS2) is C-terminal to the first linker (L1). In some embodiments, the cleavable moiety (CM) comprises the structure (CS1)-L1-(CS2) from the N-terminus to the C-terminus.

[0184] In some embodiments, the cleavable moiety (CM) further comprises at least a second linker (e.g., at least a second, at least a third, at least a fourth, at least a fifth, etc.). In some embodiments, the cleavable moiety (CM) further comprises a second linker (L2). The second linker (L2) can be any suitable linker described above. In some embodiments, the first (L1) and the second (L2) linkers are the same. In some embodiments, the first (L1) and the second (L2) linkers are different. In some embodiments, at least the second linker (L2) is C-terminal to the second cleavage site (CS2). In some embodiments, the cleavable moiety (CM) comprises the structure (CS1)-L1-(CS2)-L2 from the N-terminus to the C-terminus.

[0185] Activatable antibodies targeting CD3 The present application provides activatable antibodies, activatable antibody fragments, and polypeptides that target CD3, comprising a masking moiety (MM) comprising the amino acid sequence of SEQ ID NO:35.

[0186] In some embodiments, an antibody light chain is provided comprising a polypeptide comprising, from N-terminus to C-terminus, a MM, a cleavable moiety (CM), and a target binding moiety (TBM), wherein the MM comprises the amino acid sequence of SEQ ID NO: 35; the CM comprises at least a first cleavage site; and the TBM comprises a VL of an anti-CD3 antibody. In some embodiments, the anti-CD3 antibody comprises a VL comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 64, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 65, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 66.

[0187] In some embodiments, an antibody heavy chain is provided comprising a polypeptide comprising, from N-terminus to C-terminus, a MM, a CM, and a TBM, wherein the MM comprises the amino acid sequence of SEQ ID NO: 35; the CM comprises at least a first cleavage site; and the TBM comprises a VH of an anti-CD3 antibody. In some embodiments, the anti-CD3 antibody comprises a VH comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 61 and a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 62.

[0188] In some embodiments, an activatable antibody targeting CD3 is provided, comprising a first polypeptide comprising, from N-terminus to C-terminus, a MM, a CM, and a TBM, wherein the MM comprises the amino acid sequence of SEQ ID NO: 35, and when the CM is not cleaved, the MM inhibits binding of the activatable antibody to CD3; the CM comprises at least a first cleavage site; the TBM comprises a VL, and the activatable antibody further comprises a second polypeptide comprising a VH; and when the CM is cleaved, the activatable antibody binds to CD3 via the VH and VL. In some embodiments, the activatable antibody comprises a VH comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 61, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 62, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 63; and / or a VL comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 64, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 65, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 66. In some embodiments, the activatable antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:67 and / or a VL comprising the amino acid sequence of SEQ ID NO:68.

[0189] In some embodiments, an activatable antibody targeting CD3 is provided, comprising a first polypeptide comprising, from N-terminus to C-terminus, a MM, a CM, and a TBM, wherein the MM comprises the amino acid sequence of SEQ ID NO: 35, and when the CM is not cleaved, the MM inhibits binding of the activatable antibody to CD3; the CM comprises at least a first cleavage site; the TBM comprises a VH, and the activatable antibody further comprises a second polypeptide comprising a VL; and when the CM is cleaved, the activatable antibody binds to CD3 via the VH and VL. In some embodiments, the activatable antibody comprises a VH comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 61, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 62, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 63; and / or a VL comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 64, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 65, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 66. In some embodiments, the activatable antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:67 and / or a VL comprising the amino acid sequence of SEQ ID NO:68.

[0190] In some embodiments, an activatable antibody targeting CD3 is provided, comprising a first polypeptide comprising, from N-terminus to C-terminus, a MM, a CM, and an scFv, wherein the MM comprises the amino acid sequence of SEQ ID NO: 35, and when the CM is not cleaved, the MM inhibits binding of the activatable antibody to CD3; the CM comprises at least a first cleavage site; and when the CM is cleaved, the activatable antibody binds to CD3 via the scFv. In some embodiments, the scFv comprises a VH comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 61, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 62, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 63; and / or a VL comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 64, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 65, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 66. In some embodiments, the scFv comprises a VH comprising the amino acid sequence of SEQ ID NO: 67 and / or a VL comprising the amino acid sequence of SEQ ID NO: 68. In some embodiments, the scFv comprises, from N- to C-terminus, a VL and a VH. In some embodiments, the scFv comprises, from N- to C-terminus, a VH and a VL. In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 79.

[0191] In some embodiments, the activatable antibody that targets CD3 is a multispecific antibody, such as a bispecific antibody, hi some embodiments, the activatable antibody that targets CD3 is a bispecific T cell engager (BiTE) molecule that also targets a tumor antigen, such as HER2.

[0192] In some embodiments, an activatable bispecific T cell engager molecule is provided, comprising: a first polypeptide comprising a first CH3 domain; a second polypeptide comprising a second CH3 domain; and a third polypeptide; (i) the first polypeptide comprises a structure represented by the following formula: VH-CH1-hinge-CH2-1st CH3, (ii) the second polypeptide comprises a structure represented by the formula: MM1-CM1-scFv-hinge-CH2-2nd CH3, (iii) the third polypeptide comprises a structure represented by the following formula: MM2-CM2-VL-CL, VL is an immunoglobulin light chain variable domain; VH is an immunoglobulin heavy chain variable domain; scFv is a single chain variable fragment; CL is an immunoglobulin light chain constant domain; CH1 is immunoglobulin heavy chain constant domain 1; CH2 is immunoglobulin heavy chain constant domain 2; The hinge is an immunoglobulin hinge region that connects the CH1 and CH2 domains, MM1 is the first masking peptide, MM2 is a second masking peptide, CM1 is the first cleavable peptide, CM2 is a second cleavable peptide, The VL and VH associate to form a first Fv that specifically binds to a tumor antigen (e.g., HER2); the scFv specifically binds to CD3; if CM1 is not cleaved, MM1 inhibits binding of the scFv to CD3; and if CM2 is not cleaved, MM2 inhibits binding of the first Fv to a tumor antigen (e.g., HER2). In some embodiments, MM1 comprises the amino acid sequence of SEQ ID NO: 35. In some embodiments, the scFv comprises a VH comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 61, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 62, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 63; and / or a VL comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 64, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 65, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 66. In some embodiments, the scFv comprises a VH comprising the amino acid sequence of SEQ ID NO: 67 and / or a VL comprising the amino acid sequence of SEQ ID NO: 68. In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO:79.

[0193] In some embodiments, the activatable BiTE molecule targets HER2. In some embodiments, MM2 comprises the amino acid sequence of SEQ ID NO: 36. In some embodiments, the VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 69, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 70, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 71. In some embodiments, the VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 72, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 73, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 74. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 75. In some embodiments, the VL comprises the amino acid sequence of SEQ ID NO: 76.

[0194] In some embodiments of any one of the activatable antibodies (including BiTE molecules) targeting CD3 described herein, the activatable antibody comprises a first CH3 domain and a second CH3 domain that do not comprise any one or combination of engineered disulfide bonds or salt bridges described herein. In some embodiments, the activatable antibody comprises a first CH3 domain and a second CH3 domain that comprise any one or combination of engineered disulfide bonds or salt bridges described herein. In some embodiments, the first CH3 domain comprises D356K, E357K, S364K, and S400C substitutions and the second CH3 domain comprises L351D, K370D, N390C, and K439D substitutions, or the first CH3 domain comprises L351D, K370D, N390C, and K439D substitutions and the second CH3 domain comprises D356K, E357K, S364K, and S400C substitutions. In some embodiments, the activatable antibody comprises an IgG1 Fc region, such as an IgG1 Fc with an N297A substitution.

[0195] Exemplary BiTE molecules are shown, for example, in Tables 10 and 11. In some embodiments, an activatable bispecific T cell engager molecule is provided that comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 115, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 116, and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 117.

[0196] Activatable antibodies targeting HER2 The present application provides activatable antibodies, activatable antibody fragments, and polypeptides that target HER2, comprising a masking moiety (MM) comprising the amino acid sequence of SEQ ID NO:36.

[0197] In some embodiments, an antibody light chain is provided comprising a polypeptide comprising, from N-terminus to C-terminus, a MM, a cleavable moiety (CM), and a target binding moiety (TBM), wherein the MM comprises the amino acid sequence of SEQ ID NO: 36; the CM comprises at least a first cleavage site; and the TBM comprises a VL of an anti-HER2 antibody. In some embodiments, the anti-HER2 antibody comprises a VL comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 72, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 73, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 74.

[0198] In some embodiments, an antibody heavy chain is provided comprising a polypeptide comprising, from N-terminus to C-terminus, a MM, a CM, and a TBM, wherein the MM comprises the amino acid sequence of SEQ ID NO: 36; the CM comprises at least a first cleavage site; and the TBM comprises a VH of an anti-HER2 antibody. In some embodiments, the anti-HER2 antibody comprises a VH comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 69, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 70, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 71.

[0199] In some embodiments, an activatable antibody targeting HER2 is provided, comprising a first polypeptide comprising, from N-terminus to C-terminus, a MM, a CM, and a TBM, wherein the MM comprises the amino acid sequence of SEQ ID NO: 36, and when the CM is not cleaved, the MM inhibits binding of the activatable antibody to HER2; the CM comprises at least a first cleavage site; the TBM comprises a VL, and the activatable antibody further comprises a second polypeptide comprising a VH; and when the CM is cleaved, the activatable antibody binds to HER2 via the VH and VL. In some embodiments, the activatable antibody comprises a VH comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 69, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 70, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 71; and / or a VL comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 72, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 73, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 74. In some embodiments, the activatable antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:75 and / or a VL comprising the amino acid sequence of SEQ ID NO:76.

[0200] In some embodiments, an activatable antibody targeting HER2 is provided, comprising a first polypeptide comprising, from N-terminus to C-terminus, a MM, a CM, and a TBM, wherein the MM comprises the amino acid sequence of SEQ ID NO: 36, and when the CM is not cleaved, the MM inhibits binding of the activatable antibody to HER2; the CM comprises at least a first cleavage site; the TBM comprises a VH, and the activatable antibody further comprises a second polypeptide comprising a VL; and when the CM is cleaved, the activatable antibody binds to HER2 via the VH and VL. In some embodiments, the activatable antibody comprises a VH comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 69, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 70, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 71; and / or a VL comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 72, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 73, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 74. In some embodiments, the activatable antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:75 and / or a VL comprising the amino acid sequence of SEQ ID NO:76.

[0201] In some embodiments, an activatable antibody targeting HER2 is provided, comprising a first polypeptide comprising, from N-terminus to C-terminus, a MM, a CM, and an scFv, wherein the MM comprises the amino acid sequence of SEQ ID NO: 36, and when the CM is uncleaved, the MM inhibits binding of the activatable antibody to HER2; the CM comprises at least a first cleavage site; and when the CM is cleaved, the activatable antibody binds to HER2 via the scFv. In some embodiments, the scFv comprises a VH comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 69, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 70, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 71; and / or a VL comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 72, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 73, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 74. In some embodiments, the scFv comprises a VH comprising the amino acid sequence of SEQ ID NO: 75 and / or a VL comprising the amino acid sequence of SEQ ID NO: 76. In some embodiments, the scFv comprises, from N- to C-terminus, a VL and a VH. In some embodiments, the scFv comprises, from N- to C-terminus, a VH and a VL. In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 79.

[0202] In some embodiments, the activatable antibody that targets HER2 is a multispecific antibody, such as a bispecific antibody, hi some embodiments, the activatable antibody that targets HER2 is a bispecific T cell engager (BiTE) molecule that also targets CD3.

[0203] In some embodiments of any one of the activatable antibodies (including BiTE molecules) targeting HER2 described herein, the activatable antibody comprises a first CH3 domain and a second CH3 domain that do not comprise any one or combination of engineered disulfide bonds or salt bridges described herein. In some embodiments, the activatable antibody comprises a first CH3 domain and a second CH3 domain that comprise any one or combination of engineered disulfide bonds or salt bridges described herein. In some embodiments, the first CH3 domain comprises D356K, E357K, S364K, and S400C substitutions and the second CH3 domain comprises L351D, K370D, N390C, and K439D substitutions, or the first CH3 domain comprises L351D, K370D, N390C, and K439D substitutions and the second CH3 domain comprises D356K, E357K, S364K, and S400C substitutions. In some embodiments, the activatable antibody comprises an IgG1 Fc region, such as an IgG1 Fc with an N297A substitution.

[0204] V. Variants and Derivatives Variants and derivatives of any one of the heterodimeric proteins, multispecific antibodies, and activatable antibodies described herein are also contemplated herein.

[0205] In some embodiments, heterodimeric protein or antibody derivatives are derived from modifications of the amino acid sequence of the parent heterodimeric protein or antibody while preserving the overall molecular structure of the parent heterodimeric protein or antibody. The amino acid sequence of any region of the parent heterodimeric protein or antibody chain, such as the framework region, CDR region, or constant region, may be modified. The type of modification includes substitution, insertion, deletion, or a combination of one or more amino acids of the parent heterodimeric protein or antibody.

[0206] In some embodiments, an antibody (e.g., a multispecific antibody or an activatable antibody) derivative comprises a polypeptide that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in any of SEQ ID NOs: 84-143. In some embodiments, an antibody (e.g., a multispecific antibody or an activatable antibody) derivative comprises a VL or VH region that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in any of SEQ ID NOs: 43, 44, 51, 52, 59, 60, 67, 68, 75, and 76. In some embodiments, the antibody derivative comprises a CDR-H1 amino acid sequence region that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in any of SEQ ID NOs: 37, 45, 53, 61, and 69. In some embodiments, the antibody derivative comprises a CDR-H2 amino acid sequence region that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in any of SEQ ID NOs: 38, 46, 54, 62, and 70. In some embodiments, the antibody derivative comprises a CDR-H3 amino acid sequence region that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in any of SEQ ID NOs: 39, 47, 55, 63, and 71.In some embodiments, the antibody derivative comprises a CDR-L1 amino acid sequence region that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in any of SEQ ID NOs: 40, 48, 56, 64, and 72. In some embodiments, the antibody derivative comprises a CDR-L2 amino acid sequence region that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in any of SEQ ID NOs: 41, 49, 57, 65, and 73. In some embodiments, the antibody derivative comprises a CDR-L3 amino acid sequence region that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in any of SEQ ID NOs: 42, 50, 58, 66, and 74.

[0207] In some embodiments, the heterodimeric protein or antibody derivative comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 conservative or non-conservative substitutions and / or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 additions and / or deletions to the amino acid sequence of a heterodimeric protein or antibody described herein.

[0208] Amino acid substitutions include both conservative and non-conservative substitutions. The term "conservative amino acid substitution" refers to the replacement of one amino acid with another, where the two amino acids have similarities in certain physicochemical properties of the involved residues, such as polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathicity. For example, substitutions may generally be made within each of the following groups: (a) nonpolar (hydrophobic) amino acids, such as alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; (b) polar neutral amino acids, such as glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; (c) positively charged (basic) amino acids, such as arginine, lysine, and histidine; and (d) negatively charged (acidic) amino acids, such as aspartic acid and glutamic acid.

[0209] Modifications may be made anywhere in the antibody amino acid sequence, including the CDRs, framework regions, or constant regions. In some embodiments, the present application provides antibody derivatives containing the VH and VL CDR sequences of the exemplary antibodies described herein but containing framework sequences that differ from those of the exemplary antibodies. Such framework sequences can be obtained from public DNA databases or published references containing germline antibody gene sequences. For example, germline DNA sequences for human heavy and light chain variable region genes can be found in the Genbank database or the "VBase" human germline sequence database (Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, USDapartment of Health and Human Services, NIH Publication No. 91-3242 (1991); Tomlinson et al., J. Mal. Biol. 227:776-798 (1992); and Cox et al., Eur. J. Immunol. 24:827-836 (1994)). Framework sequences that can be used in constructing antibody derivatives include those structurally similar to the framework sequences used by the exemplary antibodies of the present application. For example, the CDR-H1, CDR-H2, and CDR-H3 sequences, and the CDR-L1, CDR-L2, and CDR-L3 sequences of the exemplary antibodies can be grafted into framework regions having the same sequences as found in the germline immunoglobulin gene from which the framework sequences are derived, or the CDR sequences can be grafted into framework regions containing one or more mutations compared to the germline sequences.

[0210] In some embodiments, the antibody derivative is a chimeric antibody comprising the amino acid sequence of an exemplary antibody described herein. In one example, one or more CDRs from one or more exemplary antibodies are combined with CDRs from an antibody derived from a non-human animal, such as a mouse or rat. In another example, all of the CDRs of the chimeric antibody are derived from one or more exemplary antibodies. In some specific embodiments, the chimeric antibody comprises one, two, or three CDRs from the heavy chain variable region and / or one, two, or three CDRs from the light chain variable region of an exemplary antibody. Chimeric antibodies can be generated using conventional methods known in the art.

[0211] Another type of modification is to mutate amino acid residues within the CDR regions of the VH and / or VL chain. Site-directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce the mutation(s), and the effect on antibody binding or other functional properties of interest can be assessed using in vitro or in vivo assays known in the art. Typically, conservative substitutions are introduced. The mutations may be amino acid additions and / or deletions. Furthermore, typically, no more than one, two, three, four, or five residues within the CDR regions are altered. In some embodiments, the antibody derivative contains one, two, three, or four amino acid substitutions in the heavy chain CDR and / or light chain CDR. In another embodiment, the amino acid substitutions are changes to one or more cysteines of the antibody to another residue, such as, but not limited to, alanine or serine. The cysteines may be standard or non-standard cysteines. In some embodiments, the antibody derivative has one, two, three, or four conservative amino acid substitutions in the heavy chain CDR regions compared to the amino acid sequence of an exemplary antibody.

[0212] Modifications may also be made to framework residues within the VH and / or VL regions. Typically, such framework variants are made to reduce the immunogenicity of the antibody. One approach is to "backmutate" one or more framework residues to the corresponding germline sequence. An antibody that has undergone somatic mutation may contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody framework sequence with the germline sequence from which the antibody is derived. To return the framework region sequences to their germline configuration, somatic mutations can be "backmutated" to the germline sequence by, for example, site-directed mutagenesis or PCR-mediated mutagenesis.

[0213] Additionally, modifications may also be made within the Fc region of an exemplary antibody, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cellular cytotoxicity. In one example, the hinge region of CH1 is modified to alter, e.g., increase or decrease, the number of cysteine ​​residues in the hinge region. This approach is further described in U.S. Patent No. 5,677,425. The number of cysteine ​​residues in the hinge region of CH1 is altered, for example, to facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody. In another case, the Fc hinge region of an antibody is mutated to decrease the biological half-life of the antibody.

[0214] In some embodiments, the Fc region of a heterodimeric protein or antibody described herein has at least one (e.g., at least one, two, or more) amino acid substitution compared to the Fc region of a wild-type IgG or wild-type antibody, in addition to the engineered disulfide bond or salt bridge forming amino acid substitutions described herein. In some embodiments, the Fc region has at least 80%, at least 85%, at least 90%, at least 95%, or more homology to a native sequence Fc region and / or the Fc region of a parent polypeptide.

[0215] Additionally, the Fc region may be modified to alter potential glycosylation sites or patterns, according to routine experimentation known in the art. In another aspect, the application provides derivatives of the heterodimeric proteins or antibodies described herein containing at least one mutation in the light or heavy chain variable region that alters the glycosylation pattern in the variable region. Such antibody derivatives may have increased affinity and / or altered specificity for binding to the antigen. The mutations may add new glycosylation sites to the V region, change the position of one or more V region glycosylation site(s), or remove existing V region glycosylation sites. In some embodiments, the application provides derivatives of the antibodies described herein with a potential N-linked glycosylation site at an asparagine in the heavy chain variable region, thereby removing a potential N-linked glycosylation site in one heavy chain variable region. In some embodiments, the application provides derivatives of the antibodies described herein with a potential N-linked glycosylation site at an asparagine in the heavy chain variable region, thereby removing a potential N-linked glycosylation site in both heavy chain variable regions. Methods for altering the glycosylation pattern of antibodies are known in the art, such as those described in US Pat. No. 6,933,368 (which application is incorporated herein by reference).

[0216] In some embodiments, the antibodies described herein (e.g., multispecific antibodies and activatable antibodies) can be of any class, such as IgG, IgM, IgE, IgA, or IgD. In some embodiments, the activatable antibodies described herein (e.g., CD3 and / or HER2 antibodies) are of the IgG class, such as the IgG1, IgG2, IgG3, or IgG4 subclass. The antibodies described herein can be converted from one class or subclass to another using methods known in the art. An exemplary method for generating antibodies of a desired class or subclass includes isolating nucleic acid encoding the heavy chain of an antibody described herein (e.g., a multispecific or activatable antibody) and nucleic acid encoding the light chain of an antibody described herein (e.g., a multispecific or activatable antibody), isolating a sequence encoding the VH region, ligating the VH sequence to a sequence encoding a heavy chain constant region of the desired class or subclass, expressing the light chain gene and heavy chain construct in a cell, and harvesting the antibody.

[0217] Heterodimeric proteins or antibody variants can also be provided with amino-terminal leader extensions, e.g., one or more amino acid residues of an amino-terminal leader sequence are present at the amino terminus of any one or more heavy or light chains of the antibody.

[0218] The heterodimeric proteins or antibodies (e.g., multispecific or activatable antibodies) described herein may be further modified. In some embodiments, the heterodimeric proteins or antibodies are linked to additional molecular entities. Examples of additional molecular entities include pharmaceutical agents, peptides or proteins, detection agents or labels, and antibodies.

[0219] In some embodiments, the heterodimeric protein or antibody of the present application is linked to a pharmaceutical agent. Examples of pharmaceutical agents include cytotoxic agents or other cancer therapeutic agents and radioisotopes. Specific examples of cytotoxic agents include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, as well as analogs or homologs thereof. Therapeutic agents also include, for example, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepachlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamineplatinum(II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and antimitotic agents (e.g., vincristine and vinblastine). Examples of radioisotopes that can be conjugated to antibodies for diagnostic or therapeutic use include, but are not limited to, iodine-131, indium, yttrium-90, and lutetium-177. Methods for linking polypeptides to pharmaceutical agents, for example, using various linker technologies, are known in the art. Examples of linker types include hydrazones, thioethers, esters, disulfides, and peptide-containing linkers.For further discussion of linkers and methods for linking therapeutic agents to antibodies, see, e.g., Saito et al., Adv. Drug De / iv. Rev. 55:199-215 (2003); Trail, et al., Cancer Immunol. Immunother. 52:328-337 (2003); Payne, Cancer Cell 3:207-212 (2003); Allen, Nat. Rev. Cancer 2:750-763 (2002); Pastan and Kreitman, Curr. Opin. Investig. Drugs 3:1089-1091 (2002); Senter and Springer (2001) Adv. Drug De / iv. Rev. 53:247-264.

[0220] In some embodiments, the heterodimeric protein or antibody of the present application is conjugated to a label and / or a cytotoxic agent. As used herein, a label is a moiety that facilitates the detection of the antibody and / or the detection of the molecule to which the antibody binds. Non-limiting examples of labels include, but are not limited to, radioisotopes, fluorescent groups, enzyme groups, chemiluminescent groups, biotin, epitope tags, metal-binding tags, etc. Those skilled in the art can select a suitable label according to the intended application.

[0221] As used herein, a cytotoxic agent is a moiety that reduces the proliferative capacity of one or more cells. A cell's proliferative capacity is reduced when it has a reduced potential for proliferation, for example, because the cell undergoes apoptosis or otherwise dies, the cell is unable to progress through the cell cycle and / or divide, the cell differentiates, etc. Non-limiting examples of cytotoxic agents include, but are not limited to, radioisotopes, toxins, and chemotherapeutic agents. One skilled in the art can select an appropriate cytotoxic agent according to the intended application.

[0222] In some embodiments, the label and / or cytotoxic agent is conjugated to the heterodimeric protein or antibody using chemical methods in vitro. Non-limiting examples of chemical methods of conjugation include services, methods, and / or reagents known in the art and commercially available from, for example, Thermo Scientific Life Science Research Produces (formerly Pierce; Rockford, Ill.), Prozyme (Hayward, Calif.), SACRI Antibody Services (Calgary, Canada), AbD Serotec (Raleigh, NC), etc. In some embodiments, when the label and / or cytotoxic agent is a polypeptide, the label and / or cytotoxic agent can be expressed from the same expression vector with at least one antibody chain, producing a polypeptide comprising the label and / or cytotoxic agent fused to the antibody chain. One of skill in the art can select a suitable method for conjugating the label and / or cytotoxic agent to the antibody according to the intended use.

[0223] VI. Preparation method In one aspect, the application provides methods for preparing a heterodimeric protein, multispecific antibody, or activatable antibody described herein. For example, a method for preparing a heterodimeric protein (e.g., a multispecific antibody) or activatable antibody is provided, the method comprising culturing a host cell containing one or more nucleic acid(s) or vector(s) encoding the heterodimeric protein (e.g., a multispecific antibody) or activatable antibody polypeptide under conditions allowing expression of the nucleic acid(s) or vector(s), and recovering the heterodimeric protein polypeptide or activatable antibody polypeptide from the host cell culture.

[0224] Polypeptides of the present application (e.g., any of the heterodimeric proteins, multispecific antibodies, or activatable antibodies described above) may be produced using recombinant methods and compositions, for example, as described in U.S. Pat. No. 4,816,567. In some embodiments, isolated nucleic acids encoding any of the polypeptides (e.g., any of the heterodimeric proteins, multispecific antibodies, or activatable antibodies described above) are provided. In some embodiments, one or more nucleic acids encoding the first and / or second polypeptides of a heterodimeric protein are provided. In some embodiments, one or more nucleic acids encoding an amino acid sequence comprising the VL(s) and / or VH(s) of a multispecific antibody or activatable antibody (e.g., the antibody light chain and / or heavy chain) are provided. In some embodiments, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided herein. In some embodiments, host cells comprising (e.g., transformed with) one or more vectors comprising nucleic acid(s) encoding a heterodimeric protein, multispecific antibody, or activatable antibody described herein. In some embodiments, the host cell is a eukaryotic cell, such as a yeast cell, an insect cell, a Chinese hamster ovary (CHO) cell, or a lymphoid cell (eg, a YO, NS0, Sp20 cell).

[0225] For recombinant production of a polypeptide of the present application (e.g., any of the heterodimeric proteins, multispecific antibodies, or activatable antibodies described above), for example, nucleic acids encoding the polypeptide (e.g., the heterodimeric proteins, multispecific antibodies, or activatable antibodies described above) are isolated and inserted into one or more vectors for further cloning and / or expression in host cells, as described above. Such nucleic acids can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to the genes encoding the polypeptide(s)).

[0226] Suitable host cells for cloning or expressing vectors encoding the polypeptide include prokaryotic or eukaryotic cells. For example, polypeptides can be produced in bacteria, particularly if glycosylation and Fc effector functions are not required (see, e.g., U.S. Pat. Nos. 5,648,237, 5,789,199, and 5,840,523; see also Charlton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes the expression of antibody fragments in E. coli). After expression, the polypeptide can be isolated from the bacterial cell paste in a soluble fraction and further purified.

[0227] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for polypeptide-encoding vectors, including fungal and yeast strains in which the glycosylation pathway has been "humanized," resulting in the production of polypeptides with partially or completely human glycosylation patterns. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li et al., Nat. Biotech. 24:210-215 (2006).

[0228] Suitable host cells for the expression of glycosylated polypeptides are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. In particular, numerous baculovirus strains have been identified that can be used with insect cells for transfection of Spodoptera frugiperda cells.

[0229] Plant cell cultures can also be used as hosts. See, e.g., U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES™ technology for producing antibodies in transgenic plants).

[0230] Vertebrate cells may also be used as hosts. For example, mammalian cell lines suitable for growth in suspension may be useful. Other examples of useful mammalian host cell lines include the SV40 (COS-7) transformed monkey kidney CV1 line; human embryonic kidney lines (e.g., 293 or 293 cells described in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (e.g., TM4 cells described in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK); buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor (MMT 060562); TRI cells (e.g., Mather et al., Annals NY Acad. Sci. 383:44-68 (1982); MRC5 cells; and FS4 cells. Other useful mammalian host cell lines include DHFR - and Chinese hamster ovary (CHO) cells, including CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines, such as Chinese hamster ovary (CHO) cells, including Y0, NS0, and Sp2 / 0. For a review of specific mammalian host cell lines suitable for antibody production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0231] For the large-scale expression and secretion of some secreted proteins, a leader sequence from a heterologous protein may be desirable. In some embodiments, using a heterologous leader sequence may be advantageous in that the leader sequence is removed in the ER during the secretion process, so the resulting mature polypeptide may remain unchanged. For the expression and secretion of some proteins, the addition of a heterologous leader sequence may be necessary.

[0232] Certain exemplary leader sequences are described, for example, in the online leader sequence database maintained by the Department of Biochemistry at the National University of Singapore. See Choo et al., BMC Bioinformatics, 6:249 (2005); and PCT Publication No. WO2006 / 081430.

[0233] VII. Compositions and Kits In some embodiments, the present application provides a pharmaceutical composition comprising any one of the heterodimeric proteins, multispecific antibodies, or activatable antibodies disclosed herein and a pharmaceutically acceptable carrier. The composition can be prepared by conventional methods known in the art.

[0234] The term "pharmaceutically acceptable carrier" refers to any inert substance suitable for use in a formulation for delivery of a polypeptide (e.g., a heterodimeric protein, a multispecific antibody, or an activatable antibody). Carriers can be antiadherents, binders, coatings, disintegrants, fillers, or diluents, preservatives (e.g., antioxidants, antibacterial agents, or antifungal agents), sweeteners, absorption delaying agents, wetting agents, emulsifiers, buffers, and the like. Examples of suitable pharmaceutically acceptable carriers include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), dextrose, vegetable oils (e.g., olive oil), saline, buffers, buffered saline, and isotonic agents, such as sugars, polyalcohols, sorbitol, and sodium chloride.

[0235] The composition may be in any suitable form, such as liquid, semisolid, and solid dosage forms. Examples of liquid dosage forms include solutions (e.g., injectable and infusible solutions), microemulsions, liposomes, dispersions, or suspensions. Examples of solid dosage forms include tablets, pills, capsules, microcapsules, and powders. A particular form of composition suitable for delivery of a polypeptide (e.g., a heterodimeric protein, a multispecific antibody, or an activatable antibody) is a sterile liquid, such as an injectable or infusible solution, suspension, or dispersion. Sterile solutions can be prepared by incorporating the required amount of the polypeptide (e.g., a heterodimeric protein, a multispecific antibody, or an activatable antibody) into an appropriate carrier, followed by sterile microfiltration. Dispersions may also be prepared by incorporating the polypeptide into a sterile vehicle containing a basic dispersion medium and other carriers. In the case of sterile powders for preparing sterile liquids, methods of preparation include vacuum drying and freeze-drying (lyophilization) to obtain a powder of the active ingredient and any additional desired ingredients from a previously sterile-filtered solution thereof. The various dosage forms of the composition can be prepared by conventional methods known in the art.

[0236] The relative amount of polypeptide (e.g., heterodimeric protein, multispecific antibody, or activatable antibody) included in a composition will vary depending on numerous factors, such as the particular polypeptide and carrier used, the dosage form, and the desired release and pharmacodynamic properties. The amount of polypeptide (e.g., heterodimeric protein, multispecific antibody, or activatable antibody) in a single dosage form will generally be that amount that produces a therapeutic effect, although lesser amounts may also be used. Generally, this amount will range from about 0.01 percent to about 99 percent, from about 0.1 percent to about 70 percent, or from about 1 percent to about 30 percent, based on the total weight of the dosage form.

[0237] In addition to the polypeptide (e.g., a heterodimeric protein, a multispecific antibody, or an activatable antibody), one or more additional therapeutic agents may be included in the composition. The appropriate amount of additional therapeutic agent included in the composition can be readily selected by one of ordinary skill in the art and will vary depending on numerous factors, such as the particular agent and carrier used, the dosage form, and the desired release and pharmacodynamic properties. The amount of additional therapeutic agent included in a single dosage form will generally be that amount of agent that produces a therapeutic effect, although lesser amounts may be used.

[0238] Any of the polypeptides (e.g., heterodimeric proteins, multispecific antibodies, or activatable antibodies) and / or compositions (e.g., pharmaceutical compositions) described herein may be used in the preparation of a medicament (e.g., a medicament used to treat or delay the progression of cancer in a subject in need thereof).

[0239] In some embodiments, provided herein are kits comprising any one of the heterodimeric proteins, multispecific antibodies, activatable antibodies, and / or compositions described herein. In some embodiments, the kits further comprise a package insert containing instructions for use of the heterodimeric proteins, multispecific antibodies, activatable antibodies, and / or compositions. The package insert may contain information regarding indications, usage, dosages, administration, combination therapies, contraindications, and / or warnings regarding use of the therapeutic product. In some embodiments, the kits further comprise one or more buffers, e.g., for storing, transporting, administering, or otherwise using the heterodimeric proteins, multispecific antibodies, activatable antibodies, and / or compositions. In some embodiments, the kits further comprise one or more containers (e.g., syringes, etc.) for storing or administering the heterodimeric proteins, multispecific antibodies, activatable antibodies, and / or compositions. Articles of manufacture comprising any one of the heterodimeric proteins, multispecific antibodies, activatable antibodies, and / or compositions described herein are also provided.

[0240] VIII.How to use The heterodimeric proteins, multispecific antibodies, activatable antibodies, and pharmaceutical compositions described herein are useful for therapeutic, diagnostic, or other purposes, such as modulating the immune response, treating cancer, enhancing the effectiveness of other cancer therapies, enhancing the effectiveness of vaccines, or treating autoimmune diseases.

[0241] In some embodiments, a method is provided for treating a disease or condition in a subject in need thereof, the method comprising administering to the subject an effective amount of a pharmaceutical composition comprising any one of the heterodimeric proteins, multispecific antibodies, or activatable antibodies (e.g., activatable BiTE molecules) described herein. In some embodiments, the disease or condition is cancer. Various cancers can be treated or prevented using the methods, uses, or pharmaceutical compositions provided by the present application.

[0242] In some embodiments, methods are provided for treating cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of a pharmaceutical composition comprising any one of the multispecific antibodies targeting one or more immune checkpoint molecules described herein (e.g., any one of PDL1xCD137, CD137xPDL1, or PDL1xCD137xCTLA4 antibodies). In some embodiments, the cancer is lung cancer. In some embodiments, the cancer is prostate cancer. In some embodiments, the cancer is melanoma. In some embodiments, the cancer is an advanced stage cancer.

[0243] In some embodiments, methods are provided for treating cancer in a subject in need thereof, the methods comprising administering to the subject an effective amount of a pharmaceutical composition comprising any one of the BiTE or activatable BiTE molecules described herein (e.g., any one of a HER2xCD3 antibody or an activatable HER2xCD3 antibody). In some embodiments, the cancer is a HER2-positive cancer. In some embodiments, the cancer is ovarian cancer.

[0244] In some embodiments, a method of enhancing an immune response in a mammal is provided, comprising administering to the mammal an effective amount of a pharmaceutical composition comprising any one of the heterodimeric proteins, multispecific antibodies, or activatable antibodies (e.g., activatable BiTE molecules) described herein. The term "enhancing an immune response" or grammatical variations thereof means stimulating, eliciting, increasing, improving, or augmenting the response of a subject's immune system. The immune response may be a cellular response (i.e., cell-mediated, e.g., cytotoxic T lymphocyte-mediated) or a humoral response (i.e., antibody-mediated), and may be a primary or secondary immune response. Examples of an enhanced immune response include activation of PBMCs and / or T cells (including increased secretion of one or more cytokines, such as IL-2 and / or IFNγ). The enhancement of the immune response can be assessed using a number of in vitro or in vivo measurements known to those skilled in the art, including, but not limited to, cytotoxic T lymphocyte assays, cytokine release, tumor regression, survival of tumor-bearing animals, antibody production, immune cell proliferation, cell surface marker expression, and cytotoxicity. Typically, the methods of the present application enhance the immune response by a mammal when compared to the immune response by an untreated mammal or a mammal not treated using the recited method.

[0245] In practicing the therapeutic method, the heterodimeric protein, multispecific antibody, or activatable antibody may be administered alone as a monotherapy or in combination with one or more additional therapeutic agents or therapies. Accordingly, in another aspect, the present application provides combination therapies comprising the heterodimeric protein, multispecific antibody, or activatable antibody described herein in combination with one or more additional therapies or therapeutic agents for separate, sequential, or simultaneous administration. The term "additional therapeutic agent" may refer to any therapeutic agent other than the heterodimeric protein, multispecific antibody, or activatable antibody provided by the present application.

[0246] A wide variety of cancer therapeutic agents can be used in combination with the heterodimeric proteins, multispecific antibodies, or activatable antibodies provided by the present application. Those skilled in the art will recognize the existence and development of other cancer therapies that can be used in combination with the methods and heterodimeric proteins, multispecific antibodies, or activatable antibodies of the present application and will not be limited to those forms of therapy described herein. Examples of categories of additional therapeutic agents that can be used in combination therapy to treat cancer include (1) chemotherapeutic agents, (2) immunotherapeutic agents, and (3) hormonal therapy agents. In some embodiments, the additional therapy is viral gene therapy, immune checkpoint inhibitors, targeted therapy, radiation therapy, and / or chemotherapy. In some embodiments, the combination therapy includes surgery to remove the tumor.

[0247] The dosage, frequency of administration, and route of administration for the therapies described herein will depend on numerous factors, including, for example, the type and severity of the disorder to be treated, the particular heterodimeric protein, multispecific antibody, or activatable antibody administered, the timing of administration, the duration of treatment, any particular additional therapy administered, the age, sex, weight, condition, health, and past medical history of the patient being treated, and similar factors known in the medical arts.

[0248] Cancer treatment can be evaluated, for example, by tumor regression, reduction in tumor weight or size, time to progression, survival, progression-free survival, overall response rate, duration of response, quality of life, protein expression, and / or activity. Approaches to determining the effectiveness of therapy can be used, including, for example, measuring response by radioimaging. [Example]

[0249] The following examples are intended to be purely illustrative of the present invention and therefore should not be construed as limiting the present invention in any way. The following examples and detailed description are offered by way of illustration and not by way of limitation.

[0250] Example 1. Design of Fc domain mutations Novel Fc mutations were designed, including disulfide bond mutations, charge mutations, and combinations thereof, as shown in Tables 1A and 1B. [Table 5] [Table 6]

[0251] Example 2. Evaluation of heterodimer purity To test the novel Fc mutations, heterodimers TYM01 to TYM013 and a reference heterodimer were constructed. The corresponding novel Fc mutations are shown in Table 2. Fab-Fc / Fc1 arm constructs with mutations at the CH3 domain interface were designed (Figure 1A). To evaluate the effect of mutations on CH3 domain heterodimerization and homodimerization, the constructed CH3_A domain was cloned into a mammalian expression vector so that it could be expressed in a light chain-heavy chain ("LC-HC") half-body format, and the CH3_B domain could be expressed in an Fc-only format. Plasmids encoding the light chain ("LC"), heavy chain ("HC"), and Fc at a molar ratio of 2:1:1 were co-transfected into HEK293 cells for transient expression. An excess of LC relative to HC chain DNA was used to avoid LC becoming limiting. Cell culture supernatants were filtered through a 0.45 μm sterile filter. The antibody was purified by Protein A affinity chromatography using a HiTrap MabSelect SuRe pre-packed column (GE Healthcare) followed by buffer exchange. To assess the heterodimer yield, the product was evaluated by SDS-PAGE and SEC-HPLC.

[0252] In the Fab-Fc / Fc1 arm construct system, the heterodimer and two homodimers differ in size and molecular weight, facilitating identification of the various pairings by SDS-PAGE electrophoresis and size-exclusion high-performance liquid chromatography (SEC-HPLC). The proteins were visualized by electrophoresis under reducing and non-reducing conditions. Under reducing conditions, three bands were observed, corresponding to the HC monomer, Fc monomer, and LC monomer. Figure 6 shows that under non-reducing conditions, there were three bands corresponding to the LC-HC homodimer, LC-HC-Fc heterodimer, and LC-HC half-body. Under these conditions, the Fc homodimer was not detected. The yield of the heterodimer was also assessed by SEC-HPLC. As shown in Figure 7, three peaks were detected overall. The first peak in the spectrum corresponds to the homodimer, the second peak at 46.6 min corresponds to the heterodimer, and the third peak corresponds to the LC-HC half-body. Quantification of peak areas by SEC-HPLC identified variant pairs that stabilized the heterodimer relative to the homodimer. The first and second peaks were used to calculate purity, and the results are shown in Table 2. [Table 7]

[0253] Example 3. Evaluation of heterodimer stability Furthermore, the stability of the heterodimers was evaluated by incubation under forced degradation conditions. Purified heterodimer samples were diluted to 1 mg / mL in the appropriate buffer and heated at different temperatures for 1 hour (Figure 8) or incubated at 37°C for up to 4 weeks (Figure 9). The treated samples were analyzed by SEC-HPLC. Figure 8 shows the different resistances of protein aggregation and precipitation at high temperatures. The changes in the SEC-HPLC spectra after storage at 37°C are shown in Figure 9. Proteins TYM10, TYM11, and TYM013 exhibited relatively good stability.

[0254] Example 4. Generation of heterodimers targeting CD137 and PDL1 We constructed a bispecific antibody with Fc mutations using anti-CD137 and anti-PDL1 antibodies. The pharmacokinetics of a monoclonal antibody can be tuned by altering its interaction with the neonatal Fc receptor FcRn. Improving the affinity of the FcRn-IgG interaction can extend the half-life of the engineered IgG. FcRn binds to the Fc region of IgG in a strictly pH-dependent manner. At physiological pH 7.4, FcRn does not bind to IgG, but at the acidic pH of the endosome (pH 6-6.5), FcRn exhibits low affinity (micromolar to nanomolar) for the Fc region of IgG. Therefore, the FcRn binding properties may reflect how the mutations affect the PK of the Fc region. Table 3 shows that the pH-dependent FcRn binding of the bispecific antibody was unaffected at both acidic and physiological pH. [Table 8]

[0255] Anti-CD137 and anti-PDL1 antibodies with a common light chain were used to construct bispecific antibodies with a common light chain and various Fc mutations (Figure 1B). The bispecific antibodies were also tested in a 293T-CD137-NFκB reporter assay. Briefly, 50x10 4 / ml 293T-CD137 cells and 50x10 4 Mix 293T-PDL1 cells at 5x10 / ml, then mix the mixed cells at 5x10 4 The cells were divided into wells of a 96-well plate at a density of 100 μl / well. 50 μl of diluted antibody solution was added to the corresponding wells and incubated for 18 hours. After incubation, the medium was aspirated, and then 50 μl of Passive Lysis Buffer (Promega E1980) was added and incubated at 37°C for 30 minutes. 20 μl of the supernatant was transferred to a white plate (Costar, 3912), and then 40 μl of Firefly substrate and 40 μl of Renina substrate were added, and the luminescence signal was read (Promega E1980).

[0256] As shown in FIG. 10, TYM10 and TYM11 had relatively high activity in the NFκB reporter assay.

[0257] Example 5. Generation and characterization of bispecific antibodies targeting CD137 and PDL1 A. Generation of Bispecific Antibodies The following example describes the development of a viable and effective format for a bispecific antibody targeting CD137 and PDL1. The format was optimized based on the "Morrison format" (Figure 2). DNA encoding an anti-CD137 Fv and an anti-PDL1 Fv was used to construct an expression plasmid in the form of a Fab or scFv. Because scFvs have reduced affinity compared to Fabs, the orientation of the two Fvs (i.e., CD137 x PDL1 or PDL1 x CD137 in the form of a Fab x scFv) can affect the efficacy of the bispecific antibody.

[0258] IgG1 or IgG4 (S228P) isotypes were used. The scFvs were linked to the C-terminus of the Fc in a VH to VL orientation with a linker of SGGGS (SEQ ID NO: 80) or GGGSGGGGS (SEQ ID NO: 81). The C-terminus of the VH of the scFv was linked to the N-terminus of the VL with a (G4S)4 (SEQ ID NO: 82) linker. A disulfide bond from VH-44 to VL-100 was also incorporated into the scFv to stabilize the format. A newly engineered N390C in the CH3 domain CH3A -S400'C CH3B Disulfide bond pairs were also tested (SEQ ID NOS: 23-24). In addition, the N297A mutation was introduced to silence effector functions mediated by the Fc region. Table 4 provides the eight scaffold designs that were developed. Table 5 lists the SEQ ID NOS corresponding to the first heavy chain, first light chain, second heavy chain, and second light chain of exemplary CD137xPDL1 and PDL1xCD137 antibodies. [Table 9]

[0259] B. CMC Characterization of Bispecific Antibodies Plasmids encoding the heavy and light chains of the bispecific antibody were transiently transfected into mammalian cells. Seven days after transfection, the bispecific antibody-containing cell culture supernatant was collected by centrifugation at 14,000 g for 30 minutes and filtered through a sterile filter (0.22 μm). The antibody was purified by Protein A affinity chromatography using a MabSelect SuRe prepacked column (GE Healthcare), followed by buffer exchange into 20 mM histidine (pH 5.5) buffer.

[0260] The aggregation ratio of the purified bispecific antibody after purification was assessed by analytical size exclusion chromatography ("SEC"). The analysis was performed as follows: SEC was performed on a Waters 2695 coupled with a Waters 2996 UV detector. A TSK Gel g3000 SWXL column (300 mm x 7.8 mm) equipped with a TSK Gel g3000 SWXL precolumn (Tosoh Bioscience) was used. 10 μg of each sample was injected and separation was performed at a flow rate of 0.5 mL / min. The elution buffer consisted of 200 mM sodium phosphate, pH 7.0. UV detection was performed at 214 nm. 100 μL of sample (1 mg / mL) was eluted at -80 o Freeze-thaw stability was tested by freezing at 4°C for 30 minutes followed by thawing at room temperature for 60 minutes. Six freeze-thaw cycles were performed and aggregation rates were also measured by analytical size exclusion chromatography.

[0261] Table 5 provides the yield of bispecific antibodies after purification, and Table 6 provides the aggregation rate of bispecific antibodies after purification and freeze-thaw. TYF05 was the optimal format, with low aggregate formation during expression and no tendency to aggregate during the freeze-thaw process. The disulfide bond in CH3 and replacement of the SGGGS (SEQ ID NO: 80) linker with the 9-amino acid linker GGGSGGGGS (SEQ ID NO: 81) both improved colloidal stability. [Table 10] [Table 11]

[0262] Six cycles of freezing and thawing were tested using 1 mg / mL of purified protein (incubated at 40°C for 28 days) (Figures 11A-11B). Four purified proteins were also heated at high temperatures to confirm their thermal stability (Figure 11C). Figures 11A-11B show the quality of the proteins as assessed by analytical size-exclusion chromatography. All formats demonstrated good long-term storage stability with little aggregation or degradation under accelerated storage conditions. As shown in Figure 11C, all tested proteins aggregated and precipitated at 60°C, with the CD137xPDL1 bispecific antibody exhibiting superior colloidal stability compared with the PDL1xCD137 bispecific antibody. These data indicated CD137xPDL1 as the most suitable antibody format for targeting CD137 and PDL1.

[0263] C. Bispecific Antibody Binding Affinity A Biacore T200 (GE Healthcare) was used as a high-performance system for real-time biomolecular interaction analysis using surface plasmon resonance technology ("SPR"). For measurements, an anti-human IgG monoclonal antibody from the Human Antibody Capture Kit provided by Biacore was immobilized on a CM5 chip, and the IgG sample was injected onto the sensor chip. For binding kinetics analysis in HBS-EP buffer, the sample was injected into the IgG capture flow cell. The data were fitted according to a 1:1 Langmuir model, and the K D The values ​​were determined (Table 7). Format 5 showed the highest affinity of all bispecific antibodies.

[0264] The affinity of the antibodies was also evaluated against human, monkey, and mouse CD137 or PDL1 transiently expressed on the surface of yeast or HEK293F cells. Briefly, yeast or HEK293F cells were transfected with plasmids expressing human, monkey, or mouse CD137 or PDL1. After 48 hours, the transfected cells were harvested and then washed. The cells were then incubated with IgG (100 nM each) for 1 hour at 4°C, protected from light and shaking at 300 rpm in a shaker bed. For simultaneous binding, the cells were incubated with biotinylated human CD137 or PDL1 protein fused to a human Fc fragment and SA-PE (streptavidin, phycoerythrin conjugate). For cross-reactivity, the cells were incubated with an Alexa Fluor 647-conjugated mouse anti-human Fc antibody. The mixture was incubated for 30 minutes at 4°C in a shaker bed, protected from light and shaking at 300 rpm. Cells were washed once before analysis by flow cytometry (Beckman CytoFlex). Figure 12 shows that the bispecific antibody simultaneously binds to human PDL1 and CD137. In addition, Figure 13 shows that the bispecific antibody maintained the cross-reactivity of the parent antibody with PDL1 or CD137 of human, mouse, or monkey origin. [Table 12]

[0265] D. In vitro and in vivo efficacy The effects of PDL1xCD137 and CD137xPDL1 bispecific antibodies on in vitro reporter gene assays were examined (Figure 14). Anti-PDL1-based bispecific antibodies were evaluated in a PDL1-blocking bioassay. Briefly, Jurkat T cells expressing human PD-1 and a luciferase reporter driven by the NFAT response element (NFAT-RE) were used as PD-1 effector cells. CHO-K1 cells expressing human PDL1 and an engineered cell surface protein designed to activate the cognate TCR in an antigen-independent manner were used as PDL1 aAPC / CHO-K1 cells. As shown in Figure 14, PD-1 effector cells were incubated with PDL1 aAPC / CHO-K1 cells in the absence or presence of anti-PDL1-based bispecific antibodies or PDL1 monomer-blocking antibodies. BIO-GLO™ reagent was added, and luminescence was quantified. Data were analyzed using GraphPad Prism® software. Anti-CD137-based bispecific antibodies were evaluated in an NFκB reporter assay. CD137-NFκB-293T stable cells were recovered, cultured, and split into 96-well plates (50 μL / well, density 6x10). 5 After 5.5 hours of incubation, diluted test antibodies (shown in Figure 14) premixed with the crosslinker at a 1:5 ratio were added. Luciferase levels were measured 18 hours later. Renilla luciferase activity was used to normalize relative luciferase units (RLU) to the blank control (no antibody treatment) for transfection efficiency, and results are expressed as the mean ± standard error of triplicates.

[0266] As shown in the top panel of Figure 14, the PDL1 reporter gene assay indicates that the PDL1xCD137 bispecific antibody had similar activity to the PDL1 monomer, and both were more potent than the CD137xPDL1 bispecific antibody. Similarly, in the CD137 reporter gene assay in the bottom panel of Figure 14, the CD137xPDL1 bispecific antibody had more potent activity than the PDL1xCD137 bispecific antibody. These results indicated that the Fab had superior activity to the scFv. Without wishing to be bound by theory, this may be due to differences in affinity.

[0267] Because the anti-CD137 and anti-PDL1 Fvs cross-reacted with mouse and monkey antigens, the in vivo efficacy of the bispecific antibodies was studied in a 3LL syngeneic mouse tumor model (Figures 15A-15C). Both the PDL1xCD137 and CD137xPDL1 bispecific antibodies inhibited tumor growth. The CD137xPDL1 bispecific antibody was slightly less effective than the combination of the CD137 and PDL1 parental antibodies, but was significantly more effective than either of the two parental antibodies alone. The PDL1xCD137 bispecific antibody was less effective than the CD137xPDL1 antibody, indicating that the orientation of the two antigens in this bispecific format is important for efficacy.

[0268] Example 6. Generation of trispecific antibodies The following example provides a trispecific antibody capable of binding to CD137, PDL1, and CTLA4 (Figure 3).

[0269] Three trispecific antibodies combining the Fc variant TYM11 with the bispecific formats TYF01, TYF02, and TYF04 were constructed and purified, as shown in Table 8. TYF02 showed the highest quality, and all three formats were stable under freeze-thaw cycles and storage at 40°C.

[0270] Because the anti-CD137 and anti-PDL1 Fvs cross-reacted with mouse and monkey antigens, the in vivo efficacy of the bispecific antibodies was studied in the 3LL syngeneic mouse tumor model. C57BL / 6 mice were injected with 2 × 10 6 3LL lung cancer cells were subcutaneously implanted. When tumors were established (70 mm 3 Treatment was initiated with isotype control IgG (n = 6), PDL1xCD137 bispecific (10 mg / kg, n = 8), CD137xPDL1 bispecific (10 mg / kg, n = 8), CD137 monomer (7.5 mg / kg, n = 6), PDL1 monomer (7.5 mg / kg, n = 6), or CD137 monomer + PDL1 monomer (both 7.5 mg / kg, n = 8) by intraperitoneal injection for up to six doses. Tumor growth was monitored three times weekly and reported as mean tumor volume ± SEM over time. As shown in Figures 15A-15B, both PDL1xCD137 and CD137xPDL1 bispecific antibodies inhibited tumor growth. The CD137xPDL1 bispecific antibody was slightly less effective than the combination of the CD137 and PDL1 parental antibodies, but was significantly more effective than either of the two parental antibodies alone. The PDL1xCD137 bispecific antibody was not as effective as the CD137xPDL1 antibody, indicating that the orientation of the two antigens in this bispecific format is important for efficacy.

[0271] The TYF02 trispecific antibody was also selected for in vivo efficacy testing using the 3LL syngeneic mouse tumor model. C57BL / 6 mice were injected with 2 × 10 6 3LL lung cancer cells were subcutaneously implanted. When tumors were established (65 mm 3Treatment was initiated by intraperitoneal injection for up to five doses with isotype control IgG (n=6), CD137xPDL1xCTLA4 trispecific (10 mg / kg, n=8), CD137xPDL1xCTLA4 trispecific (5 mg / kg, n=8), CD137xPDL1 bispecific (10 mg / kg, n=6), CD137xCTLA4 bispecific (10 mg / kg, n=6), CD137 monomer (7.5 mg / kg, n=6), PDL1 (3.75 mg / kg, n=6), CTLA4 (3.75 mg / kg, n=6), or CD137 monomer (7.5 mg / kg) + PDL1 (3.75 mg / kg) + CTLA4 (3.75 mg / kg) (n=6). Tumor growth was monitored every two days and reported as mean tumor volume ± SEM over time. As shown in Figure 15C, this trispecific antibody exhibited superior tumor growth inhibition to the corresponding bispecific antibody or the combination of the three mono-IgG parent antibodies. [Table 13]

[0272] Example 7. Biophysical characterization of heterodimeric HER2xCD3 T cell-inducing bispecific antibodies We designed a heterodimeric bispecific scaffold using the TYM13 Fc variant. Light-heavy half-antibodies and scFv-Fc chains were combined to form bispecific antibodies with the TYM13 mutation in the heterodimeric Fc domain (Figure 4). Using this scaffold, we constructed a HER2xCD3 bispecific T cell-inducing antibody. For comparison, we also constructed a corresponding antibody with the knob-into-hole mutations Y394C, T366S, L368A, Y407V-S354C T366W, and the "Xencor mutations" E357Q, S364K-L368D, K370S.

[0273] Plasmids encoding the heavy, light, and scFv-Fc chains of the bispecific antibody were transiently transfected into mammalian cells. Seven days after transfection, the bispecific antibody-containing cell culture supernatant was collected by centrifugation at 14,000 g for 30 minutes and filtered through a sterile filter (0.22 μm). The antibody was purified by Protein A affinity chromatography using a MabSelect SuRe prepacked column (GE Healthcare), followed by buffer exchange into 20 mM histidine (pH 5.5) buffer.

[0274] The biophysical purity of the heterodimeric bispecific antibodies was assessed by SEC-HPLC and SDS-PAGE. As shown in Figures 16 and 17, TY24051, which has the TYM13 mutation, exhibited very good heterodimer purity with no detectable homodimers, whereas TY24105 and TY24106, which have the knob-into-hole and Xencor mutations, both contained a homodimer of approximately 150 kDa (as shown in the SDS-PAGE and SEC-HPLC graphs in Figures 16 and 17, respectively). Furthermore, TY24051 contained fewer aggregates than TY24105 and TY24106.

[0275] When TY24051 was converted to the activatable antibody, TY24052, some aggregates were produced (see Table 9). TY24052 can be purified by cation exchange chromatography (CEX). [Table 14]

[0276] Example 8. Construction and functional characterization of activatable bispecific antibodies Activatable HER2xCD3 bispecific antibodies (also referred to herein as "SAFE bodies" or "SAFE bispecifics") were constructed (Figure 5). The constructs are shown in Tables 10 and 11. [Table 15] [Table 16]

[0277] The affinities of the bispecific antibody (TY24051) and its SAFEbody version (TY24052) were analyzed by enzyme-linked immunosorbent assay (ELISA). Human HER2 or CD3 (ε and δ chain heterodimer) fused to a human Fc fragment was prepared at 2 μg / mL and used to coat ELISA plates overnight at 2–8°C. After washing and blocking, 50 μL of serially diluted IgG was added and incubated at 37°C for 1 hour. The plate was washed three times and then incubated with 50 μL / well of TMB substrate for approximately 20 minutes at room temperature. After the reaction was stopped, the absorbance at 450 nm was measured. Data were analyzed using GraphPad Prism 6 with nonlinear fitting. As shown in Figures 18A–18B, TY24051 bound to both HER2 and CD3, while TY24052 showed significantly lower affinity than TY24051. After activation, the affinity of TY24052 was fully restored.

[0278] To compare the functional activity of TY24051 and TY24052, the antibodies were expressed, purified, and evaluated for antigen-dependent bispecific antibody-mediated tumor cell killing activity (Figure 19). For the in vitro cytotoxicity assay, naive human pan-T cells were isolated from fresh human blood and mixed with HER2-positive tumor cells (SKOV3) for 24 hours in the presence of increasing amounts of bispecific antibody (target cells: 1x10). 4 As shown in Figure 19, dose-dependent killing was observed for TY24051 and TY24052, with TY24052 showing a significantly higher EC 50 The isotype control showed an approximately 800-fold increase in specific killing.

Claims

1. 1. A heterodimeric protein comprising a first polypeptide comprising a first immunoglobulin heavy chain constant domain 3 (CH3) domain and a second polypeptide comprising a second CH3 domain, the first polypeptide and the second polypeptide each comprise, from N-terminus to C-terminus, at least a portion of an immunoglobulin hinge region, an immunoglobulin heavy chain constant domain 2 (CH2 domain), and the CH3 domain, the CH2 domain and the CH3 domain forming an IgG Fc region; the first CH3 domain comprises positively charged residues at positions 357 and 364, and the second CH3 domain comprises negatively charged residues at positions 351 and 370, or the first CH3 domain comprises negatively charged residues at positions 351 and 370, and the second CH3 domain comprises positively charged residues at positions 357 and 364; Amino acid residue numbering is based on EU numbering. Heterodimeric protein.

2. The first CH3 domain further comprises a charged residue at position 356, and the second CH3 domain further comprises a negatively charged residue at position 439, or the first CH3 domain comprises a negatively charged residue at position 439, and the second CH3 domain comprises a positively charged residue at position 356, and the amino acid residue numbering is based on EU numbering. The heterodimeric protein of claim 1.

3. i) the positively charged residue is a lysine (K) residue and the negatively charged residue is an aspartic acid (D) residue; or ii) the positively charged residue is a lysine (K) residue and the negatively charged residue is a glutamic acid (E) residue; or iii) the positively charged residue is an arginine (R) residue and the negatively charged residue is an aspartic acid (D) residue; or iv) the positively charged residue is an arginine (R) residue and the negatively charged residue is a glutamic acid (E) residue; The heterodimeric protein according to claim 1 or 2.

4. i) the first CH3 domain comprises E357K and S364K substitutions and the second CH3 domain comprises L351D and K370D substitutions, or the first CH3 domain comprises L351D and K370D substitutions and the second CH3 domain comprises E357K and S364K substitutions; or ii) the first CH3 domain comprises D356K or E356K, E357K, and S364K substitutions and the second CH3 domain comprises L351D, K370D, and K439D substitutions, or the first CH3 domain comprises L351D, K370D, and K439D substitutions and the second CH3 domain comprises D356K or E356K, E357K, and S364K substitutions; The heterodimeric protein according to claim 3.

5. i) the first CH3 domain further comprises a K392C substitution and the second CH3 domain further comprises a D399C substitution, or the first CH3 domain further comprises a D399C substitution and the second CH3 domain further comprises a K392C substitution; or ii) the first CH3 domain further comprises a Y394C substitution and the second CH3 domain further comprises a S354C substitution, or the first CH3 domain further comprises a S354C substitution and the second CH3 domain further comprises a Y394C substitution; or iii) the first CH3 domain further comprises a D356C or E356C substitution and the second CH3 domain further comprises a Y349C substitution, or the first CH3 domain further comprises a Y349C substitution and the second CH3 domain further comprises a D356C or E356C substitution; The heterodimeric protein according to claim 1 or 2.

6. The heterodimeric protein according to claim 1 or 2, wherein the first CH3 domain and the second CH3 domain are human CH3 domains.

7. The heterodimeric protein according to claim 1 or 2, wherein the Fc region is of the human IgG1 subclass.

8. The heterodimeric protein according to claim 1 or 2, wherein the Fc region is of the human IgG4 subclass.

9. The heterodimeric protein of claim 8, wherein the Fc region further comprises an S228P substitution.

10. The heterodimeric protein of claim 7, wherein the Fc region further comprises an N297A substitution.

11. The heterodimeric protein according to claim 1 or 2, wherein the first polypeptide and the second polypeptide are antibody heavy chains, and the heterodimeric protein further comprises one or more antibody light chains.

12. The heterodimeric protein of claim 11, wherein the heterodimeric protein is a multispecific antibody.

13. further comprising a third polypeptide and a fourth polypeptide; (i) the first polypeptide comprises a structure represented by the following formula: VH1-CH1-hinge-CH2-1st CH3-L1-scFv1(Ia), (ii) the second polypeptide comprises a structure represented by the following formula: VH2-CH1-hinge-CH2-2nd CH3-L2-scFv2(IIa), (iii) the third polypeptide comprises a structure represented by the following formula: VL1-CL(Ib), (iv) the fourth polypeptide comprises a structure represented by the following formula: VL2-CL(IIb), VL1 is the first immunoglobulin light chain variable domain, VH1 is the first immunoglobulin heavy chain variable domain; VL2 is a second immunoglobulin light chain variable domain, VH2 is a second immunoglobulin heavy chain variable domain; scFv1 is the first single chain variable fragment; scFv2 is a second single chain variable fragment; CL is an immunoglobulin light chain constant domain; CH1 is immunoglobulin heavy chain constant domain 1; CH2 is immunoglobulin heavy chain constant domain 2; the hinge is an immunoglobulin hinge region connecting the CH1 and CH2 domains; L1 and L2 are each independently a bond or a peptide linker; VL1 and VH1 associate to form a first Fv that specifically binds to a first target; VL2 and VH2 associate to form a second Fv that specifically binds to a second target; scFv1 specifically binds to a third target; The scFv2 specifically binds to a fourth target; The heterodimeric protein according to claim 12.

14. The heterodimeric protein of claim 13, wherein scFv1 and scFv2 are identical.

15. The heterodimeric protein of claim 13, wherein VL1 and VL2 are identical.

16. The heterodimeric protein according to claim 14, wherein the first Fv specifically binds to PDL1, the second Fv specifically binds to CD137, and scFv1 and scFv2 specifically bind to CTLA-4.

17. further comprising a third polypeptide; (i) the first polypeptide comprises a structure represented by the following formula: VH-CH1-hinge-CH2-1st CH3(IIIa), (ii) the second polypeptide comprises a structure represented by the following formula: scFv-hinge-CH2-second CH3 (IVa), (iii) the third polypeptide comprises a structure represented by the following formula: VL-CL (IIIb), VL is an immunoglobulin light chain variable domain; VH is an immunoglobulin heavy chain variable domain; scFv is a single chain variable fragment; CL is an immunoglobulin light chain constant domain; CH1 is immunoglobulin heavy chain constant domain 1; CH2 is immunoglobulin heavy chain constant domain 2; the hinge is an immunoglobulin hinge region connecting the CH1 and CH2 domains; the VL and VH associate to form an Fv that specifically binds to a first target; the scFv specifically binds to a second target; The heterodimeric protein according to claim 12.

18. The heterodimeric protein of claim 17, wherein the Fv specifically binds to CD137 and the scFv specifically binds to PDL1.

19. the heterodimeric protein is an activatable antibody, and the heterodimeric protein comprises a third polypeptide; (i) the first polypeptide comprises a structure represented by the following formula: VH-CH1-hinge-CH2-1st CH3 (Va), (ii) the second polypeptide comprises a structure represented by the following formula: MM1-CM1-scFv-hinge-CH2-second CH3 (VIa), (iii) the third polypeptide comprises a structure represented by the following formula: MM2-CM2-VL-CL (IVb), VL is an immunoglobulin light chain variable domain; VH is an immunoglobulin heavy chain variable domain; scFv is a single chain variable fragment; CL is an immunoglobulin light chain constant domain; CH1 is immunoglobulin heavy chain constant domain 1; CH2 is immunoglobulin heavy chain constant domain 2; the hinge is an immunoglobulin hinge region connecting the CH1 and CH2 domains; MM1 is the first masking peptide; MM2 is a second masking peptide; CM1 is the first cleavable peptide; CM2 is a second cleavable peptide; the VL and VH associate to form a first Fv that specifically binds to a first target; the scFv specifically binds to a second target; When CM1 is not cleaved, MM1 inhibits the binding of the scFv to the second target; When CM2 is not cleaved, MM2 inhibits the binding of the first Fv to the first target. The heterodimeric protein according to claim 12.

20. 20. The heterodimeric protein of claim 19, wherein the first target is a tumor antigen and the second target is CD3.

21. The heterodimeric protein of claim 19, wherein MM1 comprises the amino acid sequence of SEQ ID NO:

35.

22. The heterodimeric protein of claim 20, wherein the first target is HER2.

23. The heterodimeric protein of claim 19, wherein MM2 comprises the amino acid sequence of SEQ ID NO:

36.

24. The scFv comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 61, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 62, and / or CDR-H3 comprising the amino acid sequence of SEQ ID NO: 63 VH, and CDR-L1 comprising the amino acid sequence of SEQ ID NO: 64, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 65, and / or CDR-L3 comprising the amino acid sequence of SEQ ID NO: 66 VL, the heterodimeric protein according to claim 20.

25. The first Fv comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 69, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 70, and / or CDR-H3 comprising the amino acid sequence of SEQ ID NO: 71, VH, and CDR-L1 comprising the amino acid sequence of SEQ ID NO: 72, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 73, and / or CDR-L3 comprising the amino acid sequence of SEQ ID NO: 74, VL, the heterodimeric protein according to claim 22.

26. 20. The heterodimeric protein of claim 19, wherein the activatable antibody comprises an Fc region comprising a first CH3 domain and a second CH3 domain, wherein the first CH3 domain comprises D356K or E356K, E357K, and S364K substitutions, and the second CH3 domain comprises L351D, K370D, and K439D substitutions, or wherein the first CH3 domain comprises L351D, K370D, and K439D substitutions, and the second CH3 domain comprises D356K or E356K, E357K, and S364K substitutions.

27. One or more nucleic acids encoding the heterodimeric protein of claim 1 or 2.

28. 28. A vector comprising one or more nucleic acids of claim 27.

29. 28. A host cell comprising one or more nucleic acids of claim 27.

30. A host cell comprising the vector of claim 28.

31. 1. A method for preparing a heterodimeric protein, comprising: (a) culturing the host cell of claim 30 under conditions that allow expression of the one or more nucleic acid(s) or vectors; and (b) recovering the heterodimeric protein from the host cell culture; A method comprising:

32. A pharmaceutical composition comprising the heterodimeric protein of claim 1 or 2 and a pharmaceutically acceptable carrier.

33. A pharmaceutical for treating a disease or condition, comprising a heterodimeric protein according to any one of claims 16, 18, 20, 22, 24, and 25, or a pharmaceutical composition comprising a heterodimeric protein according to any one of claims 16, 18, 20, 22, 24, and 25 and a pharmaceutically acceptable carrier.

34. The pharmaceutical of claim 33, wherein the disease or condition is cancer.

35. The pharmaceutical composition of claim 34, wherein the cancer is lung cancer.

36. The pharmaceutical composition of claim 34, wherein the cancer is ovarian cancer.

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