Heterodimeric protein having an FC mutation

Abstract

The present application provides heterodimeric proteins comprising polypeptides having a CH3 domain with engineered residues that form disulfide bonds and / or salt bridges. Activatable antibodies targeting CD3 and / or HER2 are also provided. Additionally, compositions, methods of manufacture, and methods of treatment using the heterodimeric proteins and activatable antibodies are provided.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims priority to International Application No. PCT / CN2020 / 073960, filed on January 23, 2020, which is hereby incorporated by reference in its entirety.

[0002] Field of the Invention This application relates to heterodimer proteins (e.g., bispecific antibodies) and activatable antibodies, methods for their preparation, and methods for their use.

[0003] Reference to Sequence Listing The following submission in ASCII text file is hereby incorporated by reference in its entirety: Sequence Listing in computer - readable format (CRF) (filename: 695402001041SEQLIST.txt, date of record: January 21, 2020, size: 656KB).

Background Art

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

[0005] An activatable antibody exhibits an "activatable" conformation in which the antigen-binding portion contained therein has a lower potential for binding to a target when intact than after cleavage in the presence of one or more specific proteases. Thus, an activatable antibody provides an antigen-specific binding protein capable of binding to a target only in a specific situation (e.g., a protease-rich tumor microenvironment). A bispecific T cell engager is a bispecific antibody (BiTE) capable of binding to both target cells such as T cells and tumor cells. Due to the on-target off-tumor effect, BiTE molecules are associated with high cytotoxicity including toxicity to the central nervous system (CNS) and cytokine storm. There is a need for activatable BiTE molecules with improved specificity and reduced side effects.

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

Summary of the Invention

[0007] This application provides a heterodimeric protein comprising a CH3 domain having engineered residues that form disulfide bonds and / or salt bridges. Also provided are activatable antibodies that target CD3 and / or HER2.

[0008] Accordingly, one aspect of the present application provides a heterodimer 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 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; and the 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 by 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 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 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), and 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; the 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 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.

[0010] In some embodiments by 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 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 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 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 by 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 the first CH3 domain comprises L351D, K370D, and S400C substitutions, and the second CH3 domain comprises E357K, S364K, and N390C substitutions; 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 by 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 T3 6 6S, 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 T3 6comprise the 6S, L368A, and Y407V substitutions; or ii) the first CH3 domain comprises the L368V and Y407V substitutions and the second CH3 domain comprises the T366W substitution, or the first CH3 domain comprises the T366W substitution and the second CH3 domain comprises the L368V and Y407V substitutions

[0013] Another aspect of the present application provides a heterodimer 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 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, and the 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; and the 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 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.

[0014] In some embodiments with 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 an S354C substitution, or the first CH3 domain further comprises an S354C substitution, and the second CH3 domain further comprises a Y394C substitution; or iii) 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.

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

[0016] In some embodiments by any one of the above heterodimeric proteins, the first polypeptide and the second polypeptide each comprise, from the N-terminus to the 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 an S228P substitution. In some embodiments, the Fc region further comprises an N297A substitution.

[0017] In some embodiments by 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 a heterodimeric protein further comprising one or more antibody light chains. In some embodiments, the heterodimeric protein is a multispecific antibody.

[0018] In some embodiments by 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-first CH3-L1-scFv1(Ia), (ii) The second polypeptide comprises a structure represented by the following formula: VH2-CH1-hinge-CH2-second 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 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 the second single-chain variable fragment; CL is the immunoglobulin light chain constant domain; CH1 is the immunoglobulin heavy chain constant domain 1; CH2 is the immunoglobulin heavy chain constant domain 2; the hinge is the immunoglobulin hinge region that links the CH1 and CH2 domains; L1 and L2 are each independently a linker 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; 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, from the N-terminus to the C-terminus, comprise 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, and the first cysteine residue and the second cysteine residue 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 by 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-first 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 the variable domain of the immunoglobulin light chain; VH is the variable domain of the immunoglobulin heavy chain; scFv is the single-chain variable fragment; CL is the constant domain of the immunoglobulin light chain; CH1 is the first constant domain of the immunoglobulin heavy chain; CH2 is the second constant domain of the immunoglobulin heavy chain; the hinge is the immunoglobulin hinge region that links the CH1 and CH2 domains; 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 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 contains, from the N-terminus to the C-terminus: VH-L-VL, where L is a peptide linker. In some embodiments, the scFv contains a first cysteine residue at position 44 of VH and a second cysteine residue at position 100 of VL, and 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 containing the amino acid sequence of SEQ ID NO: 80 or SEQ ID NO: 81.

[0020] In some embodiments with 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-first 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 contains a structure represented by the following formula: MM2-CM2-VL-CL(IVb), VL is the variable domain of the immunoglobulin light chain; VH is the variable domain of the immunoglobulin heavy chain; scFv is the single-chain variable fragment; CL is the constant domain of the immunoglobulin light chain; CH1 is the first constant domain of the immunoglobulin heavy chain; CH2 is the second constant domain of the immunoglobulin heavy chain; the hinge is the immunoglobulin hinge region that links the CH1 and CH2 domains; MM1 is the first masking peptide; MM2 is the second masking peptide; CM1 is the first cleavable peptide; CM2 is the second cleavable peptide; VL and VH associate to form a first Fv that specifically binds to a first target; scFv specifically binds to a second target; when CM1 is not cleaved, MM1 inhibits the binding of the first Fv to the first target; when CM2 is not cleaved, MM2 inhibits the binding of 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 VL 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 / 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 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 / 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 that, from the N-terminus to the C-terminus, comprises a masking moiety (MM), a cleavable moiety (CM), and a target binding moiety (TBM), wherein MM comprises the amino acid sequence of SEQ ID NO: 35; when CM is not cleaved, MM inhibits the binding of the activatable antibody to human CD3; CM comprises at least a first cleavage site; a) TBM comprises VL, and the activatable antibody further comprises a second polypeptide comprising VH; b) TBM comprises VH, and the activatable antibody further comprises a second polypeptide comprising VL; c) TBM comprises VL and VH from the N-terminus to the C-terminus; or d) TBM comprises VH and VL from the N-terminus to the C-terminus; when CM is cleaved, the activatable antibody binds to human CD3 via VH and VL. In some embodiments, 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, 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 that includes, from the N-terminus to the C-terminus, a masking moiety (MM), a cleavable moiety (CM), and a target binding moiety (TBM), where MM includes the amino acid sequence of SEQ ID NO: 36; when CM is not cleaved, MM inhibits the binding of the activatable antibody to human HER2; CM includes at least a first cleavage site; a) TBM includes VL, and the activatable antibody further includes a second polypeptide that includes VH; b) TBM includes VH, and the activatable antibody further includes a second polypeptide that includes VL; c) TBM includes VL and VH from the N-terminus to the C-terminus; or d) TBM includes VH and VL from the N-terminus to the C-terminus; when CM is cleaved, the activatable antibody binds to human HER2 via VH and VL. In some embodiments, TBM includes a VH that includes CDR-H1 having the amino acid sequence of SEQ ID NO: 69, CDR-H2 having the amino acid sequence of SEQ ID NO: 70, and / or CDR-H3 having the amino acid sequence of SEQ ID NO: 71. In some embodiments, TBM includes a VL that includes CDR-L1 having the amino acid sequence of SEQ ID NO: 72, CDR-L2 having the amino acid sequence of SEQ ID NO: 73, and / or CDR-L3 having the amino acid sequence of SEQ ID NO: 74.

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

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

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

[0026] One aspect of the present application provides a pharmaceutical composition comprising a heterodimeric protein by any one of the above heterodimeric proteins, or an activatable antibody by 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 a pharmaceutical composition by any one of the above pharmaceutical compositions. 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 products containing any one of the above heterodimeric proteins are also provided.

Brief Description of the Drawings

[0029]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11A

Figure 11B

Figure 11C

Figure 12

Figure 13

Figure 14

Figure 15A

Figure 15B

Figure 15C

Figure 16

Figure 17

Figure 18

Figure 19

BRIEF DESCRIPTION OF THE INVENTION

[0030] The present application provides a heterodimeric protein comprising a CH3 domain having engineered disulfide bond(s) and / or salt bridge(s), which includes multispecific antibodies, e.g., bispecific antibodies comprising an Fc region having 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 reconfigured salt bridge network, e.g., 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), as compared to the wild-type CH3 domain. 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’) as compared to the wild-type CH3 domain. The heterodimeric proteins described herein provide a platform for preparing multispecific proteins and antibodies in various formats that have high yields, excellent stability (e.g., resistance to aggregation and precipitation upon exposure to high temperatures or freeze-thaw cycles), and potent activity.

[0031] I. Definitions Unless otherwise defined herein, terms are used in this specification in the meanings generally used in the relevant art.

[0032] As used herein, the term "antibody" is used in the broadest sense and includes, without limitation, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments that exhibit the desired antigen-binding activity, encompassing various antibody structures.

[0033] The term "antibody" includes, but is not limited to, fragments that can bind to an antigen, such as Fv, Fab, Fab', and (Fab')2. Papain digestion of an antibody produces two identical antigen-binding fragments called "Fab" fragments, each having a single antigen-binding site and the remaining "Fc" fragment, the name of which reflects the ability to readily crystallize. Pepsin treatment yields an F(ab')2 fragment that has two antigen-binding sites and can still cross-link antigens. 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 to bind to the antigen of the antibody. Examples of "antigen-binding fragments" of an antibody include (i) a Fab fragment (a monovalent fragment consisting of the VL, VH, CL, and CH1 domains); (ii) an F(ab')2 fragment (a divalent fragment containing two Fab fragments linked by a disulfide bridge in the hinge region); (iii) an Fv fragment consisting of the VL and VH domains of a single arm of the antibody; (v) a single-chain Fv fragment containing the VH and VL domains of the antibody (where the VH and VL domains are fused to each other); and (vi) a single-chain Fab fragment containing a single polypeptide including the VL, VH, CL, and CH1 domains, but are not limited thereto.

[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. However, variants that are contemplated, such as those containing naturally occurring mutations or arising during the production of a monoclonal antibody preparation, are generally present in minor amounts and are excluded. In contrast to polyclonal antibody preparations, which typically contain 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 made by a variety of techniques including, but not limited to, the hybridoma method, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals that contain all or part of the human immunoglobulin loci, and such methods and other exemplary methods for making monoclonal antibodies are described herein.

[0036] As used herein, the terms "hypervariable region" or "HVR" refer to each of the regions of an antibody variable domain where the sequence is hypervariable. The HVRs may form structurally defined loops ("hypervariable loops"). Generally, native four-chain antibodies contain six HVRs: three in VH (H1, H2, H3) and three in VL (L1, L2, L3). HVRs generally contain amino acid residues derived from hypervariable loops and / or "complementary determining regions" (CDRs), which have the highest sequence variability and / or are involved in antigen recognition. Exemplary hypervariable loops are present 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 present 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)). Except for CDR1 of VH, CDRs generally contain amino acid residues that form hypervariable loops. CDRs also include "specificity determining residues" or "SDRs", which are residues that contact the antigen. SDRs are contained within regions of CDRs called shortened 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 present 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, the HVR residues of the variable domain and other residues (e.g., FR residues) are numbered herein according to Kabat et al., supra.

[0037] The terms "variable region" or "variable domain" refer to the domain of the heavy or light chain of an antibody that is involved in binding of the antibody to an antigen. The variable domains of the heavy and light chains of a native antibody (VH and VL, respectively) generally have similar structures, and each domain comprises four framework regions (FRs) and three hypervariable regions (HVRs), which are arranged in the following order from the amino-terminus to the carboxy-terminus: FR1, HVR1, FR2, HVR2, FR3, HVR3, FR4. (See, e.g., 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 a particular antigen may be isolated using VH or VL domains from antibodies that bind the antigen to screen libraries 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 antibody editing in Edelman, G.M. et al., Proc. Natl. Acad. USA, 63, 78-85 (1969). The EU numbering of residues can be determined for a given antibody by aligning in the region of homology between the "standard" EU numbering sequence and the sequence of the antibody.

[0039] The Kabat numbering system is generally used when referring to residues within the variable domain (corresponding roughly to residues 1-107 of the light chain and 1-113 of 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 deletions or insertions in the FR or HVR of the variable domain. For example, the heavy chain variable domain may contain a single amino acid insertion (residue 52a according to Kabat) after residue 52 of H2, residues inserted 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 in regions of homology between the "standard" Kabat numbering sequence and the sequence of the antibody.

[0040] For a heterodimeric protein having two CH3 domains, a given amino acid position in the first CH3 domain is referred to as X and the corresponding amino acid position in the second CH3 domain is referred to as 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 relative to the wild-type, naturally occurring CH3 domains.

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

[0042] As used herein, the term "heavy chain constant region" refers to a region that includes 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 γ, δ, and α. Non-limiting examples of heavy chain constant regions also include ε and μ. Each heavy chain constant region corresponds to an antibody isotype. For example, an antibody that includes a γ constant region is an IgG antibody, an antibody that includes a δ constant region is an IgD antibody, and an antibody that includes an α constant region is an IgA antibody. Furthermore, an antibody that includes a μ constant region is an IgM antibody, and an antibody that includes an ε 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 (including the γ1 constant region), IgG2 (including the γ2 constant region), IgG3 (including the γ3 constant region), and IgG4 (including the γ4 constant region) antibodies; IgA antibodies include, but are not limited to, IgA1 (including the α1 constant region) and IgA2 (including the α2 constant region) antibodies; IgM antibodies include, but are not limited to, IgM1 and IgM2.

[0043] The term "CH2 domain" of the human IgG Fc region generally extends from about residue 231 to about residue 340 of IgG in the EU numbering system. The CH2 domain is unique in that it is not closely paired with another domain. Rather, two N-linked branched sugar chains are inserted between the two CH2 domains of an intact native IgG molecule. It is speculated that the carbohydrate provides an alternative to domain-domain pairing and may assist in the stabilization of the CH2 domain. Burton, Molec. Immunol. 22:161-206 (1985).

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

[0045] As used herein, the term "heavy chain" refers to a polypeptide that includes at least a heavy chain variable region, regardless of the presence or absence of a leader sequence. In some embodiments, the heavy chain includes at least a portion of the heavy chain constant region. As used herein, the term "full-length heavy chain" refers to a polypeptide that includes a heavy chain variable region and a heavy chain constant region, regardless of the presence or absence of a leader sequence.

[0046] As used herein, the term "light chain constant region" refers to a region that includes the 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 that includes at least a light chain variable region, regardless of the presence or absence of a leader sequence. In some embodiments, the light chain includes at least a portion of the light chain constant region. As used herein, the term "full-length light chain" refers to a polypeptide that includes a light chain variable region and a light chain constant region, regardless of the presence or absence of a leader sequence.

[0048] "Affinity" refers to the total strength 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 can generally be expressed 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 a multispecific antibody (e.g., a bispecific or trispecific antibody), the affinity for each binding specificity (i.e., target) of the antibody can be measured.

[0049] The terms "bind", "specifically bind", or "is specific for" refer to a measurable and reproducible interaction such as the binding between a target and an antibody, which determines the presence of the target in the presence of a heterogeneous population of molecules including biomolecules. For example, an antibody that binds or specifically binds to a target (which can be an epitope) is an antibody that binds to this target with greater affinity, binding strength, more readily, and / or for a longer duration than it binds to other targets. In some embodiments, the degree of binding of the antibody to an irrelevant 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, the antibody specifically binds to an epitope on a protein that is conserved between proteins from different species. In some embodiments, specific binding may include, but does not require, exclusive binding.

[0050] The term "multispecific" as used with respect to an antibody refers to an antibody having polyepitope specificity (i.e., capable of specifically binding to two, three, or more different epitopes on a single biomolecule or capable of specifically binding to epitopes on two, three, or more different biomolecules).

[0051] An "affinity matured" antibody is an antibody that has one or more modifications in one or more hypervariable regions (HVRs) compared to a parental antibody (which does not have such modifications), where such modifications result in an improvement in the affinity of the antibody for the antigen. In some examples, an affinity matured antibody is an antibody that has one or more modifications in one or more complementarity determining regions (CDRs) compared to a parental antibody (which does not have such modifications), where such modifications result in an improvement in the affinity of the antibody for the antigen.

[0052] As used herein, "chimeric antibody" refers to an antibody in which a portion of the heavy chain and / or light chain is derived from a particular source or species, while the remaining portion of the heavy chain and / or light chain is derived from a different source or species. In some embodiments, a chimeric antibody refers to an antibody comprising 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 the 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 derived from a human variable region. In some embodiments, a humanized antibody comprises at least one human constant region or a fragment thereof. In some embodiments, a humanized antibody is Fab, (Fab’)2, 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 into the framework region (FR) of a second (human) species. In some examples, as used herein, "CDR-grafted antibody" refers to a humanized antibody in which one or more complementarity-determining regions (CDRs) of a first (non-human) species have been grafted into the framework region (FR) of a second (human) species.

[0055] As used herein, "human antibody" refers to an antibody produced in a human, an antibody produced in a non-human animal comprising a human immunoglobulin gene, e.g., XENOMOUSE®, and an antibody selected using in vitro methods such as phage display, wherein 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 a polymer of nucleotides may contain natural and / or non-natural nucleotides and includes, but is not limited to, DNA, RNA, and PNA. "Nucleic acid sequence" refers to the linear sequence of nucleotides that includes 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 non-natural amino acid residues. Both full-length proteins and fragments thereof are included in the definition. The terms also include post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, etc. Further, "polypeptide" includes modifications to the native sequence, such as deletions, additions, and substitutions (generally conservative in nature), as long as the polypeptide maintains the desired activity. These modifications may be intentional (e.g., via site-directed mutagenesis) or accidental (e.g., via host mutations), which may occur during protein or error by PCR amplification.

[0058] A polypeptide "variant" means 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 (not 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-terminus or C-terminus of the polypeptide. In some embodiments, the variant has at least about 80% amino acid sequence identity. In some embodiments, the variant has at least about 90% amino acid sequence identity. In some embodiments, the variant has at least about 95% amino acid sequence identity with the 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 maximum percent sequence identity (without considering any conservative substitutions as part of the sequence identity) and introducing gaps, if necessary. Alignments for determining percent amino acid sequence identity can be achieved in various ways within the skill in the art, e.g., 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 the alignment, including any algorithms necessary to achieve the maximum alignment over the full length of the sequences being compared.

[0060] Amino acid substitutions may, but are not limited to, include replacing one amino acid in a polypeptide with another amino acid. Exemplary substitutions are shown in Table A. Amino acid substitutions may be introduced into the antibody of interest, and the product may be screened for the desired activity, e.g., retention / improvement of antigen binding, reduction of immunogenicity, or improvement of ADCC or CDC. [Table 1]

[0061] Amino acids may be grouped according to common side-chain characteristics: (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile, (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln, (3) Acidic: Asp, Glu, (4) Basic: His, Lys, Arg, (5) Residues that affect chain orientation: Gly, Pro, (6) Aromatic: Trp, Tyr, Phe. Non-conservative substitutions would require the replacement of a member of one of these classes with a member of another class.

[0062] The term "vector" is used to describe a polynucleotide that can be manipulated to contain a 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., a promoter and / or enhancer) that regulate the expression of the polypeptide of interest, and / or one or more selectable marker genes (e.g., an antibiotic resistance gene and a gene that can be used in a colorimetric assay, e.g., β-galactosidase). The term "expression vector" refers to a vector used to express a polypeptide of interest in a host cell.

[0063] "Host cell" refers to a cell that can be or is a recipient of a vector or an isolated polynucleotide. A host cell can be a prokaryotic or eukaryotic cell. Exemplary eukaryotic cells include mammalian cells, e.g., primate or non-primate animal cells; fungal cells, e.g., yeast; plant cells; and insect cells. Non-limiting examples of mammalian cells include NSO cells, PER.C6® cells (Crucell), as well as 293 and CHO cells, and their derivatives, e.g., 293-6E and DG44 cells, respectively, but are not limited thereto. The term "cell" includes primary subject cells and their 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 called "isolated" if it is separated from at least some of the components of the cell in which it is produced. When a polypeptide is secreted by a cell after expression, physically separating the supernatant containing the polypeptide from the cell that produced it is considered "isolating" the polypeptide. Similarly, a polynucleotide is called "isolated" if it is not part of a larger polynucleotide (e.g., genomic DNA or mitochondrial DNA in the case of a DNA polynucleotide) normally found in nature, or if it is separated from at least some of the components of the cell in which it is produced, e.g., in the case of an RNA polynucleotide. Thus, a DNA polynucleotide contained in a vector within a host cell can be called "isolated".

[0065] The terms "individual" or "subject" are used interchangeably herein to refer to a mammal. In some embodiments, but not limited to, 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 livestock, mammalian sport animals, and mammalian pets. In some examples, an "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 for obtaining a beneficial or desirable result, including clinical outcomes. For purposes of the present invention, beneficial or desirable clinical outcomes include, but are not limited to, one or more of the following: reducing one or more symptoms attributable to a disease, alleviating the extent of the disease, stabilizing the disease (e.g., preventing or delaying disease progression), preventing or delaying the spread (e.g., metastasis) of the disease, preventing or delaying recurrence of the disease, delaying or slowing the progression of the disease, improving the medical condition, providing (partial or complete) remission of the disease, reducing the dosage of one or more other therapeutic agents required for treatment of the disease, delaying the progression of the disease, improving quality of life, and / or extending 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. denote 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, "prevent" and similar terms also include reducing the extent, impact, symptoms, and / or burden of a disease or condition prior to recurrence of the disease or condition.

[0068] As used herein, "delaying" the onset of cancer means extending, interfering with, slowing down, suppressing, stabilizing, and / or postponing the onset of the disease. This delay can be of various durations depending on the disease history and / or the individual being treated. A method of "delaying" the onset of cancer is a method that reduces the likelihood of disease onset and / or reduces the extent of the disease within a given time frame as compared to not using the method. Such comparisons are typically based on clinical studies using a statistically significant number of individuals. The onset of cancer 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 and includes occurrence, recurrence, and onset.

[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 such that, for example, one or more of the symptoms are improved, alleviated, reduced, and / or delayed. 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 unwanted 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 doses. The effective amount of a drug or composition: (i) reduces the number of cancer cells; (ii) reduces the tumor size; (iii) inhibits, suppresses, slows to some extent, preferably stops, the invasion of cancer cells into peripheral organs; (iv) inhibits tumor metastasis (i.e., slows to some extent, preferably stops); (v) inhibits tumor growth; (vi) prevents or delays the occurrence and / or recurrence of tumors; and / or (vii) can alleviate to some extent one or more of the symptoms associated with cancer.

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

[0071] References in this specification to "about" a value or parameter include (and describe) variations that are part of the value or parameter itself. For example, a description that refers to "about X" includes a description of "X".

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

[0073] The term "about X - Y" as used herein has the same meaning as "from 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] The term "and / or" as used herein, expressions such as "A and / or B" are intended to include both A and B; A or B; A alone; and B alone. Similarly, the term "and / or" as used herein, expressions 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] For clarity, it is understood that certain features of the invention that are described in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, for brevity, the various features of the invention that are described in the context of a single embodiment may also be provided separately or in any suitable sub-combination. All combinations of embodiments related to heterodimer proteins are specifically encompassed by the present invention and are disclosed herein such that every possible combination is individually and explicitly disclosed. In addition, all sub-combinations of heterodimer proteins listed in the embodiments that describe such variations are also specifically encompassed by the present invention and are disclosed herein such that every possible partial combination of heterodimer proteins is individually and explicitly disclosed herein.

[0077] II. Heterodimer Protein The present application provides a heterodimer protein comprising a CH3 domain having any one or combination of engineered residues, which promotes heterodimer formation as described in the subsection "CH3 Domain Mutations". Also contemplated herein are heteromultimers comprising a plurality of heterodimers formed by a first polypeptide comprising a first engineered CH3 domain and a second polypeptide comprising a second engineered CH3 domain.

[0078] 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 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; the amino acid residue numbering is based on EU numbering.

[0079] 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 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 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 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), and 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, and the amino acid residue numbering is based on EU numbering.

[0080] In some embodiments, a heterodimeric protein (e.g., a multispecific antibody) is provided that includes 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; and 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), and 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, with amino acid residue numbering 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, there is provided a heterodimeric protein (e.g., a multispecific antibody) 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 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.

[0083] In some embodiments, a heterodimeric protein (e.g., a multispecific antibody) is provided that includes 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 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 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 includes 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 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 includes 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 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 includes 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 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 includes 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 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 enumerate 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 polypeptide of Fc regions 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 that comprises 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 that comprises 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 that comprises 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 that comprises 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 that comprises 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 that comprises 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 that comprises 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 that comprises 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 that comprises 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 that comprises 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 comprises 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 comprises 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 mutation 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 combinations thereof. Unless otherwise indicated, 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 hereby incorporated 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., the IgG1 allotypes G1m, 1(a), 2(x), 3(f), or 17(z). In some embodiments, the amino acid mutations or substitutions described herein are related to the wild-type CH3 domain sequence of IgG4. For example, the D356K substitution relative to the wild-type CH3 domain of one human IgG1 allotype (Uniprot ID P01857; SEQ ID NO: 29) is equivalent to the E356K substitution relative to the wild-type CH3 domain of a second human IgG1 allotype (SEQ ID NO: 30) or 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 the wild-type Fc region sequence, e.g., the IgG1 Fc region (SEQ ID NO: 32 or 33) or the IgG4 Fc region (SEQ ID NO: 34).

[0092] Novel cysteine mutations In some embodiments, the heterodimeric protein described herein comprises a first polypeptide comprising a first CH3 domain and a second polypeptide comprising a second CH3 domain, the first CH3 domain comprises a first engineered cysteine residue, the second CH3 domain comprises a second engineered cysteine residue, and 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, the first CH3 domain comprises engineered positively charged residues, the second CH3 domain comprises engineered negatively charged residues, and the engineered positively charged residues and the engineered negatively charged residues 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., “invert” the salt bridge). In some embodiments, the engineered positively charged residues substitute negatively charged residues of the wild-type CH3 domain with positively charged residues. In some embodiments, the engineered negatively charged residues substitute positively charged residues of the wild-type CH3 domain with negatively charged residues. Rearranged and inverted salt bridges may result in a change in the isoelectric point (PI) of heterodimers and homodimers comprising the engineered CH3 domains, thereby allowing for better separation of the heterodimers from the homodimers in the purification process.

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

[0098] In some embodiments, the first CH3 domain contains a positively charged residue at position 411, and the second CH3 domain contains a negatively charged residue at position 370, or the first CH3 domain contains a negatively charged residue at position 370, and the second CH3 domain contains a positively charged residue at position 411. In some embodiments, the first CH3 domain contains K at position 411, and the second CH3 domain contains D at position 370, or the first CH3 domain contains D at position 370, and the second CH3 domain contains K at position 411. In some embodiments, the first CH3 domain contains K at position 411, and the second CH3 domain contains E at position 370, or the first CH3 domain contains E at position 370, and the second CH3 domain contains K at position 411. In some embodiments, the first CH3 domain contains R at position 411, and the second CH3 domain contains D at position 370, or the first CH3 domain contains D at position 370, and the second CH3 domain contains R at position 411. In some embodiments, the first CH3 domain contains R at position 411, and the second CH3 domain contains E at position 370, or the first CH3 domain contains E at position 370, and the second CH3 domain contains R at position 411. In some embodiments, the first CH3 domain contains a T411K substitution, and the second CH3 domain contains a K370D substitution, or the first CH3 domain contains a K370D substitution, and the second CH3 domain contains 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 K at position 364, and the second CH3 domain comprises D at position 370, or the first CH3 domain comprises D at position 370, and the second CH3 domain comprises K at position 364. In some embodiments, the first CH3 domain comprises K at position 364, and the second CH3 domain comprises E at position 370, or the first CH3 domain comprises E at position 370, and the second CH3 domain comprises K at position 364. In some embodiments, the first CH3 domain comprises R at position 364, and the second CH3 domain comprises D at position 370, or the first CH3 domain comprises D at position 370, and the second CH3 domain comprises R at position 364. In some embodiments, the first CH3 domain comprises R at position 364, and the second CH3 domain comprises E at position 370, or the first CH3 domain comprises E at position 370, and the second CH3 domain comprises 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 contains a positively charged residue at position 356, and the second CH3 domain contains a negatively charged residue at position 439, or the first CH3 domain contains a negatively charged residue at position 439, and the second CH3 domain contains a positively charged residue at position 356. In some embodiments, the first CH3 domain contains K at position 356, and the second CH3 domain contains D at position 439, or the first CH3 domain contains D at position 439, and the second CH3 domain contains K at position 356. In some embodiments, the first CH3 domain contains K at position 356, and the second CH3 domain contains E at position 439, or the first CH3 domain contains E at position 439, and the second CH3 domain contains K at position 356. In some embodiments, the first CH3 domain contains R at position 356, and the second CH3 domain contains D at position 439, or the first CH3 domain contains D at position 439, and the second CH3 domain contains R at position 356. In some embodiments, the first CH3 domain contains R at position 356, and the second CH3 domain contains E at position 439, or the first CH3 domain contains E at position 439, and the second CH3 domain contains R at position 356. In some embodiments, the first CH3 domain contains a D356K substitution, and the second CH3 domain contains a K439D substitution, or the first CH3 domain contains a K439D substitution, and the second CH3 domain contains a D356K substitution.

[0101] Any of the engineered salt bridges described herein may be combined with each other. In some embodiments, the first CH3 domain comprises a positively charged residue at position 357 and a positively charged residue at position 411, 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 411. In some embodiments, the first CH3 domain comprises the E357K and T411K substitutions, and the second CH3 domain comprises the L351D and K370D substitutions, or the first CH3 domain comprises the L351D and K370D substitutions, and the second CH3 domain comprises the 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 the E357K and S364K substitutions, and the second CH3 domain comprises the L351D and K370D substitutions, or the first CH3 domain comprises the L351D and K370D substitutions, and the second CH3 domain comprises the 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, and 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 domain or Fc region described herein may further include engineered disulfide bonds and / or salt bridges 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 comprises a C at position 399, or the first CH3 domain comprises a C at position 399, and the second CH3 domain 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 contains a C at position 394, and the second CH3 domain contains a C at position 354, or the first CH3 domain contains a C at position 354, and the second CH3 domain contains a C at position 394. In some embodiments, the first CH3 domain further contains a Y394C substitution, and the second CH3 domain further contains an S354C substitution, or the first CH3 domain further contains an S354C substitution, and the second CH3 domain further contains a Y394C substitution.

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

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

[0109] In some embodiments, the first CH3 domain further contains L368D and K370S substitutions, and the second CH3 domain further contains E357Q and S364K substitutions, or the first CH3 domain further contains E357Q and S364K substitutions, and the second CH3 domain further contains 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 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 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] The engineered CH3 domain disulfide bonds and / or salt bridges described herein in heterodimeric proteins may further comprise one or more knob-into-hole residues. "Knob-into-hole" or "KIH" refers to an approach known in the art for creating bispecific antibodies, also known as the "protruberance-into-cavity" approach (see, e.g., U.S. Patent No. 5,731,168). In this approach, two immunoglobulin polypeptides (e.g., heavy chain polypeptides) each form an interface. The interface of one immunoglobulin polypeptide interacts with the corresponding interface on the other immunoglobulin polypeptide, thereby allowing the two immunoglobulin polypeptides to associate. These interfaces may be engineered such that a "knob" or "protruberance" (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 hole is the same or similar in size to the knob and is appropriately positioned such that when the two interfaces interact, the knob of one interface can be placed into the corresponding hole of the other interface. Without wishing to be bound by theory, this is thought 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 create a bispecific antibody comprising two immunoglobulin polypeptides having binding specificities for different epitopes. In some embodiments, the CH3 domain of the heterodimeric proteins described herein does not contain KIH residues.

[0114] In some embodiments, the first CH3 domain further comprises T3 6Including 6S, L368A, and Y407V substitutions, the second CH3 domain further includes a T366W substitution, or the first CH3 domain further includes a T366W substitution, and the second CH3 domain further includes T3 6 Including 6S, L368A, and Y407V substitutions.

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

[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, provided is a multispecific antibody 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 a combination of the engineered disulfide bonds or salt bridges described herein, 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 a 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, for example, an IgG1 Fc having an N297A substitution.In some embodiments, the multispecific antibody comprises an IgG4 Fc region, e.g., an IgG4 having an S228P substitution.

[0118] In some embodiments, a multispecific antibody is provided that includes a first CH3 domain, a second polypeptide that includes a second CH3 domain, a third polypeptide, and a fourth polypeptide, wherein the first CH3 domain and the second CH3 domain include any one or combination of the engineered disulfide bonds or salt bridges described herein, 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, the fourth polypeptide is a second antibody light chain, 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 includes an N390C substitution and the second CH3 domain includes an S400C substitution, or the first CH3 domain includes an S400C substitution and the second CH3 domain includes an N390C substitution. In some embodiments, the first CH3 domain includes E357K, S364K, and S400C substitutions and the second CH3 domain includes L351D, K370D, and N390C substitutions, or the first CH3 domain includes L351D, K370D, and N390C substitutions and the second CH3 domain includes E357K, S364K, and S400C substitutions. In some embodiments, the first CH3 domain includes D356K, E357K, S364K, and N390C substitutions and the second CH3 domain includes L351D, K370D, K439D, and S400C substitutions, or the first CH3 domain includes L351D, K370D, K439D, and S400C substitutions and the second CH3 domain includes 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., IgG1 having an N297A substitution. In some embodiments, the multispecific antibody comprises an IgG4 Fc region, e.g., IgG4 having an S228P substitution.

[0119] In some embodiments, there is provided a multispecific antibody 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 the engineered disulfide bonds or salt bridges described herein. (i) The first polypeptide comprises a structure represented by the following formula: VH1-CH1-hinge-CH2-first CH3, (ii) The second polypeptide comprises a structure represented by the following formula: VH2-CH1-hinge-CH2-second 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 the immunoglobulin light chain constant domain, CH1 is the immunoglobulin heavy chain constant domain 1, CH2 is the immunoglobulin heavy chain constant domain 2, The hinge is the immunoglobulin hinge region that links 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., the 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. 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 having an N297A substitution. In some embodiments, the multispecific antibody comprises an IgG4 Fc region, e.g., an IgG4 having 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 that includes 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 include any one or a combination of the engineered disulfide bonds or salt bridges described herein, (i) The first polypeptide includes a structure represented by the following formula: VH1-CH1-hinge-CH2-first CH3-L1-scFv1, (ii) The second polypeptide includes a structure represented by the following formula: VH2-CH1-hinge-CH2-second CH3-L2-scFv2, (iii) The third polypeptide includes a structure represented by the following formula: VL1-CL, (iv) The fourth polypeptide includes 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 the second single-chain variable fragment, CL is the immunoglobulin light chain constant domain, CH1 is the immunoglobulin heavy chain constant domain 1, CH2 is the immunoglobulin heavy chain constant domain 2, The hinge is the immunoglobulin hinge region that connects the CH1 and CH2 domains; L1 and L2 are each independently a linker 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, 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, and 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 quadrispecific antibody. In some embodiments, VL1 is identical to VL2 (e.g., the 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. 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., IgG1 having an N297A substitution. In some embodiments, the multispecific antibody comprises an IgG4 Fc region, e.g., IgG4 having an S228P substitution.

[0121] In some embodiments, provided is a multispecific antibody 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 either one or a combination of the 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 a 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 having an N297A substitution.In some embodiments, the multispecific antibody comprises an IgG4 Fc region, e.g., an IgG4 having 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, there is provided a multispecific antibody 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 a combination of the engineered disulfide bonds or salt bridges described herein, (i) the first polypeptide comprises a structure represented by the following formula: VH-CH1-hinge-CH2-first CH3, (ii) the second polypeptide comprises a structure represented by the following formula: scFv-hinge-CH2-second 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 an immunoglobulin heavy chain constant domain 1, CH2 is an immunoglobulin heavy chain constant domain 2, the hinge is an immunoglobulin hinge region that links the CH1 and CH2 domains, VL and VH associate to form an Fv that specifically binds to the 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 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, for example, IgG1 having an N297A substitution. In some embodiments, the multispecific antibody comprises an IgG4 Fc region, for example, IgG4 having 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 κ and λ. 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 the S228P mutation in the human IgG4 constant region. In some embodiments, the first polypeptide and the second polypeptide further comprise the S228P substitution.

[0124] Whether effector function is desirable may depend on the particular treatment method intended for the multispecific antibody. In some embodiments, when 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, when effector function is not desirable, 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 the N297A substitution. In some embodiments, the first polypeptide and the second polypeptide further comprise the 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, the second target, the third target, and / or the fourth target are cell surface antigens. In some embodiments, the cell surface antigen is, for example, an antigen on immune effector cells such as T cells (e.g., helper T cells, cytotoxic T cells, memory T cells, etc.), B cells, macrophages, and natural killer (NK) cells. 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. A tumor antigen is a protein 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 a tumor-associated antigen (TAA). TSA is unique to tumor cells and does not exist in other cells in the body. TAA-related antigens are not unique to tumor cells and are instead expressed in normal cells under conditions that do not induce a state of immune tolerance to the antigen. The expression of the antigen on the tumor can occur under conditions that allow the immune system to respond to the antigen. TAA can be an antigen that is expressed in 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 in normal cells but are expressed at much higher levels on tumor cells.

[0128] Non-limiting examples of TSA or TAA antigens include the following: differentiation antigens such as MART-1 / MelanA (MART-1), gp100 (Pmel17), tyrosinase, TRP-1, TRP-2, and tumor-specific multi-lineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, pl5; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutant 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, BTAA, 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 VL. In some embodiments, the TBM comprises VH. In some embodiments, the TBM comprises the specificity of VL and / or VH 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 is an antigen-binding fragment comprising, but not limited to, (i) a Fab fragment that is a monovalent fragment consisting of VL, VH, CL, and CHI domains; (ii) an F(ab’)2 fragment that is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge in the hinge region; (iii) an Fd fragment consisting of VH and CHI domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a dAb fragment consisting of a VH domain (Ward et al., (1989) Nature 341:544-546); (vi) an isolated CDR, and (vii) a single-chain antibody (scFv) that is a polypeptide comprising the VL region of an antibody linked to the VH region of the antibody (see, 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 VL-L1-VH from the N-terminus to the C-terminus, where L1 is a peptide linker. In some embodiments, the TBM is an scFv comprising VH-L1-VL from the N-terminus to the C-terminus, 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, and the 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, and the first cysteine residue and the second cysteine residue form a disulfide bond, and the 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 an 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 that targets an antigen of interest. The TBMs described herein may incorporate any of the CDR sequences (e.g., one, two, or three of the heavy chain variable region CDR sequences and / or one, two, or three of the light chain variable region CDR sequences), heavy chain variable region sequences, and / or light chain variable region sequences of the antibodies described in WO2019 / 036856, WO2019 / 036842, WO2019 / 036855, WO2019148444, WO2019185035, WO2019036855, which are hereby incorporated by reference in their entirety. Table C below shows the antibody CDR, VH, VL, 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, such as 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 a complementarity-determining region (CDR-H)1 having the amino acid sequence of SEQ ID NO: 37, a CDR-H2 having the amino acid sequence of SEQ ID NO: 38, and / or a CDR-H3 having the amino acid sequence of SEQ ID NO: 39. In some embodiments, the TBM comprises a VL comprising a complementarity-determining region (CDR-L)1 having the amino acid sequence of SEQ ID NO: 40, a CDR-L2 having the amino acid sequence of SEQ ID NO: 41, and / or a CDR-L3 having the amino acid sequence of SEQ ID NO: 42. In some embodiments, the TBM comprises a VH having the amino acid sequence of SEQ ID NO: 43. In some embodiments, the TBM comprises a VL having the amino acid sequence of SEQ ID NO: 44. In some embodiments, the TBM comprises an scFv having 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, such as VH, VL, scFv, light chain, or heavy chain (e.g., IgG1, IgG2, IgG4). Any of the known anti-CD137 antibodies 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 CDR-H1 having the amino acid sequence of SEQ ID NO: 45, CDR-H2 having the amino acid sequence of SEQ ID NO: 46, and / or CDR-H3 having the amino acid sequence of SEQ ID NO: 47. In some embodiments, the TBM comprises a VL comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 48, CDR-L2 having the amino acid sequence of SEQ ID NO: 49, and / or CDR-L3 having the amino acid sequence of SEQ ID NO: 50. In some embodiments, the TBM comprises a VH having the amino acid sequence of SEQ ID NO: 51. In some embodiments, the TBM comprises a VL having the amino acid sequence of SEQ ID NO: 52. In some embodiments, the TBM comprises an scFv having 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, VH, VL, scFv, light chain, or heavy chain (e.g., IgG1, IgG2, IgG4). Without limitation, ipilimumab (see U.S. Pat. Nos. 6,984,720, 7,452,535, 7,605,238, 8,017,114, and 8,142,778), tremelimumab (see U.S. Pat. Nos. 6,688,736, 7,109,003, 7,132,281, 7,411,057, 7,807,797, 7,824,679, and 8,143,379), and other anti-CTLA-4 antibodies such as single-chain antibodies (e.g., see U.S. Pat. Nos. 5,811,097, 6,051,227, and 7,229,628, U.S. Patent Publication Nos. US20110044953, US2018037654, US2009025274, US2019127468, International Patent Publication Nos. WO2019 / 152413, WO2018209701, WO2018 / 202649, and WO2019 / 152423) can be used in the present invention. 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 CDR-H1 comprising the amino acid sequence of SEQ ID NO: 53, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 54, and / or CDR-H3 comprising the amino acid sequence of SEQ ID NO: 55. In some embodiments, the TBM comprises a VL comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 56, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 57, and / or 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 its antigen-binding domain, such as VH, VL, scFv, light chain, or heavy chain (e.g., IgG1, IgG2, IgG4). Without limitation, any of the known anti-CD3 antibodies, including but not limited to, Cris-7 monoclonal antibody (Reinherz, E.L. 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 its derivatives, such as OKT3 ala-ala (Herold et al. (2003) J. Clin. Invest. 11:409), basiliximab (Carpenter et al. (2002) Blood 99:2712), and 145-2C11 monoclonal antibody (Hirsch et al. (1988) J. Immunol. 140:3766), otelixizumab, and foralumab, can be used in the present invention. Further CD3-binding molecules contemplated herein include UCHT-1 (Beverley, P C and Callard, R.E. (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 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. In some embodiments, the TBM comprises a VL comprising 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. 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 an antigen-binding domain thereof, such as VH, VL, scFv, light chain, or heavy chain (e.g., IgG1, IgG2, IgG4). Without limitation, any of the known anti-HER2 antibodies including, but not limited to, the following may be used in the present invention: 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), 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 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. In some embodiments, the TBM comprises a VL comprising 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. 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] As used herein, the term "PDL1" 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] As used herein, the term "CTLA4" 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] As used herein, the term "CD137" includes human CD137 (e.g., GenBank accession number 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), dog 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 multi-protein complex (see Abbas and Lichtman, 2003; Janeway et al., pp172 and 178, 1999). In mammals, the complex includes the CD3 gamma chain, CD3 delta chain, two CD3 epsilon chains, and a homodimer of 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 enables these chains to associate with the 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 each CD3 zeta chain has three. Without being bound by theory, ITAM is thought to be important for the signaling ability of the TCR complex. As used herein, CD3 may be derived from a variety of 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 number P07766), as well as variants, isoforms, and species homologs thereof (e.g., mouse CD3e (UniProt accession number P22646), rat CD3e (UniProt accession number A0A0G2K986), dog CD3e (UniProt accession number P27597), and cynomolgus monkey CD3e (UniProt accession number Q95LI5)).

[0149] As used herein, the term "HER2" includes human HER2 (e.g., UniProt accession number P04626), as well as its variants, isoforms, and species homologs (e.g., mouse HER2 (UniProt accession number P70424), rat HER2 (UniProt accession number P06494), canine HER2, 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 show species or other types of cross-reactivity. In other cases, the TBMs also bind to targets of non-human species.

[0151] Linker The multispecific antibodies described herein may include one or more linkers (e.g., L1, L2, L3, etc.) disposed 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.), for example, SGGGS (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; etc. Any suitable linker known in the art (e.g., a flexible linker) may be used. The linker sequence can 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 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), but are not limited thereto. In some embodiments, the multispecific antibody comprises a CH3 domain or an Fc region comprising any one or a combination of the engineered disulfide bonds and / or salt bridges described herein. In some embodiments, the multispecific antibody does not comprise a CH3 domain or an Fc region comprising any one or a combination of the 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-first CH3-L1-scFv1, (ii) the second 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 connecting the CH1 and CH2 domains, L1 is a linker or peptide linker, 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 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 scFv 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 VH-L1-VL from the N-terminus to the C-terminus, 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 VH and a second cysteine residue at position 100 of VL, and the first and second cysteine residues form a disulfide bond, with the numbering 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 having an N297A substitution.In some embodiments, the multispecific antibody comprises an IgG4 Fc region, e.g., an IgG4 having 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 that comprises 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 that comprises 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 that comprises 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 that comprises 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 that comprises 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 that comprises 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 that comprises 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 that comprises 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 is provided that targets PDL1 and CD137 and comprises a first polypeptide and a second polypeptide, (i) the first polypeptide comprises a structure represented by the following formula: VH1-CH1-Hinge-CH2-First CH3-L1-scFv1, (ii) The second 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 an immunoglobulin heavy chain constant domain 1, CH2 is an immunoglobulin heavy chain constant domain 2, The hinge is an immunoglobulin hinge region that links the CH1 and CH2 domains, L1 is a linker or 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 having the amino acid sequence of SEQ ID NO: 37, a CDR-H2 having the amino acid sequence of SEQ ID NO: 38, and a CDR-H3 having the amino acid sequence of SEQ ID NO: 39; and / or a VL comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 40, a CDR-L2 having the amino acid sequence of SEQ ID NO: 41, and a CDR-L3 having the amino acid sequence of SEQ ID NO: 42. In some embodiments, the Fv comprises a VH having the amino acid sequence of SEQ ID NO: 43 and / or a VL having the amino acid sequence of SEQ ID NO: 44. In some embodiments, the scFv comprises a VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 45, a CDR-H2 having the amino acid sequence of SEQ ID NO: 46, and a CDR-H3 having the amino acid sequence of SEQ ID NO: 47; and / or a VL comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 48, a CDR-L2 having the amino acid sequence of SEQ ID NO: 49, and a CDR-L3 having the amino acid sequence of SEQ ID NO: 50. In some embodiments, the scFv comprises a VH having the amino acid sequence of SEQ ID NO: 51 and / or a VL having the amino acid sequence of SEQ ID NO: 52. In some embodiments, the scFv comprises VH-L1-VL from the N-terminus to the C-terminus, 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 VH and a second cysteine residue at position 100 of VL, and the first cysteine residue and the second cysteine residue 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: 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, for example, an IgG1 Fc having an N297A substitution. In some embodiments, the multispecific antibody comprises an IgG4 Fc region, for example, an IgG4 having 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, bispecific antibodies are provided that comprise 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, bispecific antibodies are provided that comprise 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, bispecific antibodies are provided that comprise 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, bispecific antibodies are provided that comprise 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, bispecific antibodies are provided that comprise 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, bispecific antibodies are provided that comprise 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, trispecific antibodies are provided that target PDL1, CD137, and CTLA4, which comprise 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-first CH3-L1-scFv1, (ii) The second polypeptide comprises a structure represented by the following formula: VH-CH1-hinge-CH2-second CH3-L2-scFv2, (iii) The third polypeptide comprises a structure represented by the following formula: VL-CL, (iv) The fourth polypeptide contains 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 an immunoglobulin heavy chain constant domain 1, CH2 is an 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 linker or a peptide linker, 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 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, scFv1 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 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, 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, 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, e.g., an IgG1 Fc having an N297A substitution.

[0159] In some embodiments, a trispecific antibody is provided, which comprises 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, which comprises 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, which comprises 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 targeting CD3 and a tumor antigen (e.g., HER2) is provided, which 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-first CH3, (ii) The second polypeptide comprises a structure represented by the following formula: scFv-hinge-CH2-second 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 an immunoglobulin heavy chain constant domain 1, CH2 is the immunoglobulin heavy chain constant domain 2, The hinge is the immunoglobulin hinge region that connects the CH1 and CH2 domains, 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 comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 61, a CDR-H2 having the amino acid sequence of SEQ ID NO: 62, and a CDR-H3 having the amino acid sequence of SEQ ID NO: 63; and / or a VL comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 64, a CDR-L2 having the amino acid sequence of SEQ ID NO: 65, and a CDR-L3 having the amino acid sequence of SEQ ID NO: 66. In some embodiments, the scFv comprises a VH having the amino acid sequence of SEQ ID NO: 67, and / or a VL having the amino acid sequence of SEQ ID NO: 68. In some embodiments, the scFv has 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 having the amino acid sequence of SEQ ID NO: 69, a CDR-H2 having the amino acid sequence of SEQ ID NO: 70, and a CDR-H3 having the amino acid sequence of SEQ ID NO: 71; and / or a VL comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 72, a CDR-L2 having the amino acid sequence of SEQ ID NO: 73, and a CDR-L3 having the amino acid sequence of SEQ ID NO: 74. In some embodiments, the Fv comprises a VH having the amino acid sequence of SEQ ID NO: 75, and / or a VL having 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 having an N297A substitution. In some embodiments, a bispecific T cell engager molecule is provided, which comprises a first polypeptide having the amino acid sequence of SEQ ID NO: 112, a second polypeptide having the amino acid sequence of SEQ ID NO: 113, and a third polypeptide having 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), antigen-binding fragments thereof that are activatable, 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 an antibody fragment thereof. In some embodiments, the TBM comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH), and VH and VL form a binding domain that binds to a target in the absence of MM. In some embodiments, VH and VL are covalently linked, for example, in an scFv. In some embodiments, VH and VL form an Fv fragment. In some embodiments, VH is linked to an antibody heavy chain constant region and 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, the activatable antibody comprises an Fc region that does not comprise any one or combination of engineered disulfide bonds or salt bridges described herein.

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

[0164] In some embodiments, the activatable antibody comprises a polypeptide having a structure of masking moiety (MM)-L1-cleavable moiety (CM)-L2-VL from the N-terminus to the C-terminus, and the activatable antibody further comprises a second polypeptide comprising VH (e.g., Fab fragment). In some embodiments, the activatable antibody comprises a polypeptide having a structure of masking moiety (MM)-L1-cleavable moiety (CM)-L2-VL-L3-VH (e.g., scFv) from the N-terminus to the C-terminus. In some embodiments, the activatable antibody comprises a polypeptide having a structure of masking moiety (MM)-cleavable moiety (CM)-L1-VH from the N-terminus to the C-terminus, and the activatable antibody further comprises a second polypeptide comprising VL (e.g., Fab fragment). In some embodiments, the activatable antibody comprises a polypeptide having a structure of masking moiety (MM)-L1-cleavable moiety (CM)-L2-VH-L3-VL (e.g., scFv) from the N-terminus to the C-terminus. 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 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 for binding, or independently.

[0165] 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, and a third polypeptide, (i) The first polypeptide comprises a structure represented by the following formula: VH-CH1-hinge-CH2-first CH3, (ii) The second polypeptide comprises a structure represented by the following formula: MM1-CM1-scFv-hinge-CH2-second CH3, (iii) The third polypeptide comprises a structure represented by the following formula: MM2-CM2-VL-CL, VL is the variable domain of the immunoglobulin light chain, VH is the variable domain of the immunoglobulin heavy chain, scFv is the single-chain variable fragment, CL is the constant domain of the immunoglobulin light chain, CH1 is the first constant domain of the immunoglobulin heavy chain, CH2 is the second constant domain of the immunoglobulin heavy chain, The hinge is the immunoglobulin hinge region that connects the CH1 and CH2 domains, MM1 is the first masking peptide, MM2 is the second masking peptide, CM1 is the first cleavable peptide, CM2 is the 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; when CM1 is not cleaved, MM1 inhibits the binding of the scFv to the first target; when CM2 is not cleaved, MM2 inhibits the binding of the first Fv to the second target. In some embodiments, the first CH3 domain and the second CH3 domain do not include any one or combination of the engineered disulfide bonds or salt bridges described herein. In some embodiments, the first CH3 domain and the second CH3 domain include any one or combination of the engineered disulfide bonds or salt bridges described herein. In some embodiments, the first CH3 domain includes an N390C substitution and the second CH3 domain includes an S400C substitution, or the first CH3 domain includes an S400C substitution and the second CH3 domain includes an N390C substitution. In some embodiments, the first CH3 domain includes E357K, S364K, and S400C substitutions and the second CH3 domain includes L351D, K370D, and N390C substitutions, or the first CH3 domain includes L351D, K370D, and N390C substitutions and the second CH3 domain includes E357K, S364K, and S400C substitutions. In some embodiments, the first CH3 domain includes D356K, E357K, S364K, and N390C substitutions and the second CH3 domain includes L351D, K370D, K439D, and S400C substitutions, or the first CH3 domain includes L351D, K370D, K439D, and S400C substitutions and the second CH3 domain includes D356K, E357K, S364K, and N390C substitutions. In some embodiments, the first CH3 domain includes D356K, E357K, S364K, and S400C substitutions and the second CH3 domain includes L351D, K370D, N390C, and K439D substitutions, or the first CH3 domain includes L351D, K370D, N390C, and K439D substitutions and the second CH3 domain includes D356K, E357K, S364K, and S400C substitutions.In some embodiments, the activatable antibody comprises an IgG1 Fc region such as an IgG1 Fc having 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, there is provided an activatable antibody 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 following formula: MM2-CM2-scFv-hinge-CH2-second 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 an immunoglobulin heavy chain constant domain 1, CH2 is an immunoglobulin heavy chain constant domain 2, The hinge is an immunoglobulin hinge region that links 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; when CM1 is not cleaved, MM1 inhibits the binding of the first Fv to the first target; when CM2 is not cleaved, MM2 inhibits the binding of the scFv to the second target. In some embodiments, the first CH3 domain and the second CH3 domain do not include any one or combination of the engineered disulfide bonds or salt bridges described herein. In some embodiments, the first CH3 domain and the second CH3 domain include any one or combination of the engineered disulfide bonds or salt bridges described herein. In some embodiments, the first CH3 domain includes an N390C substitution and the second CH3 domain includes an S400C substitution, or the first CH3 domain includes an S400C substitution and the second CH3 domain includes an N390C substitution. In some embodiments, the first CH3 domain includes E357K, S364K, and S400C substitutions and the second CH3 domain includes L351D, K370D, and N390C substitutions, or the first CH3 domain includes L351D, K370D, and N390C substitutions and the second CH3 domain includes E357K, S364K, and S400C substitutions. In some embodiments, the first CH3 domain includes D356K, E357K, S364K, and N390C substitutions and the second CH3 domain includes L351D, K370D, K439D, and S400C substitutions, or the first CH3 domain includes L351D, K370D, K439D, and S400C substitutions and the second CH3 domain includes D356K, E357K, S364K, and N390C substitutions. In some embodiments, the first CH3 domain includes D356K, E357K, S364K, and S400C substitutions and the second CH3 domain includes L351D, K370D, N390C, and K439D substitutions, or the first CH3 domain includes L351D, K370D, N390C, and K439D substitutions and the second CH3 domain includes D356K, E357K, S364K, and S400C substitutions.In some embodiments, the activatable antibody comprises an IgG1 Fc region, such as an IgG1 Fc having 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 following formula: MM2-CM2-VH2-CH1-hinge-CH2-second 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 the immunoglobulin light chain constant domain, CH1 is the immunoglobulin heavy chain constant domain 1, CH2 is the immunoglobulin heavy chain constant domain 2, The hinge is the immunoglobulin hinge region that links the CH1 and CH2 domains, MM1 is the first masking peptide, MM2 is the second masking peptide, CM1 is the first cleavable peptide, CM2 is the 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; when CM1 is not cleaved, MM1 inhibits the binding of the first Fv to the first target; when CM2 is not cleaved, MM2 inhibits the binding of the second Fv to the second target. In some embodiments, the first CH3 domain and the second CH3 domain do not include any one or combination of the engineered disulfide bonds or salt bridges described herein. In some embodiments, the first CH3 domain and the second CH3 domain include any one or combination of the engineered disulfide bonds or salt bridges described herein. In some embodiments, the first CH3 domain includes an N390C substitution and the second CH3 domain includes an S400C substitution, or the first CH3 domain includes an S400C substitution and the second CH3 domain includes an N390C substitution. In some embodiments, the first CH3 domain includes E357K, S364K, and S400C substitutions and the second CH3 domain includes L351D, K370D, and N390C substitutions, or the first CH3 domain includes L351D, K370D, and N390C substitutions and the second CH3 domain includes E357K, S364K, and S400C substitutions. In some embodiments, the first CH3 domain includes D356K, E357K, S364K, and N390C substitutions and the second CH3 domain includes L351D, K370D, K439D, and S400C substitutions, or the first CH3 domain includes L351D, K370D, K439D, and S400C substitutions and the second CH3 domain includes D356K, E357K, S364K, and N390C substitutions. In some embodiments, the first CH3 domain includes D356K, E357K, S364K, and S400C substitutions and the second CH3 domain includes L351D, K370D, N390C, and K439D substitutions, or the first CH3 domain includes L351D, K370D, N390C, and K439D substitutions and the second CH3 domain includes D356K, E357K, S364K, and S400C substitutions.In some embodiments, the activatable antibody comprises an IgG1 Fc region such as an IgG1 Fc having 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, there is provided an activatable antibody comprising 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 following formula: VH2-CH1-hinge-CH2-second 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 the immunoglobulin light chain constant domain, CH1 is the immunoglobulin heavy chain constant domain 1, CH2 is the immunoglobulin heavy chain constant domain 2, The hinge is the immunoglobulin hinge region that links the CH1 and CH2 domains, MM1 is the first masking peptide, MM2 is the second masking peptide, CM1 is the first cleavable peptide, CM2 is the 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; when CM1 is not cleaved, MM1 inhibits the binding of the first Fv to the first target; when CM2 is not cleaved, MM2 inhibits the binding of the second Fv to the second target. In some embodiments, the first CH3 domain and the second CH3 domain do not include any one or combination of the engineered disulfide bonds or salt bridges described herein. In some embodiments, the first CH3 domain and the second CH3 domain include any one or combination of the engineered disulfide bonds or salt bridges described herein. In some embodiments, the first CH3 domain includes an N390C substitution and the second CH3 domain includes an S400C substitution, or the first CH3 domain includes an S400C substitution and the second CH3 domain includes an N390C substitution. In some embodiments, the first CH3 domain includes E357K, S364K, and S400C substitutions and the second CH3 domain includes L351D, K370D, and N390C substitutions, or the first CH3 domain includes L351D, K370D, and N390C substitutions and the second CH3 domain includes E357K, S364K, and S400C substitutions. In some embodiments, the first CH3 domain includes D356K, E357K, S364K, and N390C substitutions and the second CH3 domain includes L351D, K370D, K439D, and S400C substitutions, or the first CH3 domain includes L351D, K370D, K439D, and S400C substitutions and the second CH3 domain includes D356K, E357K, S364K, and N390C substitutions. In some embodiments, the first CH3 domain includes D356K, E357K, S364K, and S400C substitutions and the second CH3 domain includes L351D, K370D, N390C, and K439D substitutions, or the first CH3 domain includes L351D, K370D, N390C, and K439D substitutions and the second CH3 domain includes D356K, E357K, S364K, and S400C substitutions.In some embodiments, the activatable antibody comprises an IgG1 Fc region such as an IgG1 Fc having 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, there is provided an activatable antibody comprising 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-first CH3, (ii) the second polypeptide comprises a structure represented by the following formula: VH2-CH1-hinge-CH2-second 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 the immunoglobulin light chain constant domain, CH1 is the immunoglobulin heavy chain constant domain 1, CH2 is the immunoglobulin heavy chain constant domain 2, The hinge is the immunoglobulin hinge region that links the CH1 and CH2 domains, MM1 is the first masking peptide, MM2 is the second masking peptide, CM1 is the first cleavable peptide, CM2 is the 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; when CM1 is not cleaved, MM1 inhibits the binding of the first Fv to the first target; when CM2 is not cleaved, MM2 inhibits the binding of the second Fv to the second target. In some embodiments, the first CH3 domain and the second CH3 domain do not include any one or combination of the engineered disulfide bonds or salt bridges described herein. In some embodiments, the first CH3 domain and the second CH3 domain include any one or combination of the engineered disulfide bonds or salt bridges described herein. In some embodiments, the first CH3 domain includes an N390C substitution and the second CH3 domain includes an S400C substitution, or the first CH3 domain includes an S400C substitution and the second CH3 domain includes an N390C substitution. In some embodiments, the first CH3 domain includes E357K, S364K, and S400C substitutions and the second CH3 domain includes L351D, K370D, and N390C substitutions, or the first CH3 domain includes L351D, K370D, and N390C substitutions and the second CH3 domain includes E357K, S364K, and S400C substitutions. In some embodiments, the first CH3 domain includes D356K, E357K, S364K, and N390C substitutions and the second CH3 domain includes L351D, K370D, K439D, and S400C substitutions, or the first CH3 domain includes L351D, K370D, K439D, and S400C substitutions and the second CH3 domain includes D356K, E357K, S364K, and N390C substitutions. In some embodiments, the first CH3 domain includes D356K, E357K, S364K, and S400C substitutions and the second CH3 domain includes L351D, K370D, N390C, and K439D substitutions, or the first CH3 domain includes L351D, K370D, N390C, and K439D substitutions and the second CH3 domain includes 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, the activatable antibody is designed based on any one of the multispecific antibodies described herein, for example, by fusing a masking moiety (MM) to the target binding moiety (TBM) of the multispecific antibody via a cleavable moiety (CM). When the CM is not cleaved, the MM inhibits the binding of the TBM to the target. The activatable antibody is described, for example, in WO2019 / 149282, the content of which is hereby incorporated by reference in its entirety. The activatable antibody may comprise any one of the TBMs described in the subsection "Target Binding Moiety (TBM)" of Section III "Multispecific Antibodies". The activatable antibodies described herein may comprise one or more linkers described in the subsection "Linker" of Section III "Multispecific Antibodies", disposed, for example, between the MM and CM of the Fc, between the CM and TBM, or between the TBM and the hinge region.

[0171] MM refers to an amino acid sequence that, when the CM of an activatable antibody is intact (e.g., not cleaved by a corresponding enzyme and / or contains non-reduced cysteine-cysteine disulfide bonds), interferes with or inhibits the binding of TBM to a target. In some embodiments, MM efficiently interferes with or inhibits the binding of TBM to a target such that the binding of TBM to the target is very low and / or below the limit of detection (e.g., the binding cannot be detected by ELISA or flow cytometry assay). The amino acid sequence of the CM may overlap with or be contained within MM. It should be noted that, for convenience, the terms "ABP" or "activatable antibody" are used herein to refer to both the uncleaved (or "native") state and the cleaved state of the ABP or activatable antibody. In some embodiments, the cleaved ABP may lack MM due to cleavage of the CM (e.g., by a protease), and it will be apparent to those skilled in the art that at least the release of MM (e.g., where MM is not covalently bound to the ABP by a covalent bond, such as a disulfide bond between cysteine residues) is effected. Exemplary ABPs are described in more detail below.

[0172] The CM generally contains a cleavable amino acid sequence and functions, for example, as a substrate for an enzyme and / or a cysteine-cysteine pair that can form a reducible disulfide bond. Thus, when terms such as "cleavage," "cleavable," "cleaved," etc. are used in relation to the CM, the terms encompass, for example, enzymatic cleavage by a protease and cleavage of the disulfide bond between cysteine-cysteine pairs via reduction of the disulfide bond that can occur upon exposure to a reducing agent.

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

[0174] In some embodiments, the activatable antibody (e.g., a 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 the activatable antibody is contacted with T cells as compared to corresponding tumor cells that have not been contacted with the activatable antibody (or as compared to corresponding tumor cells that have been contacted with an isotype control antibody and T cells). In some embodiments, the activatable antibody is capable of reducing the tumor volume of a subject when the subject is administered the activatable antibody. In some embodiments, the activatable antibody (e.g., a BiTE molecule) is capable of reducing the tumor volume of a subject 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; as compared to the 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 suffering from cancer will achieve partial or complete remission when administered the activatable antibody.

[0176] Masking moiety (MM) The activatable antibodies described herein include 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, which is hereby incorporated by reference in its entirety.

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

[0178] In some embodiments, the masking peptide (MM) interferes with, blocks, reduces, suppresses, 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, suppresses, inhibits, or competes with the target binding moiety for binding to the target only when the antibody has not been activated (e.g., activated by a change in pH (increase or decrease), activated by a change in temperature (increase or decrease), activated after contact with a second molecule (e.g., a small molecule or a protein ligand, etc.)). In some embodiments, activation induces cleavage of a cleavable moiety. In some embodiments, activation induces a conformational change (e.g., displacement of the MM) of the polypeptide(s), such that the MM does not inhibit the binding of the activatable antibody to the target. In some embodiments, the MM interferes with, blocks, reduces, suppresses, inhibits, or competes with the target binding moiety for binding to the target only when 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 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.) prior to activation. In some embodiments, the masking efficiency is the difference in affinity for binding to the target of the activatable antibody (before activation) containing the MM compared to the affinity for binding to the target of the polypeptide lacking the MM (e.g., the difference in affinity for the target antigen (e.g., CD3 or HER2) of the activatable antibody (before activation) containing the MM compared to the parental antibody lacking the MM, or the difference in affinity for the target antigen of the activatable antibody (before activation) containing the MM compared to the affinity for the target antigen of the activatable antibody after activation (e.g., CD3 or HER2)).In some embodiments, masking efficiency is measured by dividing the EC50 of the binding of an activatable antibody comprising an MM (prior to activation) by the EC50 of the parental antibody (e.g., measuring the EC50 by ELISA). In some embodiments, masking efficiency is the difference in affinity of an activatable antibody comprising an MM for the target prior to activation compared to the affinity for the target after activation of the activatable antibody comprising an MM (e.g., the difference in affinity of the activatable antibody prior to activation compared to the activatable antibody after activation for the target antigen (e.g., CD3 or HER2)). 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, suppress, inhibit, or compete with the target binding moiety (TBM) with respect to 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 in pH (increase or decrease), activated by a change in temperature (increase or decrease), activated after contact with a second molecule (e.g., an enzyme), etc.). In some embodiments, the MM does not interfere with, block, reduce, suppress, inhibit, or compete with the target binding moiety (TBM) with respect to binding to the target after the cleavable moiety (CM) has been cleaved by 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 parental antibody) of up to about 1.75 (e.g., up to about 1.75, up to about 1.5, up to about 1.4, up to about 1.3, up to about 1.2, up to about 1.1, up to about 1.0, up to about 0.9, up to about 0.8, up to about 0.7, up to about 0.6, or up to 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 include purification tags (e.g., his-tag, flag-tag, maltose binding protein, and glutathione-S-transferase tags), detection tags (e.g., tags that can be detected photometrically (e.g., red or green fluorescent proteins, etc.)), tags having detectable enzyme activity (e.g., alkaline phosphatase, etc.), tags comprising a secretion sequence, a leader sequence, and / or a stabilizing sequence, protease cleavage sites (e.g., furin cleavage site, TEV cleavage site, thrombin cleavage site), etc., but are not limited thereto. In some embodiments, the one or more additional amino acid sequences are at the N-terminus of the MM.

[0181] Cleavable moiety (CM) In some embodiments, the activatable antibody comprises one or more CMs, each of which is disposed 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 the target of the polypeptide comprising the CM) may be used, which is, for example, a protease cleavage site 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, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAMDEC1, ADAMTS1, 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 proteases (e.g., RACE and / or renin); aspartic 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 proteases (e.g., cruzipain, legumain, and / or oubain-2); KLK (e.g., KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, and / or KLK14);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, FXla, FXIIa)); elastase; granzyme B; guanidino benzoatase; HtrA1; human neutrophil elastase; lactoferrin; malapsin; 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), etc. In some embodiments, the first protease cleavage site is the cleavage site of 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 the cleavage site of a protease selected from uPA, MMP-2, MMP-9, and / or TEV protease. In some embodiments, 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 includes 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 includes a second cleavage site (CS2). In some embodiments, the second cleavage site is a second protease cleavage site. The second protease cleavage site can be any suitable protease cleavage site that is recognized and / or cleaved by any of the above proteases. In some embodiments, the first (CS1) and second (CS2) cleavage sites are protease cleavage sites that are recognized and / or cleaved by the same protease. In some embodiments, the first (CS1) and second (CS2) cleavage sites are 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.) protease cleavage sites. 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) includes a structure of (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) may be any suitable linker as described above. In some embodiments, the first (L1) and second (L2) linkers are the same. In some embodiments, the first (L1) and second (L2) linkers are different. In some embodiments, at least the second linker (L2) is at the C-terminus relative to the second cleavage site (CS2). In some embodiments, the cleavable moiety (CM) comprises a structure of (CS1)-L1-(CS2)-L2 from the N-terminus to the C-terminus.

[0185] Activatable antibody targeting CD3 The present application provides an activatable antibody targeting CD3, an activatable antibody fragment, and a polypeptide, which comprise a masking moiety (MM) comprising the amino acid sequence of SEQ ID NO: 35.

[0186] In some embodiments, an antibody light chain is provided that comprises a polypeptide comprising, from the N-terminus to the C-terminus, an 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 the 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 that comprises a polypeptide comprising, from the N-terminus to the C-terminus, an 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 the 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, there is provided an activatable antibody targeting CD3, comprising a first polypeptide comprising MM, CM, and TBM from the N-terminus to the C-terminus, wherein MM comprises the amino acid sequence of SEQ ID NO: 35 and inhibits the binding of the activatable antibody to CD3 when CM is not cleaved; CM comprises at least a first cleavage site; TBM comprises VL, and the activatable antibody further comprises a second polypeptide comprising VH; when CM is cleaved, the activatable antibody binds to CD3 via 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 that comprises a first polypeptide comprising MM, CM, and TBM from the N-terminus to the C-terminus, where MM comprises the amino acid sequence of SEQ ID NO: 35 and inhibits binding of the activatable antibody to CD3 when CM is not cleaved; CM comprises at least a first cleavage site; TBM comprises VH, and the activatable antibody further comprises a second polypeptide comprising VL; when CM is cleaved, the activatable antibody binds to CD3 via VH and VL. In some embodiments, the activatable antibody comprises a VH comprising 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 CDR-H3 comprising the amino acid sequence of SEQ ID NO: 63; and / or a VL comprising 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 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 that comprises a first polypeptide comprising, from the N-terminus to the C-terminus, MM, CM, and scFv, where MM comprises the amino acid sequence of SEQ ID NO: 35 and inhibits the binding of the activatable antibody to CD3 when CM is not cleaved; CM comprises at least a first cleavage site; and when CM is cleaved, the activatable antibody binds to CD3 via 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 the N-terminus to the C-terminus, VL and VH. In some embodiments, the scFv comprises, from the N-terminus to the C-terminus, VH and VL. In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 79.

[0191] In some embodiments, the activatable antibody targeting CD3 is a multispecific antibody such as a bispecific antibody. In some embodiments, the activatable antibody targeting 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 that comprises 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-first CH3, (ii) The second polypeptide comprises a structure represented by the following formula: MM1-CM1-scFv-Hinge-CH2-Second 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 links 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 tumor antigen (e.g., HER2); scFv specifically binds to CD3; when CM1 is not cleaved, MM1 inhibits the binding of scFv to CD3; when CM2 is not cleaved, MM2 inhibits the 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, 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, 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, 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, VH 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 CDR-H3 comprising the amino acid sequence of SEQ ID NO: 71. In some embodiments, VL comprises 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 CDR-L3 comprising the amino acid sequence of SEQ ID NO: 74. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 75. In some embodiments, VL comprises the amino acid sequence of SEQ ID NO: 76.

[0194] In some embodiments by 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 contain any one or combination of the 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 contain any one or combination of the 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 having 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, which 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 antibody targeting HER2 This application provides an activatable antibody targeting HER2, an activatable antibody fragment, and a polypeptide 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 that comprises a polypeptide comprising, from N-terminus to C-terminus, an MM, a cleavable moiety (CM), and a target binding moiety (TBM), where 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 the 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 that comprises a polypeptide comprising, from N-terminus to C-terminus, an MM, a CM, and a TBM, where 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 the 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, which comprises a first polypeptide comprising MM, CM, and TBM from the N-terminus to the C-terminus, where MM comprises the amino acid sequence of SEQ ID NO: 36 and inhibits the binding of the activatable antibody to HER2 when CM is not cleaved; CM comprises at least a first cleavage site; TBM comprises VL, and the activatable antibody further comprises a second polypeptide comprising VH; when CM is cleaved, the activatable antibody binds to HER2 via 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 MM, CM, and TBM from the N-terminus to the C-terminus, where MM comprises the amino acid sequence of SEQ ID NO: 36 and inhibits the binding of the activatable antibody to HER2 when CM is not cleaved; CM comprises at least a first cleavage site; TBM comprises VH, and the activatable antibody further comprises a second polypeptide comprising VL; when CM is cleaved, the activatable antibody binds to HER2 via VH and VL. In some embodiments, the activatable antibody comprises a VH comprising 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 CDR-H3 comprising the amino acid sequence of SEQ ID NO: 71; and / or a VL comprising 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 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, which comprises a first polypeptide comprising MM, CM, and scFv from the N-terminus to the C-terminus, wherein MM comprises the amino acid sequence of SEQ ID NO: 36 and inhibits the binding of the activatable antibody to HER2 when CM is not cleaved; CM comprises at least a first cleavage site; and when CM is cleaved, the activatable antibody binds to HER2 via 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 VL and VH from the N-terminus to the C-terminus. In some embodiments, the scFv comprises VH and VL from the N-terminus to the C-terminus. In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 79.

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

[0203] In some embodiments by 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 include any one or combination of the 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 include any one or combination of the engineered disulfide bonds or salt bridges described herein. In some embodiments, the first CH3 domain includes D356K, E357K, S364K, and S400C substitutions, and the second CH3 domain includes L351D, K370D, N390C, and K439D substitutions, or the first CH3 domain includes L351D, K370D, N390C, and K439D substitutions, and the second CH3 domain includes D356K, E357K, S364K, and S400C substitutions. In some embodiments, the activatable antibody includes an IgG1 Fc region such as an IgG1 Fc having an N297A substitution.

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

[0205] In some embodiments, the heterodimeric protein or antibody derivative is derived from a modification 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 a framework region, CDR region, or constant region, may be modified. The types of modifications include substitutions, insertions, deletions, or combinations thereof of one or more amino acids of the parent heterodimeric protein or antibody.

[0206] In some embodiments, the antibody (e.g., bispecific antibody or activatable antibody) derivative comprises a polypeptide having 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% identity to the amino acid sequence set forth in any of SEQ ID NOs: 84-143. In some embodiments, the antibody (e.g., bispecific antibody or activatable antibody) derivative comprises a VL or VH region having 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% identity 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 having 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% identity 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 having 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% identity 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 having 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% identity 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 to, and / or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 additions and / or deletions of, the amino acid sequence of the heterodimeric protein or antibody described herein.

[0208] Amino acid substitutions include both conservative and non-conservative substitutions. The term "conservative amino acid substitution" means replacing one amino acid with another, where the two amino acids have similarity in the specific physicochemical properties of the residues involved, such as polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathicity. For example, substitutions may typically 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] The modification may be made at any position of the amino acid sequence of the antibody, including the CDR, framework region, or constant region. In some embodiments, the present application provides antibody derivatives that contain the VH and VL CDR sequences of the exemplary antibodies described herein, but contain framework sequences that are different from those of the exemplary antibodies. Such framework sequences can be obtained from publicly available DNA databases or published references including germline antibody gene sequences. For example, the germline DNA sequences of the 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, U.S. Department 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)). The framework sequences that can be used in constructing antibody derivatives include those that are structurally similar to the framework sequence used by the exemplary antibodies of the present application. For example, the CDR-H1, CDR-H2, and CDR-H3 sequences, as well as the CDR-L1, CDR-L2, and CDR-L3 sequences of the exemplary antibodies, can be transplanted into a framework region having the same sequences as those found in the germline immunoglobulin genes from which the framework sequence is derived, or the CDR sequences can be transplanted into a framework region that contains 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 of an antibody 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 particular embodiments, the chimeric antibody comprises 1, 2, or 3 CDRs of the heavy chain variable region of an exemplary antibody and / or 1, 2, or 3 CDRs of the light chain variable region. 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 chains. 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 evaluated in in vitro or in vivo assays known in the art. Usually, conservative substitutions are introduced. The mutations can be amino acid additions and / or deletions. Further, usually 1, 2, 3, 4, or 5 or fewer residues within the CDR region are changed. In some embodiments, the antibody derivative comprises 1, 2, 3, or 4 amino acid substitutions in the heavy chain CDR and / or light chain CDR. In another embodiment, the amino acid substitution is to change one or more cysteines of the antibody to another residue such as, but not limited to, alanine or serine. The cysteine can be a standard or non-standard cysteine. In some embodiments, the antibody derivative has 1, 2, 3, or 4 conservative amino acid substitutions in the heavy chain CDR region compared to the amino acid sequence of the exemplary antibody.

[0212] Modifications may also be made to framework residues within the VH and / or VL regions. Typically, such framework variants are created to reduce the immunogenicity of the antibody. One approach is to "revert" 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 to the germline sequence from which the antibody is derived. Somatic mutations can be "reverted" to the germline sequence, for example, by site-directed mutagenesis or PCR-mediated mutagenesis, to return the framework region sequence to its germline configuration.

[0213] In addition, modifications may also be made within the Fc region of exemplary antibodies, typically to alter one or more functional properties of the antibody such as serum half-life, complement binding, Fc receptor binding, and / or antibody-dependent cell cytotoxicity. In one example, the hinge region of CH1 is modified such that the number of cysteine residues within the hinge region is altered, e.g., increased or decreased. 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 light and heavy chain assembly or to increase or decrease the stability of the antibody. In another case, the Fc hinge region of the antibody is mutated to decrease the biological half-life of the antibody.

[0214] In some embodiments, the Fc region of the heterodimeric protein or antibody described herein has at least one (e.g., at least 1, 2, or 3 or more) amino acid substitution compared to the Fc region of wild-type IgG or wild-type antibody, in addition to the amino acid substitutions that form engineered disulfide bonds or salt bridges described herein. In some embodiments, the Fc region has at least 80%, at least 85%, at least 90%, at least 95%, or more homology with the native sequence Fc region and / or the Fc region of the parental polypeptide.

[0215] Furthermore, the Fc region may be modified to alter potential glycosylation sites or patterns according to routine experiments known in the art. In another aspect, the present application provides a derivative of the heterodimeric protein or antibody described herein that contains at least one mutation in the variable region of the light or heavy chain that changes the pattern of glycosylation in the variable region. Such antibody derivatives may have increased affinity and / or altered specificity with respect to binding to an antigen. The mutation may add a novel glycosylation site to the V region, change the position of one or more V region glycosylation site(s), or remove an existing V region glycosylation site. In some embodiments, the present application provides a derivative of the antibody described herein that has a potential N-linked glycosylation site at asparagine in the heavy chain variable region, and the potential N-linked glycosylation site within one heavy chain variable region is removed. In some embodiments, the present application provides a derivative of the antibody described herein that has a potential N-linked glycosylation site at asparagine in the heavy chain variable region, and the potential N-linked glycosylation site within both heavy chain variable regions is removed. Methods for altering the glycosylation pattern of antibodies as described in U.S. Patent No. 6,933,368 (which application is incorporated herein by reference) are known in the art.

[0216] In some embodiments, the antibodies described herein (e.g., bispecific 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 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. Exemplary methods for generating antibodies of a desired class or subclass include isolating the nucleic acid encoding the heavy chain of the antibody described herein (e.g., bispecific or activatable antibody) and the nucleic acid encoding the light chain of the antibody described herein (e.g., bispecific or activatable antibody), isolating the sequence encoding the VH region, ligating the VH sequence to the sequence encoding the heavy chain constant region of the desired class or subclass, expressing the light chain gene and the heavy chain construct in cells, and collecting the antibody.

[0217] The heterodimeric protein or antibody variant is also provided with an amino-terminal leader extension. For example, one or more amino acid residues of the amino-terminal leader sequence are present at the amino terminus of any one or more of the heavy or light chains of the antibody.

[0218] The heterodimeric proteins or antibodies described herein (e.g., bispecific antibodies or activatable antibodies) can be further modified. In some embodiments, the heterodimeric protein or antibody is linked to an additional molecular entity. Examples of additional molecular entities include pharmaceuticals, peptides or proteins, detection agents or labels, and antibodies.

[0219] In some embodiments, the heterodimer protein or antibody of the present application is linked to a pharmaceutical. Examples of pharmaceuticals include cytotoxic agents or other cancer therapeutics, and radioisotopes. Specific examples of cytotoxic agents include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracinedione, mitoxantrone, mitramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, as well as analogs or homologs thereof. Therapeutic agents 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), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiammine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mitramycin, 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. For example, methods for linking polypeptides to pharmaceuticals using various linker technologies are known in the art. Examples of linker types include hydrazone, thioether, ester, disulfide, and peptide-containing linkers.For further discussion of linkers and methods for conjugating therapeutic agents to antibodies, see, for example, the following: Saito et al., Adv. Drug Deliv. 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 Deliv. 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, and the like. One of ordinary skill in the art can select a suitable label according to the intended use.

[0221] As used herein, a cytotoxic agent is a moiety that reduces the proliferative capacity of one or more cells. When the likelihood of cell proliferation is reduced, the proliferative capacity of the cell is reduced. The reasons for this include, for example, the cell undergoes apoptosis or dies in other ways, the cell cycle of the cell cannot progress and / or divide, the cell differentiates, and the like. Non-limiting examples of cytotoxic agents include, but are not limited to, radioisotopes, toxins, and chemotherapeutic agents. One of ordinary skill in the art can select a suitable cytotoxicity according to the intended use.

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

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

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

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

[0226] Host cells suitable for cloning or expressing a vector encoding a polypeptide include prokaryotic or eukaryotic cells. For example, the polypeptide can be produced in bacteria, particularly if glycosylation and Fc effector functions are not required (see, e.g., U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523; see also Charlton, Methods in Molecular Biology, Vol. 248 (B.K.C. 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 the soluble fraction and further purified.

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

[0228] Host cells suitable 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, a number of baculovirus strains have been identified that can be used with insect cells for the 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 (which describe the PLANTIBODIES™ technology for producing antibodies in transgenic plants).

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

[0231] In order for some secreted proteins to be expressed and secreted in large amounts, a leader sequence derived from a heterologous protein may be desirable. In some embodiments, using a heterologous leader sequence may be advantageous in that the resulting mature polypeptide can remain unchanged since the leader sequence is removed in the ER during the secretion process. In order to express and secrete some proteins, it may be necessary to add a heterologous leader sequence.

[0232] Certain exemplary leader sequences are described, for example, in the online leader sequence database maintained by the Department of Biochemistry of 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 heterodimer 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 heterodimer protein, a multispecific antibody, or an activatable antibody). The carrier can be an antiadhesive agent, binder, coating, disintegrant, filler, or diluent, preservative (e.g., antioxidant, antibacterial, or antifungal agent), sweetening agent, absorption delaying agent, wetting agent, emulsifying agent, buffer, etc. 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), physiological saline, buffers, buffered physiological saline, and isotonic agents such as sugars, polyalcohols, sorbitol, and sodium chloride.

[0235] The composition may be in any suitable form, such as liquid, semi-solid, 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. The specific form of the composition suitable for the delivery of a polypeptide (e.g., a heterodimeric protein, a multispecific antibody, or an activatable antibody) is a sterile liquid, such as a solution, suspension, or dispersion for injection or infusion. Sterile solutions can be prepared by incorporating the required amount of polypeptide (e.g., a heterodimeric protein, a multispecific antibody, or an activatable antibody) into a suitable carrier followed by sterile filtration through a fine filter. Dispersions may 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, the preparation method includes obtaining a powder of the active ingredient and any additional desired ingredients from its pre-sterile filtered solution by vacuum drying and lyophilization (freeze-drying). The various dosage forms of the composition can be prepared by conventional techniques known in the art.

[0236] The relative amount of polypeptide (e.g., a heterodimeric protein, a multispecific antibody, or an activatable antibody) contained in the composition will vary depending on a number of factors, such as the specific polypeptide and carrier used, the dosage form, and the desired release and pharmacodynamic properties. The amount of polypeptide (e.g., a heterodimeric protein, a multispecific antibody, or an activatable antibody) in a single dosage form will generally be an amount that produces a therapeutic effect, but it may also be a lesser amount. Generally, this amount will range from about 0.01 percent to about 99 percent, about 0.1 percent to about 70 percent, or 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 the additional therapeutic agent included in the composition can be readily selected by one of ordinary skill in the art and will vary depending on a number of factors, such as the particular agent and carrier used, the dosage form, and the desired release and pharmacodynamic properties. The amount of the additional therapeutic agent included in a single dosage form will generally be an amount of the agent that produces a therapeutic effect, although it may be a lesser amount.

[0238] Any of the polypeptides (e.g., a heterodimeric protein, a multispecific antibody, or an activatable antibody) and / or compositions (e.g., pharmaceutical compositions) described herein may be used in the preparation of a medicament (e.g., a medicament used therefor in a subject in need of treatment of cancer or delay of cancer progression).

[0239] In some embodiments, a kit is provided herein that includes any one of the heterodimeric proteins, multispecific antibodies, activatable antibodies, and / or compositions described herein. In some embodiments, the kit further includes a package insert that includes instructions for use of the heterodimeric protein, multispecific antibody, activatable antibody, and / or composition. The package insert may contain information regarding indications, usage, dosage, administration, combination therapy, contraindications, and / or warnings regarding the use of the therapeutic product. In some embodiments, the kit further includes one or more buffers for, e.g., storing, transporting, administering, or otherwise using the heterodimeric protein, multispecific antibody, activatable antibody, and / or composition. In some embodiments, the kit further includes one or more containers (e.g., syringes, etc.) for storing or administering the heterodimeric protein, multispecific antibody, activatable antibody, and / or composition. An article of manufacture is also provided that includes any one of the heterodimeric proteins, multispecific antibodies, activatable antibodies, and / or compositions described herein.

[0240] VIII. Methods of Use The heterodimeric proteins, multispecific antibodies, activatable antibodies, and pharmaceutical compositions described herein are useful for treatment, diagnosis, or other purposes, such as modulation of the immune response, treatment of cancer, enhancement of the effectiveness of other cancer therapies, enhancement of the effectiveness of vaccines, or treatment of autoimmune diseases.

[0241] In some embodiments, a method for treating a disease or condition in a subject in need thereof is provided, 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., an activatable BiTE molecule) described herein. In some embodiments, the disease or condition is cancer. A variety of cancers can be treated or prevented using the methods, uses, or pharmaceutical compositions provided by this application.

[0242] In some embodiments, a method for treating cancer in a subject in need thereof is provided, the method comprising administering to the subject an effective amount of a pharmaceutical composition comprising any one of the multispecific antibodies that target one or more immune checkpoint molecules described herein (e.g., any one of the PDL1×CD137, CD137×PDL1, or PDL1×CD137×CTLA4 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 advanced cancer.

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

[0244] In some embodiments, provided is a method of enhancing an immune response in a mammal, which comprises 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, evoking, increasing, improving, or augmenting the response of the immune system of a subject. 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 response), and may be a primary or secondary immune response. Examples of enhancing an immune response include activation of PBMCs and / or T cells (including an increase in the secretion of one or more cytokines such as IL-2 and / or IFNγ). Enhancement of the immune response can be evaluated 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, expression of cell surface markers, and cytotoxicity. Generally, the methods of the present application enhance the immune response in a mammal as compared to the immune response of an untreated mammal or a mammal not treated using the recited methods.

[0245] In the practice of the methods of treatment, the heterodimeric protein, multispecific antibody, or activatable antibody may be administered alone as monotherapy or in combination with one or more additional therapeutic agents or therapies. Accordingly, in another aspect, the present application provides a combination therapy comprising a 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" can refer to any therapeutic agent other than the heterodimeric protein, multispecific antibody, or activatable antibody provided by the present application.

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

[0247] The dosage, dosing frequency, and route of administration for the therapies described herein depend on a number of factors, such as the type and severity of the disorder to be treated, the specific heterodimeric protein, multispecific antibody, or activatable antibody being administered, the timing of administration, the duration of treatment, the specific additional therapy being administered, the age, sex, weight, condition, health status, and past medical history of the patient being treated, as well as similar factors known in the medical art.

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

Example

[0249] The following examples are intended to purely illustrate the present invention and should therefore in no way be considered to limit the present invention. The following examples and detailed description are provided as illustration, not limitation.

[0250] Example 1. Design of Fc domain mutations As shown in Tables 1A and 1B, novel Fc mutations were designed, including disulfide bond mutations, charge mutations, and combinations thereof.

Table 5

Table 6

[0251] Example 2. Purity Evaluation of Heterodimer To test the novel Fc mutations, heterodimers TYM01 to TYM013 and reference heterodimers were constructed. The corresponding novel Fc mutations are shown in Table 2. A Fab-Fc / Fc1 arm construct with a mutation at the CH3 domain interface was designed (Figure 1A). To evaluate the effect of the mutations on heterodimerization and homodimerization of the CH3 domain, cloning in a mammalian expression vector was performed such that the constructed CH3_A domain could be expressed in a light chain-heavy chain ("LC-HC") half-body, and the CH3_B domain could be expressed in an Fc-only format. Plasmids encoding the light chain ("LC"), heavy chain ("HC"), and Fc in a 2:1:1 molar ratio were co-transfected into HEK293 cells for transient expression. An excess of LC was used relative to the HC chain DNA to avoid limiting the LC. The cell culture supernatant was filtered through a 0.45 μm sterile filter. The antibody was purified by protein A affinity chromatography using a HiTrap MabSelect SuRe prepacked column (GE Healthcare), followed by buffer exchange. To evaluate the yield of the heterodimer, 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, which facilitates the identification of various pairings by SDS-PAGE electrophoresis and size exclusion high performance liquid chromatography ("SEC-HPLC"). Proteins were visualized by electrophoresis under reducing and non-reducing conditions. Under reducing conditions, three bands corresponding to the HC monomer, Fc monomer, and LC monomer were observed. Figure 6 shows that under non-reducing conditions, there are 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 evaluated by SEC-HPLC. As shown in Figure 7, a total of three peaks were detected. The first peak in the spectrum corresponds to the homodimer, the second peak at 46.6 minutes corresponds to the heterodimer, and the third peak corresponds to the LC-HC half body. By quantifying the peak areas in SEC-HPLC, variant pairs that stabilize the heterodimer compared to the homodimer were identified. The purity was calculated using the first and second peaks, and the results are shown in Table 2.

Table 7

[0253] Example 3. Evaluation of the stability of the heterodimer Furthermore, the stability of the heterodimer was evaluated by incubation under forced degradation conditions. The purified heterodimer sample was diluted to 1 mg / mL with an 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 different resistances to protein aggregation and precipitation at high temperatures. The changes in the SEC-HPLC spectrum after storage at 37 °C are shown in Figure 9. Proteins TYM10, TYM11, and TYM013 showed relatively excellent stability.

[0254] Example 4. Generation of heterodimers targeting CD137 and PDL1 Using anti-CD137 antibody and anti-PDL1 antibody, a bispecific antibody with an Fc mutation was constructed. The pharmacokinetics of monoclonal antibodies can be regulated by changing the interaction with the neonatal Fc receptor FcRn. Improving the affinity of the FcRn-IgG interaction can extend the half-life of the modified 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 (pH 6-6.5) of the endosome, FcRn exhibits a low affinity (micromolar to nanomolar) for the Fc region of IgG. Therefore, the FcRn binding characteristics can reflect how the mutation affects the PK of the Fc region. Table 3 shows that the pH-dependent FcRn binding of the bispecific antibody was not affected at both acidic and physiological pHs.

Table 8

[0255] Using anti-CD137 and anti-PDL1 antibodies with a common light chain, a bispecific antibody with a common light chain and various Fc mutations was constructed (Figure 1B). The bispecific antibody was also tested in a 293T-CD137-NFκB reporter assay. Briefly summarized, 50x10 4 / ml of 293T-CD137 cells and 50x10 4 / ml of 293T-PDL1 cells were mixed, and then the mixed cells were divided into the wells of a 96-well plate at a density of 5x10 4 cells / well (100 μl / well). 50 μl of the 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 the luciferase substrate and 40 μl of the Renilla substrate were added, and the luminescence signal was read (Promega E1980).

[0256] As shown in FIG. 10, TYM10 and TYM11 had relatively high activities 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 examples describe the development of a developable and effective format for bispecific antibodies targeting CD137 and PDL1. The format was optimized based on the "Morrison format" (FIG. 2). DNA encoding anti-CD137 Fv and anti-PDL1 Fv was used to construct expression plasmids in the form of Fab or scFv. Since scFv has a reduced affinity compared to Fab, the orientation of the two Fvs (i.e., CD137×PDL1 or PDL1×CD137 in the form of Fab×scFv) can affect the effectiveness of the bispecific antibody.

[0258] IgG1 or IgG4(S228P) isotype was used. The scFv was linked to the C-terminus of Fc in the orientation from VH to VL with a linker of SGGGS (SEQ ID NO:80) or GGGSGGGGS (SEQ ID NO:81). Among the scFvs, the C-terminus of VH was linked to the N-terminus of VL with a (G4S)4 (SEQ ID NO:82) linker. The disulfide bond from VH-44 to VL-100 was also incorporated into the scFv and designed to stabilize the format. The newly engineered N390C CH3A -S400’C CH3B Pairs of disulfide bonds were also tested (SEQ ID NOs:23 - 24). In addition, the N297A mutation was introduced to silence the effector function mediated by the Fc region. Table 4 provides the eight scaffold designs developed. Table 5 lists the SEQ ID NOs corresponding to the first heavy chain, the first light chain, the second heavy chain, and the second light chain of the exemplary CD137×PDL1 and PDL1×CD137 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 cell culture supernatant containing the bispecific antibody was harvested 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), and subsequently buffer-exchanged in 20 mM histidine (pH 5.5) buffer.

[0260] The aggregation ratio of the purified bispecific antibody after purification was evaluated by analytical size exclusion chromatography (“SEC”). The analysis was performed as follows. SEC was carried out on a Waters 2695 combined with a Waters 2996 UV detector. A TSK Gel g3000 SWXL column (300 mm × 7.8 mm) equipped with a TSK Gel g3000 SWXL precolumn (Tosoh Bioscience) was used. Each sample was injected at 10 μg, and separation was carried out at a flow rate of 0.5 mL / min. The elution buffer was composed of 200 mM sodium phosphate at pH 7.0. UV detection was performed at 214 nm. 100 μL of the sample (1 mg / mL) was frozen at -80 o C for 30 minutes and then thawed at room temperature for 60 minutes to test the freeze-thaw stability. Six freeze-thaw cycles were performed, and the aggregation rate was also measured by analytical size exclusion chromatography.

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

Table 10

Table 11

[0262] Using the purified protein at 1 mg / mL (incubated at 40 °C for 28 days), 6 cycles of freezing and thawing were tested (Figures 11A - 11B). Also, 4 purified proteins were heated at high temperatures to confirm their thermal stability (Figure 11C). Figures 11A - 11B show the quality of the proteins evaluated by analytical size exclusion chromatography. All formats showed good long - term storage stability with little aggregation and degradation under accelerated storage conditions. As shown in Figure 11C, all the proteins tested aggregated and precipitated at 60 °C, and the CD137xPDL1 bispecific antibody showed better colloidal stability than the PDL1xCD137 bispecific antibody. These data indicated CD137xPDL1 as the most suitable antibody format for targeting CD137 and PDL1.

[0263] C. Binding Affinity of Bispecific Antibodies Biacore T200 (GE Healthcare) was used as a high - performance system for real - time biomolecular interaction analysis using surface plasmon resonance technology (「SPR」). During measurement, the anti - human IgG monoclonal antibody of the Human Antibody Capture Kit provided by Biacore was immobilized on the CM5 chip, and the IgG sample was injected into the sensor chip. For the binding kinetics analysis in HBS - EP buffer, the analyte was injected into the IgG capture flow cell. The data was fitted according to the 1:1 Langmuir model, and the K D value was determined (Table 7). Format 5 showed the highest affinity among all the 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. Next, the cells were incubated with IgG (at 100 nM each) for 1 hour at 4°C while shaking at 300 rpm in a shaking bed protected from light. To bind simultaneously, the cells were incubated with biotinylated human CD137 or PDL1 protein fused to the human Fc fragment, and SA-PE (streptavidin, phycoerythrin conjugate). For cross-reactivity, the cells were incubated with Alexa Fluor 647-conjugated mouse anti-human Fc antibody. The mixture was incubated for 30 minutes at 4°C while shaking at 300 rpm in a shaking bed protected from light. The cells were washed once before analysis by flow cytometry (Beckman CytoFlex). Figure 12 shows that the bispecific antibody binds to human PDL1 and CD137 simultaneously. In addition, Figure 13 shows that the bispecific antibody maintained cross-reactivity with the parental antibody against PDL1 or CD137 of human, mouse, or monkey origin.

Table 12

[0265] D. Efficacy in vitro and in vivo The effects of PDL1xCD137 and CD137xPDL1 bispecific antibodies on in vitro reporter gene assays were tested (Figure 14). Anti-PDL1-based bispecific antibodies were evaluated in PDL1 blockade bioassays. Briefly, Jurkat T cells expressing a luciferase reporter driven by human PD-1 and 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 antigen-independently 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 blockade 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 divided into 96-well plates (50 μL / well, density 6x10 5 ). After 5.5 hours of incubation, diluted test antibodies pre-mixed with a crosslinker at a 1:5 ratio (shown in Figure 14) were added. After 18 hours, luciferase levels were measured. Renilla luciferase activity was used to normalize the relative luciferase units (RLU) to transfection efficiency against a blank control (no antibody treatment), and the results were expressed as the mean ± standard error of triplicates.

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

[0267] Since 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 and much more effective than only one of the two parental antibodies. 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.

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

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

[0270] Anti-CD137 and anti-PDL1 Fvs cross-reacted with mouse and monkey antigens, so the in vivo efficacy of the bispecific antibodies was studied in a 3LL syngeneic mouse tumor model. C57BL / 6 mice were subcutaneously implanted with 2×10 6 3LL lung cancer cells. When tumors were established (70 mm 3 ), treatment was initiated by intraperitoneal injection up to 6 times 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). Tumor growth was monitored three times a week 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 CD137 and PDL1 parental antibodies and much more effective than only one of the two parental antibodies. 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] Also, the TYF02 trispecific antibody was selected to test in vivo efficacy using a 3LL syngeneic mouse tumor model. C57BL / 6 mice were subcutaneously implanted with 2×10 6 3LL lung cancer cells. When tumors were established (65 mm 3) Treatment was initiated by intraperitoneal injection 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) up to a maximum of 5 doses. Tumor growth was monitored every 2 days and reported as mean tumor volume ± SEM over time. As shown in Figure 15C, this trispecific antibody demonstrated superior tumor growth inhibition compared to the corresponding bispecific antibodies or the combination of the parental antibodies of the three mono-IgGs.

Table 13

[0272] Example 7. Biophysical Characterization of Heterodimeric HER2×CD3 T Cell-Inducing Bispecific Antibodies A heterodimeric bispecific scaffold was designed using the TYM13 Fc variant. A bispecific antibody with a TYM13 mutation in the heterologous Fc domain was formed by combining a light-chain–heavy-chain half-antibody and an scFv-Fc chain (Figure 4). This scaffold was used to construct a HER2xCD3 bispecific T cell-inducing antibody. For comparison, the corresponding antibodies with the knob-into-hole mutations Y394C, T366S, L368A, Y407V-S354’C T366’W, and the “Xencor mutations” E357Q, S364K-L368’D, K370’S were also constructed.

[0273] Plasmids encoding the heavy chain, light chain, and scFv-Fc chain of the bispecific antibody were transiently transfected into mammalian cells. Seven days after transfection, the cell culture supernatant containing the bispecific antibody was recovered 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), and subsequently buffer-exchanged in 20 mM histidine (pH 5.5) buffer.

[0274] The biophysical purity of the heterodimeric bispecific antibody was evaluated by SEC-HPLC and SDS-PAGE. As shown in Figures 16 and 17, TY24051 with the TYM13 mutation showed very good heterodimer purity without detectable homodimers, while TY24105 and TY24106 with the knob-into-hole and Xencor mutations both contained a homodimer of approximately 150 kDa (shown in the SDS-PAGE and SEC-HPLC graphs of Figures 16 and 17, respectively). Furthermore, TY24051 contained fewer aggregates than TY24105 and TY24106.

[0275] When TY24051 was converted to TY24052, an activatable antibody, some aggregates were generated (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 An activatable HER2×CD3 bispecific antibody (also referred to herein as "SAFE body" or "SAFE bispecific") was constructed (Figure 5). The constructs are shown in Tables 10 and 11. [Table 15]

Table 16

[0277] The affinity of the bispecific antibody (TY24051) and its SAFE body version (TY24052) was analyzed by an enzyme-linked immunosorbent assay (ELISA). Human HER2 or CD3 (ε and δ chain heterodimer) fused with the human Fc fragment at 2 μg / mL was prepared 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 plates were washed three times and then incubated with 50 μL / well of TMB substrate at room temperature for approximately 20 minutes. After the reaction was stopped, the absorbance at 450 nm was measured. The data were analyzed using GraphPad Prism 6 with nonlinear fitting. As shown in FIGS. 18A-18B, TY24051 bound to both HER2 and CD3, while TY24052 showed a significantly lower affinity than TY24051. After activation, the affinity of TY24052 was fully restored.

[0278] To compare the functional activities of TY24051 and TY24052, the antibodies were expressed, purified, and evaluated for antigen-dependent bispecific antibody-mediated tumor cell killing activity (FIG. 19). In an 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 while increasing the amount of the bispecific antibody (target cells: 1x104 cells / well, E:T = 10:1). As shown in FIG. 19, dose-dependent killing was observed for both TY24051 and TY24052, and TY24052 showed an approximately 800-fold increase in EC50 compared to TY24051. No specific killing was observed with the isotype control. FC1 represents the first polypeptide chain in a heterodimeric protein such as a multispecific antibody, and FC2 represents the second polypeptide chain. H1 or HC1 refers to the first heavy chain of the antibody, H2 or HC2 refers to the second heavy chain of the antibody, L1 or LC1 refers to the first light chain of the antibody, and L2 or LC2 refers to the second light chain of the antibody. The above annotations also apply to the sequences in 695402001040SEQLIST.txt. >SEQ ID NO: 1 - IgG1_FC1_CH3_T366S,L368A,Y407V,N390C GQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENCYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK > SEQ ID NO: 2 - IgG1_FC2_ CH3_T366’W,S400’C GQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDCDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >SEQ ID NO: 3 - IgG1_FC1_ CH3_T366S,L368A,Y407V,S400C GQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDCDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >SEQ ID NO: 4 - IgG1_FC2_ CH3_T366’W,N390’C GQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENCYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >SEQ ID NO: 5 - IgG1_FC1_ CH3_L368V,Y407V,N390C GQPREPQVYTLPPSRDELTKNQVSLTCVVKGFYPSDIAVEWESNGQPENCYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >SEQ ID NO: 6 - IgG1_FC2_ CH3_T366’W,S400’C GQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDCDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >SEQ ID NO: 7 - IgG1_FC1_ CH3_L368V,Y407V,S400C GQPREPQVYTLPPSRDELTKNQVSLTCVVKGFYPSDIAVEWESNGQPENNYKTTPPVLDCDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >SEQ ID NO: 8 - IgG1_FC2_ CH3_T366’W,N390’C GQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENCYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >SEQ ID NO: 9 - IgG1_FC1_ CH3_E357K:T411K GQPREPQVYTLPPSRDKLTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLKVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >SEQ ID NO: 10 - IgG1_FC2_ CH3_L351’D:K370’D GQPREPQVYTDPPSRDELTKNQVSLTCLVDGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >SEQ ID NO: 11 - IgG1_FC1_ CH3_E357K:S364K GQPREPQVYTLPPSRDKLTKNQVKLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >SEQ ID NO: 12 - IgG1_FC2_ CH3_L351’D:K370’D GQPREPQVYTDPPSRDELTKNQVSLTCLVDGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >SEQ ID NO: 13 - IgG1_FC1_ CH3_D356K:E357K:S364K GQPREPQVYTLPPSRKKLTKNQVKLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >SEQ ID NO: 14 - IgG1_FC2_ CH3_L351’D:K370’D:K439’D GQPREPQVYTDPPSRDELTKNQVSLTCLVDGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQDSLSLSPGK >SEQ ID NO: 15 - IgG1_FC1_ CH3_E357K:S364K:N390C GQPREPQVYTLPPSRDKLTKNQVKLTCLVKGFYPSDIAVEWESNGQPENCYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >SEQ ID NO: 16 - IgG1_FC2_ CH3_L351’D:K370’D:S400’C GQPREPQVYTDPPSRDELTKNQVSLTCLVDGFYPSDIAVEWESNGQPENNYKTTPPVLDCDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >SEQ ID NO: 17 - IgG1_FC1_ CH3_E357K:S364K:S400C GQPREPQVYTLPPSRDKLTKNQVKLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDCDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK > SEQ ID NO: 18 - IgG1_FC2_ CH3_L351’D:K370’D:N390’C GQPREPQVYTDPPSRDELTKNQVSLTCLVDGFYPSDIAVEWESNGQPENCYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >SEQ ID NO: 19 - IgG1_FC1_ CH3_D356K:E357K:S364K:N390C GQPREPQVYTLPPSRKKLTKNQVKLTCLVKGFYPSDIAVEWESNGQPENCYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >SEQ ID NO: 20 - IgG1_FC2_ CH3_L351’D:K370’D:S400’C:K439’D GQPREPQVYTDPPSRDELTKNQVSLTCLVDGFYPSDIAVEWESNGQPENNYKTTPPVLDCDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQDSLSLSPGK >SEQ ID NO: 21 - IgG1_FC1_ CH3_D356K:E357K:S364K:S400C GQPREPQVYTLPPSRKKLTKNQVKLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDCDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >SEQ ID NO: 22 - IgG1_FC2_ CH3_L351’D:K370’D:N390’C:K439’D GQPREPQVYTDPPSRDELTKNQVSLTCLVDGFYPSDIAVEWESNGQPENCYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQDSLSLSPGK >SEQ ID NO: 23 - IgG1_FC1_CH3_S400C GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDCDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK > SEQ ID NO: 24- IgG1_FC2_CH3_N390’C GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENCYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >SEQ ID NO: 25-IgG1_FC1_CH3_K392C GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYCTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >SEQ ID NO: 26-IgG1_FC2_CH3_V397’C GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPCLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >SEQ ID NO: 27- IgG1_FC1_CH3_K392C GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYCTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >SEQ ID NO: 28 - IgG1_FC2_CH3_S400’C GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDCDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >SEQ ID NO: 29 - IgG1_WT_CH3_356D,358L GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >SEQ ID NO: 30 - IgG1_WT_CH3_356E,358M GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >SEQ ID NO: 31 - IgG4_WT_CH3 GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK >SEQ ID NO: 32-IgG1_WT 356D,358L ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >SEQ ID NO: 33-IgG1_WT 356E,358M ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >SEQ ID NO: 34-IgG4_WT ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK >SEQ ID NO: 84 - TY22121_LC1|aa DIQLTQSPSSLSASVGDRVTITCRASQSIPSFLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHYISWPRQFTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >SEQ ID NO: 85 - TY22121_HC1|aa EVQLVESGGGLVQPGGSLRLSCAASGYSISSGYYWGWIRQAPGKGLEWIGIIYPSGGGTNYAQKFQGRVTISRDNSKNTLYLQLNSLRAEDTAVYYCARGGGLGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFSLSTGGVGVGWIRQAPGKCLEWLALIDWADDKYYSPSLKSRLTISRDNSKNTLYLQLNSLRAEDTAVYYCARGGSDTVIGDWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVTITCRASQSIGSYLAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGYYLWTFGCGTKVEIKR >SEQ ID NO: 86 - TY22148_LC1|aa DIQLTQSPSSLSASVGDRVTITCRASQSIPSFLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHYISWPRQFTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC > SEQ ID NO: 87 - TY22148_HC1|aa EVQLVESGGGLVQPGGSLRLSCAASGYSISSGYYWGWIRQAPGKGLEWIGIIYPSGGGTNYAQKFQGRVTISRDNSKNTLYLQLNSLRAEDTAVYYCARGGGLGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENCYKTTPPVLDCDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFSLSTGGVGVGWIRQAPGKCLEWLALIDWADDKYYSPSLKSRLTISRDNSKNTLYLQLNSLRAEDTAVYYCARGGSDTVIGDWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVTITCRASQSIGSYLAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGYYLWTFGCGTKVEIKR >SEQ ID NO:88 - TY22172_LC1|aa DIQLTQSPSSLSASVGDRVTITCRASQSIPSFLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHYISWPRQFTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >SEQ ID NO: 89 - TY22172_HC1|aa EVQLVESGGGLVQPGGSLRLSCAASGYSISSGYYWGWIRQAPGKGLEWIGIIYPSGGGTNYAQKFQGRVTISRDNSKNTLYLQLNSLRAEDTAVYYCARGGGLGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFSLSTGGVGVGWIRQAPGKCLEWLALIDWADDKYYSPSLKSRLTISRDNSKNTLYLQLNSLRAEDTAVYYCARGGSDTVIGDWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVTITCRASQSIGSYLAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGYYLWTFGCGTKVEIKR >SEQ ID NO: 90 - TY22176_LC1|aa DIQLTQSPSSLSASVGDRVTITCRASQSIPSFLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHYISWPRQFTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC > SEQ ID NO: 91 - TY22176_HC1|aa EVQLVESGGGLVQPGGSLRLSCAASGYSISSGYYWGWIRQAPGKGLEWIGIIYPSGGGTNYAQKFQGRVTISRDNSKNTLYLQLNSLRAEDTAVYYCARGGGLGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENCYKTTPPVLDCDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFSLSTGGVGVGWIRQAPGKCLEWLALIDWADDKYYSPSLKSRLTISRDNSKNTLYLQLNSLRAEDTAVYYCARGGSDTVIGDWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVTITCRASQSIGSYLAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGYYLWTFGCGTKVEIKR >SEQ ID NO: 92 - TY22161_LC1|aa DIQLTQSPSSLSASVGDRVTITCRASQSIPSFLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHYISWPRQFTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC > SEQ ID NO: 93 - TY22161_HC1|aa EVQLVESGGGLVQPGGSLRLSCAASGYSISSGYYWGWIRQAPGKGLEWIGIIYPSGGGTNYAQKFQGRVTISRDNSKNTLYLQLNSLRAEDTAVYYCARGGGLGFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFSLSTGGVGVGWIRQAPGKCLEWLALIDWADDKYYSPSLKSRLTISRDNSKNTLYLQLNSLRAEDTAVYYCARGGSDTVIGDWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVTITCRASQSIGSYLAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGYYLWTFGCGTKVEIKR >SEQ ID NO: 94 - TY22165_LC1|aa DIQLTQSPSSLSASVGDRVTITCRASQSIPSFLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHYISWPRQFTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC > SEQ ID NO: 95 - TY22165_HC1|aa EVQLVESGGGLVQPGGSLRLSCAASGYSISSGYYWGWIRQAPGKGLEWIGIIYPSGGGTNYAQKFQGRVTISRDNSKNTLYLQLNSLRAEDTAVYYCARGGGLGFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENCYKTTPPVLDCDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFSLSTGGVGVGWIRQAPGKCLEWLALIDWADDKYYSPSLKSRLTISRDNSKNTLYLQLNSLRAEDTAVYYCARGGSDTVIGDWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVTITCRASQSIGSYLAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGYYLWTFGCGTKVEIKR >SEQ ID NO: 96 - TY22122_LC1|aa DIQLTQSPSSLSASVGDRVTITCRASQSIGSYLAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGYYLWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >SEQ ID NO: 97 - TY22122_HC1|aa EVQLVESGGGLVQPGGSLRLSCAASGFSLSTGGVGVGWIRQAPGKGLEWLALIDWADDKYYSPSLKSRLTISRDNSKNTLYLQLNSLRAEDTAVYYCARGGSDTVIGDWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSISSGYYWGWIRQAPGKCLEWIGIIYPSGGGTNYAQKFQGRVTISRDNSKNTLYLQLNSLRAEDTAVYYCARGGGLGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVTITCRASQSIPSFLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHYISWPRQFTFGCGTKVEIKR >SEQ ID NO: 98 - TY22149_LC1|aa DIQLTQSPSSLSASVGDRVTITCRASQSIGSYLAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGYYLWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >SEQ ID NO:99 - TY22149_HC1|aa EVQLVESGGGLVQPGGSLRLSCAASGFSLSTGGVGVGWIRQAPGKGLEWLALIDWADDKYYSPSLKSRLTISRDNSKNTLYLQLNSLRAEDTAVYYCARGGSDTVIGDWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENCYKTTPPVLDCDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSISSGYYWGWIRQAPGKCLEWIGIIYPSGGGTNYAQKFQGRVTISRDNSKNTLYLQLNSLRAEDTAVYYCARGGGLGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVTITCRASQSIPSFLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHYISWPRQFTFGCGTKVEIKR >SEQ ID NO: 100 - TY22173_LC1|aa DIQLTQSPSSLSASVGDRVTITCRASQSIGSYLAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGYYLWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC > SEQ ID NO: 101 - TY22173_HC1|aa EVQLVESGGGLVQPGGSLRLSCAASGFSLSTGGVGVGWIRQAPGKGLEWLALIDWADDKYYSPSLKSRLTISRDNSKNTLYLQLNSLRAEDTAVYYCARGGSDTVIGDWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSISSGYYWGWIRQAPGKCLEWIGIIYPSGGGTNYAQKFQGRVTISRDNSKNTLYLQLNSLRAEDTAVYYCARGGGLGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVTITCRASQSIPSFLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHYISWPRQFTFGCGTKVEIKR >SEQ ID NO: 102 - TY22177_L1|aa DIQLTQSPSSLSASVGDRVTITCRASQSIGSYLAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGYYLWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >SEQ ID NO: 103 - TY22177_H1|aa EVQLVESGGGLVQPGGSLRLSCAASGFSLSTGGVGVGWIRQAPGKGLEWLALIDWADDKYYSPSLKSRLTISRDNSKNTLYLQLNSLRAEDTAVYYCARGGSDTVIGDWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENCYKTTPPVLDCDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSISSGYYWGWIRQAPGKCLEWIGIIYPSGGGTNYAQKFQGRVTISRDNSKNTLYLQLNSLRAEDTAVYYCARGGGLGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVTITCRASQSIPSFLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHYISWPRQFTFGCGTKVEIKR >SEQ ID NO: 104 - TY22359_LC1|aa DIQLTQSPSSLSASVGDRVTITCRASQSIGSYLAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGYYLWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC > SEQ ID NO: 105 - TY22359_HC1|aa EVQLVESGGGLVQPGGSLRLSCAASGFSLSTGGVGVGWIRQAPGKGLEWLALIDWADDKYYSPSLKSRLTISRDNSKNTLYLQLNSLRAEDTAVYYCARGGSDTVIGDWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENCYKTTPPVLDCDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSISSGYYWGWIRQAPGKCLEWIGIIYPSGGGTNYAQKFQGRVTISRDNSKNTLYLQLNSLRAEDTAVYYCARGGGLGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVTITCRASQSIPSFLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHYISWPRQFTFGCGTKVEIKR >SEQ ID NO: 106 - TY22162_LC1 |aa DIQLTQSPSSLSASVGDRVTITCRASQSIGSYLAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGYYLWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >SEQ ID NO: 107 - TY22162_HC1|aa EVQLVESGGGLVQPGGSLRLSCAASGFSLSTGGVGVGWIRQAPGKGLEWLALIDWADDKYYSPSLKSRLTISRDNSKNTLYLQLNSLRAEDTAVYYCARGGSDTVIGDWFAYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSISSGYYWGWIRQAPGKCLEWIGIIYPSGGGTNYAQKFQGRVTISRDNSKNTLYLQLNSLRAEDTAVYYCARGGGLGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVTITCRASQSIPSFLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHYISWPRQFTFGCGTKVEIKR >SEQ ID NO: 108 - TY22166_L1|aa DIQLTQSPSSLSASVGDRVTITCRASQSIGSYLAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGYYLWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC > SEQ ID NO: 109 - TY22166_H1|aa EVQLVESGGGLVQPGGSLRLSCAASGFSLSTGGVGVGWIRQAPGKGLEWLALIDWADDKYYSPSLKSRLTISRDNSKNTLYLQLNSLRAEDTAVYYCARGGSDTVIGDWFAYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENCYKTTPPVLDCDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSISSGYYWGWIRQAPGKCLEWIGIIYPSGGGTNYAQKFQGRVTISRDNSKNTLYLQLNSLRAEDTAVYYCARGGGLGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVTITCRASQSIPSFLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHYISWPRQFTFGCGTKVEIKR >SEQ ID NO: 110 - TY22362_LC1|aa DIQLTQSPSSLSASVGDRVTITCRASQSIGSYLAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGYYLWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >SEQ ID NO: 111 - TY22362_HC1|aa EVQLVESGGGLVQPGGSLRLSCAASGFSLSTGGVGVGWIRQAPGKGLEWLALIDWADDKYYSPSLKSRLTISRDNSKNTLYLQLNSLRAEDTAVYYCARGGSDTVIGDWFAYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENCYKTTPPVLDCDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGSGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSISSGYYWGWIRQAPGKCLEWIGIIYPSGGGTNYAQKFQGRVTISRDNSKNTLYLQLNSLRAEDTAVYYCARGGGLGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVTITCRASQSIPSFLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHYISWPRQFTFGCGTKVEIKR >SEQ ID NO: 112 TY24051_LC1|aa DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >SEQ ID NO: 113 - TY24051_HC1|aa EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRKKLTKNQVKLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDCDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >SEQ ID NO: 114 - TY24051_HC2|aa QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLTVLRGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVGRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTDPPSRDELTKNQVSLTCLVDGFYPSDIAVEWESNGQPENCYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQDSLSLSPGK >SEQ ID NO: 115 - TY24052_LC1|aa ESDACDADPFDCQAPLGLAGSGGSDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >SEQ ID NO: 116 - TY24052_HC1|aa EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRKKLTKNQVKLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDCDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >SEQ ID NO: 117 - TY24052_HC2|aa EVGSYPYDDPDCPSHDSDCDNSGRSAGGGGTPLGLAGSGGSQAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLTVLRGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVGRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTDPPSRDELTKNQVSLTCLVDGFYPSDIAVEWESNGQPENCYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQDSLSLSPGK >SEQ ID NO: 118 - TY22224_LC1|aa DIQLTQSPSSLSASVGDRVTITCRASQSIGSYLAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGYYLWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >SEQ ID NO: 119 - TY22224_HC1|aa EVQLVESGGGLVQPGGSLRLSCAASGFSLSTGGVGVGWIRQAPGKGLEWLALIDWADDKYYSPSLKSRLTISRDNSKNTLYLQLNSLRAEDTAVYYCARGGSDTVIGDWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREKMTKNQVKLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDCDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSISSGYYWGWIRQAPGKCLEWIGIIYPSGGGTNYAQKFQGRVTISRDNSKNTLYLQLNSLRAEDTAVYYCARGGGLGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVTITCRASQSIPSFLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHYISWPRQFTFGCGTKVEIKR >SEQ ID NO: 120 - TY22224_HC2|aa EVQLVESGGGLVQPGGSLRLSCAASGFSLSTGGVGVGWIRQAPGKGLEWLALIDWADDKYYSPSLKSRLTISRDNSKNTLYLQLNSLRAEDTAVYYCARGGSDTVIGDWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTDPPSREEMTKNQVSLTCLVDGFYPSDIAVEWESNGQPENCYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSISSGYHWSWIRQAPGKCLEWLARIDWDDDKYYSTSLKSRLTISRDNSKNTLYLQLNSLRAEDTAVYYCARSYVYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVTITCRASQSVRGRFLAWYQQKPGKAPKLLIYDASNRATGIPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSSSWPPTFGCGTKVEIKR >SEQ ID NO: 121 - TY22225_LC1|aa DIQLTQSPSSLSASVGDRVTITCRASQSIGSYLAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGYYLWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC > SEQ ID NO: 122 - TY22225_HC1|aa EVQLVESGGGLVQPGGSLRLSCAASGFSLSTGGVGVGWIRQAPGKGLEWLALIDWADDKYYSPSLKSRLTISRDNSKNTLYLQLNSLRAEDTAVYYCARGGSDTVIGDWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREKMTKNQVKLTCLVKGFYPSDIAVEWESNGQPENCYKTTPPVLDCDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSISSGYYWGWIRQAPGKCLEWIGIIYPSGGGTNYAQKFQGRVTISRDNSKNTLYLQLNSLRAEDTAVYYCARGGGLGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVTITCRASQSIPSFLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHYISWPRQFTFGCGTKVEIKR > SEQ ID NO: 123 - TY22225_HC2|aa EVQLVESGGGLVQPGGSLRLSCAASGFSLSTGGVGVGWIRQAPGKGLEWLALIDWADDKYY...

Claims

**Claim 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, wherein the first polypeptide and the second polypeptide each comprise, from the N-terminus to the C-terminus, at least a part of an immunoglobulin hinge region, an immunoglobulin heavy chain constant domain 2 (CH2 domain), and a CH3 domain, and the CH2 domain and the CH3 domain form an IgG Fc region, wherein the first CH3 domain contains a cysteine (C) residue at position 390, and the second CH3 domain contains a cysteine residue at position 400, or the first CH3 domain contains a cysteine residue at position 400, and the second CH3 domain contains a cysteine residue at position 390, and the amino acid residue numbering is based on EU numbering, a heterodimeric protein. **Claim 2** The heterodimeric protein according to claim 1, wherein 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. **Claim 3** i) the first CH3 domain further contains a positively charged residue at position 357, and the second CH3 domain further contains a negatively charged residue at position 351, or the first CH3 domain further contains a negatively charged residue at position 351, and the second CH3 domain further contains a positively charged residue at position 357; or ii) the first CH3 domain further contains a positively charged residue at position 411, and the second CH3 domain further contains a negatively charged residue at position 370, or the first CH3 domain further contains a negatively charged residue at position 370, and the second CH3 domain further contains a positively charged residue at position 411; or iii) the first CH3 domain further contains a positively charged residue at position 364, and the second CH3 domain further contains a negatively charged residue at position 370, or the first CH3 domain further contains a negatively charged residue at position 370, and the second CH3 domain further contains 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, The heterodimer protein according to claim 1.

4. wherein the first CH3 domain further contains a positively charged residue at position 356 and the second CH3 domain further contains a negatively charged residue at position 439, or the first CH3 domain further contains a negatively charged residue at position 439 and the second CH3 domain further contains a positively charged residue at position 356; wherein the amino acid residue numbering is based on EU numbering, The heterodimer protein according to any one of claims 1 to 3.

5. 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 heterodimer protein according to claim 4.

6. i) the first CH3 domain contains E357K and T411K substitutions, and the second CH3 domain contains L351D and K370D substitutions, or the first CH3 domain contains L351D and K370D substitutions, and the second CH3 domain contains E357K and T411K substitutions; or ii) the first CH3 domain contains E357K and S364K substitutions, and the second CH3 domain contains L351D and K370D substitutions, or the first CH3 domain contains L351D and K370D substitutions, and the second CH3 domain contains E357K and S364K substitutions; or iii) the first CH3 domain contains D356K, E357K, and S364K substitutions, and the second CH3 domain contains L351D, K370D, and K439D substitutions, or the first CH3 domain contains L351D, K370D, and K439D substitutions, and the second CH3 domain contains D356K, E357K, and S364K substitutions, The heterodimer protein according to claim 5.

7. 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 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 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 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; The heterodimer protein according to any one of claims 1 to 3. Claim 8 i) the first CH3 domain comprises E357K, S364K, and N390C substitutions, and the second CH3 domain comprises L351D, K370D, and S400C substitutions, or the first CH3 domain comprises L351D, K370D, and S400C substitutions, and the second CH3 domain comprises E357K, S364K, and N390C substitutions; 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, The heterodimeric protein according to claim 6.

9. The heterodimeric protein according to any one of claims 1 to 3, wherein the first CH3 domain and the second CH3 domain further comprise knob-into-hole residues.

10. i) the first CH3 domain comprises T366S, 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 T366S, L368A, and Y407V substitutions; or ii) the first CH3 domain contains L368V and Y407V substitutions, and the second CH3 domain contains a T366W substitution, or the first CH3 domain contains a T366W substitution and the second CH3 domain contains L368V and Y407V substitutions, The heterodimeric protein according to claim 9. **Claim 11** The heterodimeric protein according to any one of claims 1 to 3, wherein the first CH3 domain and the second CH3 domain are human CH3 domains. **Claim 12** The heterodimeric protein according to any one of claims 1 to 3, wherein the Fc region is of the human IgG1 subclass. **Claim 13** The heterodimeric protein according to any one of claims 1 to 3, wherein the Fc region is of the human IgG4 subclass. **Claim 14** The heterodimeric protein according to claim 13, wherein the Fc region further contains an S228P substitution. **Claim 15** The heterodimeric protein according to claim 12, wherein the Fc region further contains an N297A substitution. **Claim 16** The heterodimeric protein according to any one of claims 1 to 3, wherein the first polypeptide and the second polypeptide are antibody heavy chains, and the heterodimeric protein further comprises one or more antibody light chains. **Claim 17** The heterodimeric protein according to claim 16, wherein the heterodimeric protein is a multispecific antibody. **Claim 18** Further comprising a third polypeptide and a fourth polypeptide, (i) the first polypeptide contains a structure represented by the following formula: VH1-CH1-hinge-CH2-first CH3-L1-scFv1(Ia), (ii) the second polypeptide contains a structure represented by the following formula: VH2-CH1-hinge-CH2-second CH3-L2-scFv2(IIa), (iii) the third polypeptide contains a structure represented by the following formula: and VL1-CL(Ib), (iv) the fourth polypeptide contains 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 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 the second single-chain variable fragment, CL is the immunoglobulin light chain constant domain, CH1 is the immunoglobulin heavy chain constant domain 1, CH2 is the immunoglobulin heavy chain constant domain 2, the hinge is the immunoglobulin hinge region connecting the CH1 and CH2 domains, L1 and L2 are each independently a linker 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, scFv2 specifically binds to a fourth target, The heterodimeric protein according to claim 17.

19. The heterodimeric protein according to claim 18, wherein scFv1 and scFv2 are identical.

20. The heterodimeric protein according to claim 18, wherein VL1 and VL2 are identical.

21. The heterodimeric protein according to claim 19, wherein the first Fv specifically binds to PDL1, the second Fv specifically binds to CD137, and scFv1 and scFv2 specifically bind to CTLA-4.

22. Furthermore, it comprises a third polypeptide, (i) the first polypeptide comprises a structure represented by the following formula: VH-CH1-hinge-CH2-first CH3 (IIIa), (ii) the second polypeptide comprises a structure represented by the following formula: and scFv-hinge-CH2-second CH3 (IVa), (iii) the third polypeptide comprises a structure represented by the following formula: VL-CL (IIIb), VL is the immunoglobulin light chain variable domain, VH is the immunoglobulin heavy chain variable domain, scFv is the single-chain variable fragment, CL is the immunoglobulin light chain constant domain, CH1 is the immunoglobulin heavy chain constant domain 1, CH2 is the immunoglobulin heavy chain constant domain 2, the hinge is the immunoglobulin hinge region connecting the CH1 and CH2 domains, 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 17.

23. The heterodimer protein according to claim 22, wherein the Fv specifically binds to CD137 and the scFv specifically binds to PDL1.

24. The heterodimer protein is an activatable antibody, and the heterodimer protein comprises a third polypeptide, wherein (i) the first polypeptide comprises a structure represented by the following formula: VH-CH1-hinge-CH2-first CH3 (Va), wherein (ii) the second polypeptide comprises a structure represented by the following formula: and MM1-CM1-scFv-hinge-CH2-second CH3 (Via), wherein (iii) the third polypeptide comprises a structure represented by the following formula: MM2-CM2-VL-CL (IVb), wherein VL is an immunoglobulin light chain variable domain, wherein VH is an immunoglobulin heavy chain variable domain, wherein scFv is a single-chain variable fragment, wherein CL is an immunoglobulin light chain constant domain, wherein CH1 is an immunoglobulin heavy chain constant domain 1, wherein CH2 is an immunoglobulin heavy chain constant domain 2, wherein the hinge is an immunoglobulin hinge region that links the CH1 and CH2 domains, wherein MM1 is a first masking peptide, wherein MM2 is a second masking peptide, wherein CM1 is a first cleavable peptide, wherein CM2 is a second cleavable peptide, wherein VL and VH associate to form a first Fv that specifically binds to a first target, wherein the scFv specifically binds to a second target, wherein when CM1 is not cleaved, MM1 inhibits the binding of the scFv to the second target, wherein when CM2 is not cleaved, MM2 inhibits the binding of the first Fv to the first target. The heterodimer protein according to claim 17.

25. The heterodimer protein according to claim 24, wherein the first target is a tumor antigen and the second target is CD3.

26. The heterodimer protein according to claim 24, wherein MM1 comprises the amino acid sequence of SEQ ID NO:

35.

27. The heterodimer protein according to claim 25, wherein the first target is HER2.

28. The heterodimer protein according to claim 24, wherein MM2 comprises the amino acid sequence of SEQ ID NO:

36. **Claim 29** The heterodimeric protein according to claim 25, wherein the scFv that specifically binds to CD3 comprises a VH comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 61, CDR-H2 having the amino acid sequence of SEQ ID NO: 62, and / or CDR-H3 having the amino acid sequence of SEQ ID NO: 63, and a VL comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 64, CDR-L2 having the amino acid sequence of SEQ ID NO: 65, and / or CDR-L3 having the amino acid sequence of SEQ ID NO:

66. **Claim 30** The heterodimeric protein according to claim 27, wherein the first Fv that specifically binds to HER2 comprises a VH comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 69, CDR-H2 having the amino acid sequence of SEQ ID NO: 70, and / or CDR-H3 having the amino acid sequence of SEQ ID NO: 71, and a VL comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 72, CDR-L2 having the amino acid sequence of SEQ ID NO: 73, and / or CDR-L3 having the amino acid sequence of SEQ ID NO:

74. **Claim 31** The heterodimeric protein according to claim 24, wherein the activatable antibody comprises an Fc region comprising a first CH3 domain and a second CH3 domain, 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. **Claim 32** One or more nucleic acids encoding the heterodimeric protein according to any one of claims 1 to 3. **Claim 33** A vector comprising the one or more nucleic acids according to claim 32. **Claim 34** A host cell comprising the one or more nucleic acids according to claim 32. **Claim 35** A host cell comprising the vector according to claim 33. **Claim 36** A method for preparing a heterodimeric protein, comprising: (a) culturing the host cell according to claim 35 under conditions that allow expression of the one or more nucleic acids or the vector; and (b) recovering the heterodimeric protein from the culture of the host cell. **Claim 37** A pharmaceutical composition comprising the heterodimer protein according to any one of claims 1 to 3 and a pharmaceutically acceptable carrier.

38. A medicament for treating a disease or condition, comprising the heterodimer protein according to any one of claims 1 to 3 or the pharmaceutical composition according to claim 37.

39. The medicament according to claim 38, wherein the disease or condition is cancer.

40. The medicament according to claim 39, wherein the cancer is lung cancer.

41. The medicament according to claim 39, wherein the cancer is ovarian cancer.

Images