Heterodimeric proteins with FC mutations

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 of related applications This application claims priority to International Application PCT / CN2020 / 073960, filed on 23 January 2020, which is incorporated herein by reference in its entirety.

[0002] Field of Invention This application relates to heterodimer proteins (e.g., bispecific antibodies), activatable antibodies, methods for preparing them, and methods for using them.

[0003] Sequence listing reference The following submission in ASCII text file is incorporated herein by reference in its entirety: a computer-readable format (CRF) sequence listing (filename: 695402001041SEQLIST.txt, date: January 21, 2020, size: 656KB). [Background technology]

[0004] Multispecific antibodies can bind to multiple different antigens simultaneously. This property enables the development of therapeutic strategies that are not possible with conventional monoclonal antibodies. One format of multispecific antibodies is a heterodimer protein, for example, an antibody composed of separate chains that bind to different antigens. Such heterodimer multispecific antibodies can accurately target multiple antigens only when assembled with appropriate complements for their monomer components. Therefore, multispecific antibodies that heterodimerize in a specific and stable manner are needed in this field.

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

[0006] All references cited herein, including patent applications, patent publications, non-patent literature, and UniProtKB / Swiss-Prot / GenBank accession numbers, are incorporated herein by reference in whole, as is specifically and individually indicated for each individual reference. [Overview of the Initiative]

[0007] This application provides a heterodimer protein comprising a CH3 domain having manipulated residues that form disulfide bonds and / or salt bridges. Activatable antibodies targeting CD3 and / or HER2 are also provided.

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

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

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

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

[0012] In some embodiments of any one of the heterodimer proteins described above, the first CH3 domain and the second CH3 domain further include knob-into-hole residues. In some embodiments, i) the first CH3 domain includes T336S, L368A, 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 T336S, L368A, and Y407V substitutions; or ii) 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.

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

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

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

[0016] In some embodiments of any one of the heterodimer proteins described above, the first polypeptide and the second polypeptide each include, from the N-terminus to the C-terminus, at least a portion of the immunoglobulin hinge region, an immunoglobulin heavy chain constant domain 2 (CH2 domain), and a CH3 domain. In some embodiments, the CH2 and CH3 domains 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 includes an S228P substitution. In some embodiments, the Fc region further includes an N297A substitution.

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

[0018] In some embodiments using any one of the above heterodimer proteins, the heterodimer 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 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. 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 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 linking the CH1 and CH2 domains; VL and VH associate to form an Fv that specifically binds to a first target; and 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, scFv contains:VH-L-VL from the N-terminus to the C-terminus, where L is a peptide linker. In some embodiments, scFv contains a first cysteine ​​residue at position 44 of VH and a second cysteine ​​residue at position 100 of VL, with the first and second cysteine ​​residues forming a disulfide bond. In some embodiments, scFv is fused to the hinge of the 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 using any one of the heterodimer proteins described above, the heterodimer protein is an activatable antibody, and the heterodimer 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 comprises a structure represented by the following formula: MM2-CM2-VL-CL(IVb), 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 linking 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 the first Fv which specifically binds to the first target; scFv specifically binds to the second target; if CM1 is not cleaved, MM1 inhibits the binding of the first Fv to the first target; if 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 includes the amino acid sequence of SEQ ID NO: 35. In some embodiments, the first Fv includes VH, which includes CDR-H1, which includes the amino acid sequence of SEQ ID NO: 61, CDR-H2, which includes the amino acid sequence of SEQ ID NO: 62, and / or CDR-H3, which includes the amino acid sequence of SEQ ID NO: 63. In some embodiments, TBM includes VL, which includes CDR-L1, which includes the amino acid sequence of SEQ ID NO: 64, CDR-L2, which includes the amino acid sequence of SEQ ID NO: 65, and / or CDR-L3, which includes the amino acid sequence of SEQ ID NO: 66. In some embodiments, the first target is HER2. In some embodiments, MM2 includes the amino acid sequence of SEQ ID NO: 36. In some embodiments, scFv includes VH, which includes CDR-H1, which includes the amino acid sequence of SEQ ID NO: 69, CDR-H2, which includes the amino acid sequence of SEQ ID NO: 70, and / or CDR-H3, which includes the amino acid sequence of SEQ ID NO: 71. In some embodiments, the TBM includes a VL containing CDR-L1 containing the amino acid sequence of SEQ ID NO: 72, CDR-L2 containing the amino acid sequence of SEQ ID NO: 73, and / or CDR-L3 containing the amino acid sequence of SEQ ID NO: 74.

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

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

[0023] In some embodiments using any one of the above-described activatable antibodies, the activatable antibody comprises a first polypeptide, a second polypeptide, and a third polypeptide: (i) The first polypeptide comprises a structure represented by the following formula: VH-CH1-Hinge-CH2-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 comprises a structure represented by the following formula: MM2-CM2-VL-CL(IVb), VL is the variable domain of the immunoglobulin light chain. VH is a variable domain of the immunoglobulin heavy chain, scFv is a single-chain variable fragment, CL is the constant domain of the immunoglobulin light chain. CH1 is the constant domain 1 of the immunoglobulin heavy chain. CH2 is the constant domain 2 of the immunoglobulin heavy chain. The hinge is an 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 the first target; scFv specifically binds to the second target; and MM is either MM1 or MM2.

[0024] In some embodiments using any one of the above-described activatable antibodies, the activatable antibody comprises an Fc region including a first CH3 domain and a second CH3 domain, wherein 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.

[0025] One aspect of this application provides one or more nucleic acids encoding a heterodimer protein by any one of the above-mentioned heterodimer proteins, or an activatable antibody by any one of the above-mentioned activatable antibodies, a vector (may be more than one) 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 heterodimer protein or an activatable antibody is provided, the method comprising (a) culturing a host cell with any one of the above-mentioned host cells under conditions that enable the expression of the one or more nucleic acids or vector; and (b) recovering the heterodimer protein or activatable antibody from the host cell culture.

[0026] One aspect of this application provides a pharmaceutical composition comprising a heterodimer protein comprising any one of the above-mentioned heterodimer proteins, or an activatable antibody comprising any one of the above-mentioned activatable antibodies, and a pharmaceutically acceptable carrier.

[0027] One aspect of this application provides a method for treating a disease or condition in a subject requiring treatment of the disease or condition, the method comprising administering an effective amount of one of the above-described pharmaceutical compositions to the subject. 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-stage cancer.

[0028] Compositions, uses, kits, and products containing any one of the above heterodimer proteins are also provided. [Brief explanation of the drawing]

[0029] [Figure 1] This application provides schematic diagrams of exemplary antibody designs. A shows a schematic diagram of a Fab-Fc / Fc1 arm scaffold. B shows a schematic diagram of a typical light chain scaffold. [Figure 2] This invention provides a schematic diagram of an exemplary antibody design. A schematic diagram of a Morrison-format bispecificity scaffold is shown. Shown on the right are Morrison-format PD-L1 X CD137 and CD137 X PD-L1 bispecificity antibodies. [Figure 3] A schematic diagram of an exemplary antibody design of this application is provided. To the right is a schematic diagram of a triplespecific scaffold containing triplespecific antibodies against PD-L1, CD137, and CTLA4. [Figure 4] This application provides schematic diagrams of exemplary antibody designs. To the right is a schematic diagram of the ScFv bispecific scaffold, including schematic diagrams of HER2 and CD3 bispecific antibodies. [Figure 5] This application provides schematic diagrams of exemplary antibody designs. To the right, a schematic diagram of an activatable scaffold is shown, including schematic diagrams of activatable antibodies against HER2 and CD3. A masking peptide (represented as a ball) can be fused to the antigen-binding domain via a cleavable linker. [Figure 6] A 10% SDS-PAGE gel is provided showing the yield of heterodimer proteins. The three bands, as shown, correspond to light-heavy chain homodimers, light-heavy chain-Fc heterodimers, and light-heavy chain half-bodies. [Figure 7] This provides size-exclusion high-performance liquid chromatography (HCM) data for heterodimer proteins. Time is shown on the x-axis, and relative protein abundance on the y-axis. Stars indicate peaks corresponding to heterodimer proteins. [Figure 8] This provides size exclusion high-performance liquid chromatography (HCM) analysis data showing heterodimer proteins after incubation at the indicated temperatures for 1 hour. The x-axis represents temperature (from left to right: control, 40°C, 50°C, 60°C, 65°C, or 67.5°C), and the y-axis represents the peak area corresponding to the incubated heterodimer proteins compared to the control (unincubated). [Figure 9]As shown, size exclusion high-performance liquid chromatography spectra of heterodimer proteins after storage at 4°C or 37°C for 7, 14, 21, or 28 days are provided. Time is on the x-axis, and relative protein abundance is on the y-axis. [Figure 10] This graph shows the effect of a bispecific antibody in an NFκB-activated luciferase reporter assay. The x-axis represents the antibody concentration, logarithmically transformed in nM, and the y-axis represents the relative luminescence units (RLU) of the luciferase reporter. [Figure 11A] This provides an evaluation of the quality of purified anti-PDL1 and CD137 bispecific antibodies in different formats. Protein quality is shown, assessed by analytical size exclusion chromatography after 3 and 6 freeze-thaw cycles. Time is shown on the x-axis, relative protein abundance on the y-axis, and the format of the bispecific antibody is indicated. [Figure 11B] This provides an evaluation of the quality of purified anti-PDL1 and CD137 bispecific antibodies in different formats. The protein quality is shown as evaluated by analytical size exclusion chromatography after incubation at 40°C for 28 days. Time is shown on the x-axis, relative protein abundance on the y-axis, and the format of the bispecific antibody is indicated. [Figure 11C] This provides quality assessment of purified anti-PDL1 and CD137 bispecific antibodies in different formats. It also provides analysis of the thermal stability of purified anti-PDL1 and CD137 bispecific antibodies in different formats. Antibodies were incubated for 1 hour at the temperatures shown on the x-axis (from left to right: control, 50°C, 60°C, 65°C, and 70°C), and the y-axis shows the percentage of the major peak area. PDL1xCD137 TYF01 is shown as a square, CD137xPDL1 TYF01 as a circle, PDL1xCD137 TYF02 as an upward-pointing triangle, and CD137xPDL1 TYF02 as a downward-pointing triangle. [Figure 12] This report provides flow cytometry analysis of the binding of an anti-PDL1xCD137 bispecific antibody to PDL1 and CD137. The x-axis of each plot shows antigen representation in the APC channel, and the y-axis shows ligand binding in the PE channel. [Figure 13] This provides flow cytometry analysis of the binding and cross-reactivity of anti-PDL1 and CD137 bispecific antibodies. The TYF01 antibody is shown in the upper set of plots, and the TYF02 antibody is shown in the lower set of plots. The x-axis of each plot shows antigen representation in the FITC channel, and the y-axis shows antibody binding in the APC channel. As shown, the ability of bispecific antibodies in different formats to bind to PDL1 or CD137 of mouse, monkey, or human origin was tested. [Figure 14] This provides the effects of PDL1xCD137 and CD137xPDL1 bispecific antibodies on PDL1 and CD137 reporter gene assays. The upper plot shows the PDL1 reporter gene assay, with the x-axis showing logarithmically transformed antibody concentration in ng / ml units and the y-axis showing relative luminescence units ("RUL"). In the PDL1 reporter gene assay plot, squares represent PDL1xCTLA4 bispecific antibodies, downward-pointing triangles represent PDL1 monomers, circles represent PDL1xCD137 bispecific antibodies, and upward-pointing triangles represent CD137xPDL1 bispecific antibodies. The lower plot shows the CD137-NFκB reporter gene assay, with the x-axis showing antibody concentration in nM units and the y-axis showing relative luminescence units ("RLU"). In the CD137-NFκB reporter assay plot, squares represent PDL1xCD137 bispecific antibodies, upward-pointing triangles represent CD137xPDL1 bispecific antibodies, diamonds represent CD137xCTLA4 bispecific antibodies, downward-pointing triangles represent CD137 monomers, and circles represent negative controls. [Figure 15A] This figure shows the effect of anti-PDL1 and / or anti-CD137 antibodies on in vivo tumor growth in a 3LL syngeneic mouse model. The x-axis represents days after the start of treatment, and the y-axis represents tumor volume (mm3). Mono-IgG, bispecific, or triplicate antibodies were tested alone or in combination at the indicated concentrations. [Figure 15B]This figure shows the effect of anti-PDL1 and / or anti-CD137 antibodies on in vivo tumor growth in a 3LL syngeneic mouse model. The x-axis represents days after the start of treatment, and the y-axis represents tumor volume (mm3). Mono-IgG, bispecific, or triplicate antibodies were tested alone or in combination at the indicated concentrations. [Figure 15C] This figure shows the effect of anti-PDL1 and / or anti-CD137 antibodies on in vivo tumor growth in a 3LL syngeneic mouse model. The x-axis represents days after the start of treatment, and the y-axis represents tumor volume (mm3). Mono-IgG, bispecific, or triplicate antibodies were tested alone or in combination at the indicated concentrations. [Figure 16] This provides characterization of bispecific antibodies by SDS-PAGE electrophoresis. The gel on the left is a 12% SDS-PAGE gel under reducing conditions, and the gel on the right is a 4-15% SDS-PAGE gel under non-reducing conditions. The MW lanes show molecular weight markers, which are labeled in kilodaltons on the left side of each gel. In both gels, lane 1 shows antibody TY24051, lane 2 shows antibody TY24052, and lane 3 shows antibody TY24053. [Figure 17] This provides size-exclusion high-performance liquid chromatography (HCM) analysis of bispecific antibodies. The top plot shows the TY24051 antibody, the middle plot shows the TY24105 antibody, and the bottom plot shows the TY24106 antibody. In each plot, time is shown on the x-axis and relative protein abundance on the y-axis. Peaks corresponding to heterodimer proteins and aggregates are shown. [Figure 18] This document provides enzyme-linked immunosorbent assay (ELISA) analysis of antibodies TY24051 and TY24052. A shows the binding of HER2 by TY24051 (square), TY24052 (upward-pointing triangle), and activated TY24052 (downward-pointing triangle). B shows the binding of CD3 by TY24051 (square), TY24052 (upward-pointing triangle), and activated TY24052 (downward-pointing triangle). In A and B, the antibody concentration is shown on the x-axis in units of M, and the absorbance at 450 nm is shown on the y-axis. [Figure 19]This shows an assay for T cell-mediated cytotoxic killing when treated with a bispecific antibody. Antibody concentration (ng / ml) is shown on the x-axis, and the percentage of cell lysis is shown on the y-axis. Target cells were incubated with T cells for 24 hours under the conditions of TY24051 (circle), TY24052 (square), isotype control (upward triangle), or no antibody (downward triangle). [Modes for carrying out the invention]

[0030] This application provides heterodimer proteins comprising CH3 domains having manipulated disulfide bonds(s) and / or salt bridges(s), which include multispecific antibodies, such as bispecific antibodies comprising Fc regions having manipulated disulfide bonds(s) and / or salt bridges(s). In some embodiments, the heterodimer protein comprises manipulated disulfide bonds 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 heterodimer protein includes a rearranged salt bridge network compared to the wild-type CH3 domain, for example, between positions 357 and 411 of the first CH3 domain and positions 351 and 370 of the second CH3 domain (e.g., E357K:T411K-L351'D:K370'D), or between positions 357 and 364 of the first CH3 domain and positions 351 and 370 of the second CH3 domain (e.g., E357K:S364K-L351'D:K370'D). In some embodiments, the heterodimer protein includes an inverted salt bridge between position 356 of the first CH3 domain and position 439 of the second CH3 domain compared to the wild-type CH3 domain (e.g., D356-K439'). The heterodimer proteins described herein provide a platform for preparing multispecific proteins and antibodies in various formats that exhibit high yield, excellent stability (e.g., resistance to aggregation and precipitation due to high temperatures or freeze-thaw cycles), and potent activity.

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

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

[0033] The term "antibody" includes, but is not limited to, fragments capable of binding 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; its name reflects its ability to readily crystallize. Pepsin treatment produces an F(ab')2 fragment having two antigen-binding sites and still capable of crosslinking antigens. The term "antibody" also includes, but is not limited to, chimeric antibodies, humanized antibodies, and antibodies from various species such as mouse, human, and cynomolgus monkey.

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

[0035] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies constituting the population are identical and / or bind to the same epitope. However, this excludes possible variant antibodies, such as those containing naturally occurring mutations or arising during the production of monoclonal antibody preparations, although such variants are generally present in trace amounts. In contrast to polyclonal antibody preparations, which typically contain different antibodies against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is against a single determinant on an antigen. Therefore, the modifier “monoclonal” should not be interpreted as indicating an antibody characteristic such as that obtained from a substantially homogeneous population of antibodies, and should not be interpreted as requiring antibody production by any particular method. For example, monoclonal antibodies used in accordance with the present invention may be produced by a variety of methods, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of a human immunoglobulin locus, and such methods and other exemplary methods for producing monoclonal antibodies are described herein.

[0036] As used herein, the terms “hypervariable region” or “HVR” refer to each region of the antibody variable domain whose sequence is hypervariable. HVRs may form structurally defined loops (“hypervariable loops”). Generally, natural quadruple-chain antibodies have 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 “complementarity-determining regions” (CDRs), the CDRs being the most sequence-variable and / or involved in antigen recognition. Exemplary hypervariable loops are located at amino acid residues 26–32 (L1), 50–52 (L2), 91–96 (L3), 26–32 (H1), 53–55 (H2), and 96–101 (H3). (Chothia and Lesk, J.Mol.Biol.196:901–917 (1987)). Exemplary CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) are located at amino acid residues 24-34 of L1, 50-56 of L2, 89-97 of L3, 31-35B of H1, 50-65 of H2, and 95-102 of H3. (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)). Except for VH CDR1, CDRs generally contain amino acid residues that form a hypervariable loop. CDRs also contain "specificity-determining residues" or "SDRs," which are residues that come into contact with the antigen. SDRs are contained within a region of the CDR called a shortened CDR or a-CDR. Exemplary a-CDRs (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2, and a-CDR-H3) are located at amino acid residues 31-34 of L1, 50-55 of L2, 89-96 of L3, 31-35B of H1, 50-58 of H2, and 95-102 of H3. (See Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)).Unless otherwise specified, HVR residues and other residues (e.g., FR residues) in the variable domain are numbered herein in accordance with Kabat et al., cited above.

[0037] The term "variable region" or "variable domain" refers to a domain in the heavy or light chain of an antibody that is involved in the binding of the antibody to an antigen. The variable domains of the heavy and light chains of native antibodies (VH and VL, respectively) generally have similar structures, and each domain contains four framework regions (FRs) and three hypervariable regions (HVRs), arranged from the amino terminus to the carboxyl terminus in the following order: FR1, HVR1, FR2, HVR2, FR3, HVR3, FR4. (See, for example, Kindt et al. Kuby Immunology, 6th ed., WH Freeman and Co., page 91 (2007)). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a particular antigen may be isolated using VH or VL domains derived from the antigen-binding antibody, respectively, to screen for a library of complementary VL or VH domains. For example, see 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 their variations refer to the numbering system used for the heavy chain constant domain of antibody editing in Edelman, GM et al., Proc. Natl. Acad. USA, 63, 78-85 (1969). The EU numbering of residues can be determined for a given antibody by aligning the "standard" EU numbering sequence with homologous regions of the antibody sequence.

[0039] The Kabat numbering system is generally used to refer to residues within the variable domain (approximately corresponding to light chain residues 1-107 and heavy chain residues 1-113) (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 the shortening or insertion of FR or HVR in the variable domain. For example, the heavy chain variable domain may contain a single amino acid insertion after H2 residue 52 (residue 52a according to Kabat), and a residue inserted after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c according to Kabat). The Kabat numbering of residues can be determined for a given antibody by aligning the "standard" Kabat numbering sequence with homologous regions of the antibody sequence.

[0040] In relation to heterodimer proteins having two CH3 domains, a given amino acid position in the first CH3 domain is denoted as X, and the corresponding amino acid position in the second CH3 domain is denoted as X'. For example, N390C-S400'C refers to a heterodimer protein having a first CH3 domain with the N390C mutation and a second CH3 domain with the S400C mutation. All mutations or substitutions in heterodimer proteins described herein refer to in relation to the naturally occurring CH3 domain of the wild type.

[0041] Unless otherwise indicated, all polypeptide chain formulas described herein list the polypeptide components 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 containing at least three heavy chain constant domains, CH1, CH2, and CH3, as well as a hinge region between CH1 and CH2. Unrestricted heavy chain constant regions include γ, δ, and α. Unrestricted heavy chain constant regions also include ε and μ. Each heavy chain constant region corresponds to an antibody isotype. For example, an antibody containing a γ constant region is an IgG antibody, an antibody containing a δ constant region is an IgD antibody, and an antibody containing an α constant region is an IgA antibody. Furthermore, an antibody containing a μ constant region is an IgM antibody, and an antibody containing an ε constant region is an IgE antibody. Certain isotypes 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; and IgM antibodies include, but are not limited to, IgM1 and IgM2.

[0043] The term "CH2 domain" in the human IgG Fc region typically refers to approximately 231–340 residues 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 glycans are inserted between the two CH2 domains of an intact, native IgG molecule. It is hypothesized that carbohydrates may provide a substitute for domain-domain pairing and help stabilize the CH2 domain. (Burton, Molec. Immunol. 22:161-206 (1985)).

[0044] The term "CH3 domain" refers to the sequence of residues from the C-terminus of the Fc region to the CH2 domain (i.e., approximately amino acid residues 341 to 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, with or without 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 both a heavy chain variable region and a heavy chain constant region, with or without a leader sequence.

[0046] As used herein, the term “light chain steady region” refers to the region containing the light chain steady domain CL. A non-limiting exemplary light chain steady region includes λ and κ.

[0047] As used herein, the term “light chain” refers to a polypeptide that includes at least a light chain variable region, with or without 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 both a light chain variable region and a light chain constant region, with or without a leader sequence.

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

[0049] The terms “binding,” “specifically binding,” or “specific to” refer to measurable and reproducible interactions, such as binding between a target and an antibody, which determine the presence of the target in the presence of a heterogeneous population of molecules, including biomolecules. For example, an antibody that binds to or specifically binds to a target (which may be an epitope) is an antibody that binds to this target with greater affinity, binding strength, more readily, and / or for a longer duration than an antibody that binds to other targets. In some embodiments, the degree of antibody binding to unrelated targets is less than about 10% of the degree of antibody binding to the target (e.g., 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 of different species. In some embodiments, specific binding may include, but does not require, exclusive binding.

[0050] The term "multispecificity," used in conjunction with antibodies, refers to antibodies that possess polyepitope specificity (i.e., they are capable of specifically binding to two, three, or more different epitopes on a given biomolecule, or to two, three, or more different epitopes on a given biomolecule).

[0051] A "affinity-mature" antibody is an antibody that has one or more modifications in one or more hypervariable regions (HVRs) compared to a parent antibody (without such modifications), where such modifications result in an improvement in the antibody's affinity for the antigen. In some examples, affinity-mature antibodies are antibodies that have one or more modifications in one or more complementarity-determining regions (CDRs) compared to a parent antibody (without such modifications), where such modifications result in an improvement in the antibody's affinity 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 originates from a particular source or species, while the remainder of the heavy chain and / or light chain originates 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 the chimeric antibody originate from the first species, and all of the constant regions of the chimeric antibody originate from the 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 is replaced with a corresponding amino acid derived from a human variable region. In some embodiments, the humanized antibody comprises at least one human constant region or a fragment thereof. In some embodiments, the humanized antibody is Fab, (Fab')2, etc.

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

[0055] As used herein, "human antibodies" refers to antibodies produced in humans, antibodies produced in non-human animals containing human immunoglobulin genes, such as XENOMOUSE®, and antibodies selected using in vitro methods such as phage display, and 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 polymers of nucleotides. Such polymers of nucleotides may contain natural and / or non-natural nucleotides, including, but not limited to, DNA, RNA, and PNA. “Nucleic acid sequence” refers to a linear sequence of nucleotides, including a nucleic acid molecule or polynucleotide.

[0057] The terms “polypeptide” and “peptide” are used interchangeably to refer to polymers of amino acid residues and are not limited to the minimum length. Such polymers of amino acid residues may contain native or non-native amino acid residues. Both full-length proteins and their fragments are included in the definition. The term also includes post-expression modifications of polypeptides, e.g., glycosylation, sialylation, acetylation, phosphorylation, etc. Furthermore, “polypeptide” includes modifications to the native sequence, e.g., deletions, additions, and substitutions (generally inherently conserved), as long as the polypeptide maintains the desired activity. These modifications may be planned (e.g., via site-directed mutagenesis) or accidental (e.g., via host mutations), resulting in protein or error upon PCR amplification.

[0058] A polypeptide "variant" means a bioactive polypeptide having at least about 80% amino acid sequence identity with the native polypeptide after aligning the sequence to achieve the maximum percentage of sequence identity (without considering any conservative substitutions as part of sequence identity) and introducing gaps as 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 polypeptide.

[0059] As used herein, “percent (%) amino acid sequence identity” for peptide, polypeptide, or antibody sequences 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 sequence identity) and introducing gaps as necessary. Alignment for determining percent amino acid sequence identity can be achieved in various ways within the scope of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN® (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring the alignment, including any algorithm necessary to achieve maximum alignment over the entire length of the sequences being compared.

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

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

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

[0063] "Host cell" refers to a cell that can or is a recipient of a vector or isolated polynucleotide. The host cell may 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-exclusive mammalian cells include, but are not limited to, NSO cells, PER.C6® cells (Crucell), and 293 and CHO cells, as well as their derivatives, e.g., 293-6E and DG44 cells, respectively. The term "cell" includes primary target cells and their offspring.

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

[0065] The terms “individual” or “subject” are used interchangeably herein to refer to mammals. In some embodiments, methods for treating mammals are provided, including, but not limited to, humans, rodents, monkeys, cats, dogs, horses, cattle, pigs, sheep, goats, mammalian laboratory animals, mammalian livestock, mammalian sports animals, and mammalian pets. In some examples, “individual” or “subject” refers to an individual or subject requiring treatment for a disease or disorder.

[0066] As used herein, “treatment” or “to treat” is an approach to obtain beneficial or desirable outcomes, including clinical outcomes. For the 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 caused by the disease; reducing the severity of the disease; stabilizing the disease (e.g., preventing or delaying disease exacerbation); preventing or delaying the spread of the disease (e.g., metastasis); preventing or delaying disease recurrence; delaying or slowing disease progression; improving the condition; providing (partial or total) remission of the disease; reducing the dosage of one or more other therapeutic agents required to treat the disease; delaying disease progression; improving quality of life; and / or extending survival. Reduction of the pathological outcomes of cancer is also encompassed by “treatment.” The methods of the present invention aim to achieve any one or more of these aspects of treatment.

[0067] The terms “prevent,” and similar terms such as “preventable,” and “preventing,” refer to approaches to prevent, inhibit, or reduce the likelihood of recurrence of a disease or condition, such as cancer. It also refers to delaying the recurrence of a disease or condition, or delaying the recurrence of symptoms of a disease or condition. As used herein, “prevention” and similar terms also include reducing the severity, impact, symptoms, and / or burden of a disease or condition before it recurs.

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

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

[0070] Embodiments of the present invention described herein are understood to include "consisting of" and / or "essentially consisting of" embodiments.

[0071] In this specification, references to values ​​or parameters "about" include (and describe) variations in the value or parameter itself. For example, a statement referring to "about X" includes a statement of "X".

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

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

[0074] As used herein and in the appended claims, the singular forms "a," "an," and "the" refer to multiple subjects unless otherwise explicitly stated in the context.

[0075] As used herein, the terms “and / or,” expressions such as “A and / or B,” are intended to include both A and B; A or B; A (alone); and B (alone). Similarly, as used herein, the terms “and / or,” expressions such as “A, B, and / or C,” are intended to include 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 described in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, for brevity, various features of the Invention described in the context of a single embodiment may also be provided separately or in any preferred subcombination. All combinations of embodiments relating to heterodimer proteins are specifically encompassed by the Invention and are disclosed herein so as to be individually and explicitly disclosed. In addition, all subcombinations of heterodimer proteins enumerated in embodiments describing such variant forms are also specifically encompassed by the Invention and are disclosed herein so as to be individually and explicitly disclosed in this specification.

[0077] II. Heterodimer Proteins This application provides heterodimer proteins comprising a CH3 domain having any one or a combination of manipulated residues, which facilitate heterodimer formation as described in the subsection “CH3 Domain Mutations”. Heteromultimers comprising multiple heterodimers formed by a first polypeptide comprising a first manipulated CH3 domain and a second polypeptide comprising a second manipulated CH3 domain are also contemplated herein.

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

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

[0080] In some embodiments, a heterodimer protein (e.g., a multispecific antibody) is provided comprising a first polypeptide containing a first CH3 domain and a second polypeptide containing a second CH3 domain, i) the first CH3 domain containing a cysteine ​​(C) residue at position 390 and the second CH3 domain containing a cysteine ​​residue at position 400, or the first CH3 domain containing a cysteine ​​residue at position 400 and the second CH3 domain containing a cysteine ​​residue at position 390; or ii) the first CH3 domain The first CH3 domain contains a cysteine ​​residue at position 392, and the second CH3 domain contains a cysteine ​​residue at position 397, or the first CH3 domain contains a cysteine ​​residue at position 397, and the second CH3 domain contains a cysteine ​​residue at position 392; or iii) the first CH3 domain contains a cysteine ​​residue at position 392, 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 392. 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 b) 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 a) and b) or a) and c) and amino acid residue numbering is based on EU numbering.In some embodiments, the first CH3 domain further includes a positively charged residue at position 356, and the second CH3 domain further includes a negatively charged residue at position 439, or the first CH3 domain further includes a negatively charged residue at position 439, and the second CH3 domain further includes 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, IgG2, IgG3, or IgG4 molecule. In some embodiments, the CH3 domain is a human CH3 domain. In some embodiments, the CH3 domain is derived from a human IgG1 molecule.

[0082] In some embodiments, a heterodimer protein (e.g., a multispecific antibody) is provided comprising a first polypeptide containing a first CH3 domain and a second polypeptide containing a second CH3 domain, i) the first CH3 domain containing an N390C substitution and the second CH3 domain containing an S400C substitution, or the first CH3 domain containing an S400C substitution and the second CH3 domain containing an N390C substitution; or ii) the first The CH3 domain of one CH3 domain contains the K392C substitution, and the second CH3 domain contains the V397C substitution, or the first CH3 domain contains the V397C substitution, and the second CH3 domain contains the K392C substitution; or iii) the first CH3 domain contains the K392C substitution, and the second CH3 domain contains the S400C substitution, or the first CH3 domain contains the S400C substitution, and the second CH3 domain contains the K392C substitution.

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

[0084] In some embodiments, a heterodimer protein (e.g., a multispecific antibody) is provided comprising a first polypeptide containing a first CH3 domain and a second polypeptide containing 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 heterodimer protein (e.g., a multispecific antibody) is provided comprising a first polypeptide containing a first CH3 domain and a second polypeptide containing 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 heterodimer protein (e.g., a multispecific antibody) is provided comprising a first polypeptide containing a first CH3 domain and a second polypeptide containing 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 heterodimer protein (e.g., a multispecific antibody) is provided comprising a first polypeptide containing a first CH3 domain and a second polypeptide containing 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 heterodimer protein includes an IgG Fc region containing an engineered CH3 domain. The Fc region may be derived from any preferred Fc subclass, including, but not limited to, the IgG1, IgG2, IgG3, and IgG4 subclasses.

[0089] Tables 1A-1B in the Examples section list exemplary polypeptide sequences (SEQ ID NOs: 1-28) of a CH3 domain (or Fc region) containing the manipulated disulfide bond(s) and / or salt bridge(s) described herein. Other polypeptide sequences of the manipulated CH3 domain or Fc region include SEQ ID NOs: 138-365. Polypeptides containing amino acid sequences selected from the group consisting of SEQ ID NOs: 1-28 and 138-365 are also provided.

[0090] In some embodiments, a heterodimer protein (e.g., a multispecific antibody) is provided, comprising a first polypeptide containing the amino acid sequence of SEQ ID NO: 1 and a second polypeptide containing the amino acid sequence of SEQ ID NO: 2. In some embodiments, a heterodimer protein (e.g., a multispecific antibody) is provided, comprising a first polypeptide containing the amino acid sequence of SEQ ID NO: 3 and a second polypeptide containing the amino acid sequence of SEQ ID NO: 4. In some embodiments, a heterodimer protein (e.g., a multispecific antibody) is provided, comprising a first polypeptide containing the amino acid sequence of SEQ ID NO: 5 and a second polypeptide containing the amino acid sequence of SEQ ID NO: 6. In some embodiments, a heterodimer protein (e.g., a multispecific antibody) is provided, comprising a first polypeptide containing the amino acid sequence of SEQ ID NO: 7 and a second polypeptide containing the amino acid sequence of SEQ ID NO: 8. In some embodiments, a heterodimer protein (e.g., a multispecific antibody) is provided, comprising a first polypeptide containing the amino acid sequence of SEQ ID NO: 9 and a second polypeptide containing the amino acid sequence of SEQ ID NO: 10. In some embodiments, a heterodimer protein (e.g., a multispecific antibody) is provided, comprising a first polypeptide containing the amino acid sequence of SEQ ID NO: 11 and a second polypeptide containing the amino acid sequence of SEQ ID NO: 12. In some embodiments, a heterodimer protein (e.g., a multispecific antibody) is provided comprising a first polypeptide containing the amino acid sequence of SEQ ID NO: 13 and a second polypeptide containing the amino acid sequence of SEQ ID NO: 14. In some embodiments, a heterodimer protein (e.g., a multispecific antibody) is provided comprising a first polypeptide containing the amino acid sequence of SEQ ID NO: 15 and a second polypeptide containing the amino acid sequence of SEQ ID NO: 16. In some embodiments, a heterodimer protein (e.g., a multispecific antibody) is provided comprising a first polypeptide containing the amino acid sequence of SEQ ID NO: 17 and a second polypeptide containing the amino acid sequence of SEQ ID NO: 18. In some embodiments, a heterodimer protein (e.g., a multispecific antibody) is provided comprising a first polypeptide containing the amino acid sequence of SEQ ID NO: 19 and a second polypeptide containing the amino acid sequence of SEQ ID NO: 20.In some embodiments, a heterodimer protein (e.g., a multispecific antibody) is provided comprising 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 heterodimer protein (e.g., a multispecific antibody) is provided comprising 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 heterodimer proteins described herein may have one or more manipulated disulfide bonds, one or more manipulated (e.g., rearranged or inverted) salt bridges, or a combination thereof. Unless otherwise stated, all amino acid residue numbering herein is based on EU numbering, and amino acid substitutions are relative to the wild-type (or naturally occurring) sequence at the corresponding amino acid position in the wild-type (or naturally occurring) CH3 domain sequence. It is understood that the mutations or substitutions described herein are applicable to all IgG subclasses and allotypes. IgG allotypes are described, for example, in Jefferis R. and Lefranc M. mAbs 1:4,1-7 (2009), which is incorporated herein by reference in whole. In some embodiments, the amino acid mutations or substitutions described herein are relative to the wild-type CH3 domain sequence of IgG1, e.g., IgG1 allotype G1m, 1(a), 2(x), 3(f), or 17(z). In some embodiments, the amino acid mutations or substitutions described herein relate to the wild-type CH3 domain sequence of IgG4. For example, a D356K substitution in the wild-type CH3 domain of one human IgG1 allotype (Uniprot ID P01857; SEQ ID NO: 29) is equivalent to an E356K substitution in the wild-type CH3 domain of a second human IgG1 allotype (SEQ ID NO: 30) or in the wild-type CH3 domain of human IgG4 (SEQ ID NO: 31). Exemplary CH3 domain mutations are shown in Tables 1A-1B. In some embodiments, the amino acid mutations or substitutions described herein are in relation to wild-type Fc region sequences, 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 heterodimer protein described herein comprises a first polypeptide comprising a first CH3 domain and a second polypeptide comprising a second CH3 domain, wherein the first CH3 domain comprises a first manipulated cysteine ​​residue, and the second CH3 domain comprises a second manipulated cysteine ​​residue, and the first manipulated cysteine ​​residue and the second cysteine ​​residue form a disulfide bond.

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

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

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

[0096] Novel salt bridge mutation In some embodiments, the heterodimer protein described herein comprises a first polypeptide containing a first CH3 domain and a second polypeptide containing a second CH3 domain, wherein the first CH3 domain contains an engineered positively charged residue, and the second CH3 domain contains an engineered uncharged residue, and the engineered positively charged residue and the engineered uncharged residue form a salt bridge. The engineered salt bridge may be formed by introducing a new salt bridge between CH3 domains, rearranging the salt bridge network between two or more amino acid residues, or reversing the charge of the amino acid residues forming the salt bridge relative to the wild-type CH3 domain (i.e., "reversing" the salt bridge). In some embodiments, the engineered positively charged residue replaces the uncharged residue of the wild-type CH3 domain with a positively charged residue. In some embodiments, the engineered uncharged residue replaces the positively charged residue of the wild-type CH3 domain with an uncharged residue. Rearranged and inverted salt bridges can result in changes to the isoelectric point (PI) of heterodimers and homodimers containing the manipulated CH3 domain, thereby enabling better separation of heterodimers from 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 the T411K substitution and the second CH3 domain contains the K370D substitution, or the first CH3 domain contains the K370D substitution and the second CH3 domain contains the T411K substitution.

[0099] In some embodiments, the first CH3 domain contains a positively charged residue at position 364 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 364. In some embodiments, the first CH3 domain contains K at position 364 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 364. In some embodiments, the first CH3 domain contains K at position 364 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 364. In some embodiments, the first CH3 domain contains R at position 364 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 364. In some embodiments, the first CH3 domain contains R at position 364 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 364. In some embodiments, the first CH3 domain contains the S364K substitution and the second CH3 domain contains the K370D substitution, or the first CH3 domain contains the K370D substitution and the second CH3 domain contains the 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 the D356K substitution and the second CH3 domain contains the K439D substitution, or the first CH3 domain contains the K439D substitution and the second CH3 domain contains the D356K substitution.

[0101] Any of the manipulated salt bridges described herein may be combined with each other. In some embodiments, the first CH3 domain includes a positively charged residue at position 357 and a positively charged residue at position 411, and the second CH3 domain includes a loaded residue at position 351 and a loaded residue at position 370, or the first CH3 domain includes a loaded residue at position 351 and a loaded residue at position 370, and the second CH3 domain includes a positively charged residue at position 357 and a positively charged residue at position 411. In some embodiments, the first CH3 domain includes E357K and T411K substitutions, and the second CH3 domain includes L351D and K370D substitutions, or the first CH3 domain includes L351D and K370D substitutions, and the second CH3 domain includes E357K and T411K substitutions.

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

[0103] In some embodiments, the first CH3 domain includes 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 includes a loaded residue at position 351, a loaded residue at position 370, and a loaded residue at position 439, or the first CH3 domain includes a loaded residue at position 351, a loaded residue at position 370, and a loaded residue at position 439, and the second CH3 domain includes 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 includes the D356K, E357K, and S364K substitutions, and the second CH3 domain includes the L351D, K370D, and K439D substitutions, or the first CH3 domain includes the L351D, K370D, and K439D substitutions, and the second CH3 domain includes the D356K, E357K, and S364K substitutions.

[0104] Other mutations The CH3 domains or Fc regions described herein may further include the manipulated 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 includes C at position 392 and the second CH3 domain includes C at position 399, or the first CH3 domain includes C at position 399 and the second CH3 domain includes C at position 392. In some embodiments, the first CH3 domain further includes a K392C substitution and the second CH3 domain further includes a D399C substitution, or the first CH3 domain further includes a D399C substitution and the second CH3 domain further includes a K392C substitution.

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

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

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

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

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

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

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

[0113] The heterodimer proteins comprising the manipulated CH3 domain disulfide bond and / or salt bridge described herein may further comprise one or more knob-into-hole residues. "Knob-into-hole" or "KIH" refers to an approach known in the art for producing bispecific antibodies, also known as the "protuberance-into-cavity" approach (see, for example, U.S. Patent No. 5,731,168). In this approach, two immunoglobulin polypeptides (e.g., heavy chain polypeptides) each constitute an interface. The interface of one immunoglobulin polypeptide interacts with a corresponding interface on the other immunoglobulin polypeptide, thereby allowing the two immunoglobulin polypeptides to associate. These interfaces may be manipulated such that a "knob" or "protuberance" (these terms may be used interchangeably herein) located at the interface of one immunoglobulin polypeptide corresponds to a "hole" or "cavity" (these terms may be used interchangeably herein) located at the interface of the other immunoglobulin polypeptide. In some embodiments, the holes are identical or similar in size to the knobs and are appropriately positioned so that when the two interfaces interact, the knob of one interface can be positioned in the corresponding hole of the other interface. While we do not wish to be bound by theory, this is thought to stabilize the heteromultimer and promote the formation of heteromultimers more than other species, e.g., homomultimers. In some embodiments, the KIH approach is used in combination with the manipulated disulfide bonds and / or salt bridges described herein to promote the heteromultimerization of two different immunoglobulin polypeptides and to produce a bispecific antibody containing two immunoglobulin polypeptides having binding specificity to different epitopes. In some embodiments, the CH3 domain of the heterodimer protein described herein does not contain a KIH residue.

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

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

[0116] III. Multispecific antibodies In some embodiments, the heterodimer protein described herein is a multispecific antibody, such as a bispecific or tripspecific antibody.

[0117] In some embodiments, a multispecific antibody is provided comprising a first polypeptide comprising a first CH3 domain and a first target-binding moiety (TBM), and a second polypeptide comprising a second CH3 domain and a second TBM, wherein the first and second CH3 domains comprise either one or a combination of the manipulated disulfide bond or salt bridge 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 scFv or VHH. In some embodiments, the first CH3 domain comprises an N390C substitution and the second CH3 domain comprises an S400C substitution, or the first CH3 domain comprises an S400C substitution and the second CH3 domain comprises an N390C substitution. In some embodiments, the first CH3 domain includes the substitutions E357K, S364K, and S400C, and the second CH3 domain includes the substitutions L351D, K370D, and N390C, or the first CH3 domain includes the substitutions L351D, K370D, and N390C, and the second CH3 domain includes the substitutions E357K, S364K, and S400C. In some embodiments, the first CH3 domain includes the substitutions D356K, E357K, S364K, and N390C, and the second CH3 domain includes the substitutions L351D, K370D, K439D, and S400C, or the first CH3 domain includes the substitutions L351D, K370D, K439D, and S400C, and the second CH3 domain includes the substitutions D356K, E357K, S364K, and N390C. 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 multispecific antibody includes an IgG1 Fc region, for example, an IgG1 Fc having the N297A substitution.In some embodiments, the multispecific antibody comprises IgG4 having an IgG4 Fc region, for example, an S228P substitution.

[0118] In some embodiments, a multispecific antibody is provided comprising a first CH3 domain, a second polypeptide containing a second CH3 domain, a third polypeptide, and a fourth polypeptide, wherein the first CH3 domain and the second CH3 domain comprise one or a combination of either an manipulated disulfide bond or a salt bridge as described herein, the first polypeptide being a first antibody heavy chain, the second polypeptide being a second antibody heavy chain, the third polypeptide being a first antibody light chain, and the fourth polypeptide being a second antibody light chain, the first polypeptide and the third polypeptide associating to form a first antigen-binding site that specifically binds to a first target, and the second polypeptide and the fourth polypeptide associating 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 the N390C substitution and the second CH3 domain includes the S400C substitution, or the first CH3 domain includes the S400C substitution and the second CH3 domain includes the N390C substitution. In some embodiments, the first CH3 domain includes the E357K, S364K, and S400C substitutions and the second CH3 domain includes the L351D, K370D, and N390C substitutions, or the first CH3 domain includes the L351D, K370D, and N390C substitutions and the second CH3 domain includes the E357K, S364K, and S400C substitutions. In some embodiments, the first CH3 domain includes the substitutions D356K, E357K, S364K, and N390C, and the second CH3 domain includes the substitutions L351D, K370D, K439D, and S400C, or the first CH3 domain includes the substitutions L351D, K370D, K439D, and S400C, and the second CH3 domain includes the substitutions D356K, E357K, S364K, and N390C.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 multispecific antibody includes an IgG1 Fc region, e.g., IgG1 Fc having an N297A substitution. In some embodiments, the multispecific antibody includes an IgG4 Fc region, e.g., IgG4 having an S228P substitution.

[0119] In some embodiments, a multispecific antibody is provided comprising a first polypeptide containing a first CH3 domain, a second polypeptide containing a second CH3 domain, a third polypeptide, and a fourth polypeptide, wherein the first CH3 domain and the second CH3 domain comprise one or a combination of the manipulated disulfide bond or salt bridge 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 constant domain of the immunoglobulin light chain. CH1 is the constant domain 1 of the immunoglobulin heavy chain. CH2 is the constant domain 2 of the immunoglobulin heavy chain. The hinge is an immunoglobulin hinge region that connects the CH1 and CH2 domains. VL1 and VH1 associate to form a first Fv that specifically binds to a first target; VL2 and VH2 associate to form a second Fv that specifically binds to a second target. In some embodiments, VL1 is identical to VL2 (e.g., a multispecific antibody is an antibody with a common light chain). In some embodiments, VL1 is different from VL2. In some embodiments, the first CH3 domain contains an N390C substitution and the second CH3 domain contains an S400C substitution, or the first CH3 domain contains an S400C substitution and the second CH3 domain contains an N390C substitution. In some embodiments, the first CH3 domain includes the substitutions E357K, S364K, and S400C, and the second CH3 domain includes the substitutions L351D, K370D, and N390C, or the first CH3 domain includes the substitutions L351D, K370D, and N390C, and the second CH3 domain includes the substitutions E357K, S364K, and S400C. In some embodiments, the first CH3 domain includes the substitutions D356K, E357K, S364K, and N390C, and the second CH3 domain includes the substitutions L351D, K370D, K439D, and S400C, or the first CH3 domain includes the substitutions L351D, K370D, K439D, and S400C, and the second CH3 domain includes the substitutions D356K, E357K, S364K, and N390C. 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 multispecific antibody includes an IgG1 Fc region, e.g., IgG1 Fc having an N297A substitution. In some embodiments, the multispecific antibody includes an IgG4 Fc region, e.g., 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 comprising a first polypeptide containing a first CH3 domain, a second polypeptide containing a second CH3 domain, a third polypeptide, and a fourth polypeptide, wherein the first CH3 domain and the second CH3 domain comprise one or a combination of the manipulated disulfide bond or salt bridge described herein. (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: VH2-CH1-Hinge-CH2-Second CH3-L2-scFv2, (iii) The third polypeptide comprises a structure represented by the following formula: VL1-CL, (iv) The fourth polypeptide comprises a structure represented by the following formula: VL2-CL, VL1 is the first immunoglobulin light chain variable domain, VH1 is the first immunoglobulin heavy chain variable domain, VL2 is the second immunoglobulin light chain variable domain, VH2 is the second immunoglobulin heavy chain variable domain, scFv1 is the first single-chain variable fragment, scFv2 is the second single-chain variable fragment, CL is the constant domain of the immunoglobulin light chain. CH1 is the constant domain 1 of the immunoglobulin heavy chain. CH2 is the constant domain 2 of the immunoglobulin heavy chain. The hinge is an immunoglobulin hinge region that links the CH1 and CH2 domains; L1 and L2 are, independently, binding or peptide linkers. 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 heterodimer protein is a bispecific antibody, where the first and second targets are the same, and the third and fourth targets are the same. In some embodiments, the heterodimer protein is a triplicate antibody, where the third and fourth targets are the same, or the first and second targets are the same. In some embodiments, the heterodimer protein is a quadruplicate antibody. In some embodiments, VL1 is identical to VL2 (for example, a multispecific antibody is an antibody with a common light chain). In some embodiments, VL1 is different from VL2. In some embodiments, the first CH3 domain includes the N390C substitution and the second CH3 domain includes the S400C substitution, or the first CH3 domain includes the S400C substitution and the second CH3 domain includes the N390C substitution. In some embodiments, the first CH3 domain includes the E357K, S364K, and S400C substitutions and the second CH3 domain includes the L351D, K370D, and N390C substitutions, or the first CH3 domain includes the L351D, K370D, and N390C substitutions and the second CH3 domain includes the E357K, S364K, and S400C substitutions. In some embodiments, the first CH3 domain includes the substitutions D356K, E357K, S364K, and N390C, and the second CH3 domain includes the substitutions L351D, K370D, K439D, and S400C, or the first CH3 domain includes the substitutions L351D, K370D, K439D, and S400C, and the second CH3 domain includes the substitutions D356K, E357K, S364K, and N390C.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 multispecific antibody includes an IgG1 Fc region, e.g., IgG1 Fc having an N297A substitution. In some embodiments, the multispecific antibody includes an IgG4 Fc region, e.g., IgG4 having an S228P substitution.

[0121] In some embodiments, a multispecific antibody is provided comprising a first polypeptide containing a first CH3 domain, a second polypeptide containing 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 and second CH3 domains contain either one or a combination of the manipulated disulfide bonds or salt bridges described herein, and the first and third polypeptides 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 scFv or VHH. In some embodiments, the first CH3 domain contains an N390C substitution and the second CH3 domain contains an S400C substitution, or the first CH3 domain contains an S400C substitution and the second CH3 domain contains an N390C substitution. In some embodiments, the first CH3 domain includes the substitutions E357K, S364K, and S400C, and the second CH3 domain includes the substitutions L351D, K370D, and N390C, or the first CH3 domain includes the substitutions L351D, K370D, and N390C, and the second CH3 domain includes the substitutions E357K, S364K, and S400C. In some embodiments, the first CH3 domain includes the substitutions D356K, E357K, S364K, and N390C, and the second CH3 domain includes the substitutions L351D, K370D, K439D, and S400C, or the first CH3 domain includes the substitutions L351D, K370D, K439D, and S400C, and the second CH3 domain includes the substitutions D356K, E357K, S364K, and N390C. 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 multispecific antibody includes an IgG1 Fc region, for example, an IgG1 Fc having the N297A substitution.In some embodiments, the multispecific antibody comprises IgG4 having an IgG4 Fc region, for example, an S228P substitution. In some embodiments, the first target is PDL1 and the second target is CD137. In some embodiments, the first target is CD137 and the second target is PDL1. In some embodiments, the first target is CD137 and the second target is CTLA4. In some embodiments, the first target is CTLA4 and the second target is PDL1.

[0122] In some embodiments, a multispecific antibody is provided comprising a first polypeptide, a second polypeptide, and a third polypeptide, each comprising a first CH3 domain, wherein the first CH3 domain and the second CH3 domain comprise one or a combination of the manipulated disulfide bond or salt bridge 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 the variable domain of the immunoglobulin light chain. VH is a variable domain of the immunoglobulin heavy chain, scFv is a single-chain variable fragment, CL is the constant domain of the immunoglobulin light chain. CH1 is the constant domain 1 of the immunoglobulin heavy chain. CH2 is the constant domain 2 of the immunoglobulin heavy chain. The hinge is an immunoglobulin hinge region that connects the CH1 and CH2 domains. VL and VH associate to form Fv, which specifically binds to the first target. scFv specifically binds to a second 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 the substitutions D356K, E357K, S364K, and N390C, and the second CH3 domain includes the substitutions L351D, K370D, K439D, and S400C, or the first CH3 domain includes the substitutions L351D, K370D, K439D, and S400C, and the second CH3 domain includes the substitutions D356K, E357K, S364K, and N390C. In some embodiments, the first CH3 domain includes the D356K, E357K, S364K, and S400C substitutions, and the second CH3 domain includes the L351D, K370D, N390C, and K439D substitutions, or the first CH3 domain includes the L351D, K370D, N390C, and K439D substitutions, and the second CH3 domain includes the D356K, E357K, S364K, and S400C substitutions. In some embodiments, the multispecific antibody includes an IgG1 Fc region, e.g., IgG1 Fc having the N297A substitution. In some embodiments, the multispecific antibody includes an IgG4 Fc region, e.g., IgG4 having the S228P substitution. In some embodiments, the scFv is linked to the hinge of the second polypeptide via a linker, such as a peptide linker containing 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).

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

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

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

[0126] In some embodiments, the first, second, third, and / or fourth target are cell surface antigens. In some embodiments, the cell surface antigen is 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, cell surface antigens are tumor antigens. Tumor antigens are proteins produced by tumor cells that can trigger an immune response, particularly a T-cell-mediated immune response. In some embodiments, tumor antigens are tumor-specific antigens (TSAs) or tumor-associated antigens (TAAs). TSAs are specific to tumor cells and are not present in other cells in the body. TAA-associated antigens are not specific to tumor cells and are instead expressed on normal cells under conditions that do not induce a state of immune tolerance to the antigen. Antigen expression on tumors can occur under conditions that allow the immune system to respond to the antigen. TAAs may be antigens expressed on normal cells during fetal development when the immune system is immature and unable to respond, or they may be antigens that are normally present at very low levels on normal cells but are expressed at much higher levels on tumor cells.

[0128] Non-limiting examples of TSA or TAA antigens include: differentiation antigens, e.g., MART-1 / MelanA (MART-1), gp100 (Pmel17), tyrosinase, TRP-1, TRP-2, and tumor-specific multiseries antigens, e.g., MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, pl5; overexpressed embryonic antigens, e.g., CEA; overexpressed oncogenes and mutated tumor suppressor genes, e.g., p53, Ras, HER2 / neu; unique tumor antigens resulting from chromosomal translocations, e.g., BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens, e.g., 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, and BT. Examples include AA, CA125, CA15-3, CA27, 29, BCAA, CA195, CA242, CA-50, CAM43, CD68, P1, CO-029, FGF-5, G250, Ga733, EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90, Mac-2 binding protein, cyclophyllin C-related protein, TAAL6, TAG72, TLP, and TPS.

[0129] In some embodiments, the first, second, third, and / or fourth targets are immune checkpoint molecules. In some embodiments, the immune checkpoint molecule is a stimulating immune checkpoint molecule. Exemplary stimulating 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) includes an antibody light chain variable region (VL) and / or an antibody heavy chain variable region (VH). In some embodiments, the TBM includes the VL. In some embodiments, the TBM includes the VH. In some embodiments, the TBM includes specificity of the VL and / or VH to 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, but is not limited to, (i) a Fab fragment which is a monovalent fragment consisting of VL, VH, CL, and CHI domains; (ii) a F(ab')2 fragment which is a bivalent fragment containing two Fab fragments linked by disulfide crosslinking at the hinge region; (iii) an Fd fragment consisting of VH and CHI domains; (iv) an Fv fragment consisting of VL and VH domains of a single arm of the 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) which is a polypeptide containing the VL region of the antibody linked to the VH region of the antibody (e.g., Bird et al. (1988) Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad. Sci. USA It is an antigen-binding fragment containing (see 85:5879-5883).

[0132] In some embodiments, the TBM is an scFv containing 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 containing VH-L1-VL from the N-terminus to the C-terminus, where L1 is a peptide linker. In some embodiments, L1 contains the amino acid sequence of SEQ ID NO: 82. In some embodiments, the TBM is an scFv containing an manipulated 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 contains a first cysteine ​​residue at position 44 of VH and a second cysteine ​​residue at position 100 of VL, where the first and second cysteine ​​residues form a disulfide bond, 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 any class, such as IgG, IgM, IgE, IgA, or IgD. In some embodiments, the antibody heavy chain is of the IgG class, such as the IgG1, IgG2, IgG3, or IgG4 subclass. The antibody heavy chains described herein may be converted from one class or subclass to another using methods known in the art.

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

[0135] In some embodiments, the TBM is an anti-PD-L1 antibody, or its antigen-binding domain, which includes, for example, VH, VL, scFv, light chain, or heavy chain (e.g., IgG1, IgG2, IgG4). Any known anti-PD-L1 antibody 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. Examples of 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 includes a VH comprising an antibody heavy chain complementarity determining region (CDR-H) 1 containing the amino acid sequence of SEQ ID NO: 37, a CDR-H2 containing the amino acid sequence of SEQ ID NO: 38, and / or a CDR-H3 containing the amino acid sequence of SEQ ID NO: 39. In some embodiments, the TBM includes a VL comprising an antibody light chain complementarity determining region (CDR-L) 1 containing the amino acid sequence of SEQ ID NO: 40, a CDR-L2 containing the amino acid sequence of SEQ ID NO: 41, and / or a CDR-L3 containing the amino acid sequence of SEQ ID NO: 42. In some embodiments, the TBM includes a VH comprising the amino acid sequence of SEQ ID NO: 43. In some embodiments, the TBM includes a VL comprising the amino acid sequence of SEQ ID NO: 44. In some embodiments, the TBM includes an scFv comprising the amino acid sequence of SEQ ID NO: 77.

[0137] In some embodiments, the TBM is an anti-CD137 antibody, or its antigen-binding domain, which includes, for example, VH, VL, scFv, light chain, or heavy chain (e.g., IgG1, IgG2, IgG4). Any known anti-CD137 antibody may 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), utomirumab (also known as PF-05082566), CTX-471, ATOR-1017, and AGEN2373.

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

[0139] In some embodiments, the TBM is an anti-CTLA4 antibody, or its antigen-binding domain, which includes, for example, VH, VL, scFv, light chain, or heavy chain (e.g., IgG1, IgG2, IgG4). This includes, but is not limited to, ipilimumab (see U.S. Patent Nos. 6,984,720, 7,452,535, 7,605,238, 8,017,114, and 8,142,778), toremilimumab (see U.S. Patent Nos. 6,68,736, 7,109,003, 7,132,281, 7,411,057, 7,807,797, 7,824,679, and 8,143,379), and other anti-CTLA-4 antibodies such as single-chain antibodies (e.g., U.S. Patent No. 5,811,097 Any known anti-CTLA4 antibody, including (see Patent Nos. 6051,227 and 7,229,628, U.S. Patent Publication No. US20110044953, U.S. Patent Publication No. US2018037654, U.S. Patent Publication No. US2009025274, U.S. Patent Publication No. US2019127468, International Patent Publication No. WO2019 / 152413, International Patent Publication No. WO2018209701, International Patent Publication No. WO2018 / 202649 and International Patent Publication No. WO2019 / 152423), may 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 includes VH, which includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 53, CDR-H2 containing the amino acid sequence of SEQ ID NO: 54, and / or CDR-H3 containing the amino acid sequence of SEQ ID NO: 55. In some embodiments, the TBM includes VL, which includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 56, CDR-L2 containing the amino acid sequence of SEQ ID NO: 57, and / or CDR-L3 containing the amino acid sequence of SEQ ID NO: 58. In some embodiments, the TBM includes VH, which includes the amino acid sequence of SEQ ID NO: 59. In some embodiments, the TBM includes VL, which includes the amino acid sequence of SEQ ID NO: 60. In some embodiments, the TBM includes scFv, which includes 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, which includes, for example, VH, VL, scFv, light chain, or heavy chain (e.g., IgG1, IgG2, IgG4). While not limited to these, examples include Cris-7 monoclonal antibody (Reinherz, E 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 their derivatives, such as OKT3 ala-ala (Herold et al. (2003) J. Clin. Invest. 11:409), bicilizumab (Carpenter et al. (2002) Blood 99:2712), and 145-2C11 monoclonal antibody (Hirsch et al. Any known anti-CD3 antibody, including al. (1988) J. Immunol. 140:3766), otelexizumab, and foralumab, may be used in the present invention. Further CD3-binding molecules considered herein include UCHT-1 (Beverley, PC and Callard, RE (1981) Eur. J. Immunol. 11:329-334), SP34 (Silvana et. al. (1985) The EMBO Journal. 4:337-344), and the CD3-binding molecules described in WO2004 / 106380; WO2010 / 037838; WO2008 / 119567; WO2007 / 042261; WO2010 / 0150918; WO2018 / 052503; WO2016 / 204966.

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

[0143] In some embodiments, the TBM is an anti-HER2 antibody, or its antigen-binding domain, which includes, for example, VH, VL, scFv, light chain, or heavy chain (e.g., IgG1, IgG2, IgG4). Any known anti-HER2 antibody, 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), disitamab (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 includes VH, which includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 69, CDR-H2 containing the amino acid sequence of SEQ ID NO: 70, and / or CDR-H3 containing the amino acid sequence of SEQ ID NO: 71. In some embodiments, the TBM includes VL, which includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 72, CDR-L2 containing the amino acid sequence of SEQ ID NO: 73, and / or CDR-L3 containing the amino acid sequence of SEQ ID NO: 74. In some embodiments, the TBM includes VH, which includes the amino acid sequence of SEQ ID NO: 75. In some embodiments, the TBM includes VL, which includes the amino acid sequence of SEQ ID NO: 76.

[0145] As used in this application, the term "PDL1" includes human PDL1 (e.g., UniProt accession number Q9NZQ7), as well as its variants, isoforms, and species homologs (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 in this application, the term "CTLA4" includes human CTLA4 (e.g., UniProt accession number P16410), as well as its variants, isoforms, and species homologs (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 in this application, the term "CD137" includes human CD137 (e.g., GenBank accession numbers NM_001561; NP_001552), as well as its variants, isoforms, and species homologs (e.g., mouse CD137 (GenBank gene ID 21942), rat CD137 (GenBank gene ID 500590), canine CD137 (GenBank gene ID 608274), cynomolgus monkey CTLA4 (GenBank gene ID 102127961), etc.).

[0148] The term "CD3" is known in the art as a six-chain polyprotein complex (see Abbas and Lichtman, 2003; Janeway et al., pp. 172 and 178, 1999). In mammals, the complex includes a CD3 gamma chain, a 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 relevant cell surface proteins of the immunoglobulin superfamily, each containing a single immunoglobulin domain. The transmembrane regions of the CD3 gamma, CD3 delta, and CD3 epsilon chains are charged, a property that allows these chains to associate with positively charged T cell receptor chains. The intracellular tails of the CD3 gamma, CD3 delta, and CD3 epsilon chains each contain a single conserved motif known as the immunoreceptor tyrosine-based activation motif or ITAM, while each CD3 zeta chain has three. While not theoretically bound, ITAM is considered important for the signaling capability of the TCR complex. The CD3 used herein may be derived from various animal species, including humans, primates, mice, rats, or other mammals. For example, the CD3 used herein includes human CD3e (i.e., CD3 epsilon; e.g., UniProt accession number P07766), as well as its variants, isoforms, and species homologs (e.g., mouse CD3e (UniProt accession number P22646), rat CD3e (UniProt accession number A0A0G2K986), canine CD3e (UniProt accession number P27597), and cynomolgus monkey CD3e (UniProt accession number Q95LI5)).

[0149] As used in this application, 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 may bind to human targets (e.g., PDL1, CTLA4, CD137, CD3, or HER2). In some cases, the TBMs may be perfectly specific to human targets and may not exhibit cross-reactivity with species or other types of organisms. In other cases, the TBMs may 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.) positioned between various regions of the polypeptide.

[0152] For example, glycine polymer (G)n (wherein 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 polymer (GS)n (wherein 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 ( Any suitable linker known in the art (e.g., a flexible linker) may be used, including sequence number 80), GGGSGGGS (sequence number 81), (G4S)4 (sequence number 82), GGGGS (sequence number 130), SGGS (sequence number 131), GGSG (sequence number 132), GGSGG (sequence number 133), GSGSG (sequence number 134), GSGGG (sequence number 135), GGGSG (sequence number 136), and / or GSSSG (sequence number 137); glycine-alanine polymer; alanine-serine polymer; etc. The linker sequence may be of any length, for example, about 1 amino acid (e.g., glycine or serine) to about 20 amino acids (e.g., 20 amino acid glycine polymer or glycine-serine polymer), about 1 amino acid to about 15 amino acids, about 3 amino acids to about 12 amino acids, about 4 amino acids to about 10 amino acids, about 5 amino acids to about 9 amino acids, about 6 amino acids to about 8 amino acids, etc. In some embodiments, the linker is any of approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid lengths.

[0153] A schematic example of a multi-specific antibody Exemplary multispecific antibodies described herein include, but are not limited to, bispecific antibodies targeting PDL1 and CD137 (e.g., PDL1×CD137 and CD137×PDL1 antibodies), bispecific antibodies targeting CD137 and CTLA4 (e.g., CD137×CTLA4 antibody), bispecific T cell engagers (BiTEs) targeting CD3 and cell surface antigens, and triplicate antibodies targeting PDL1, CD137, and CTLA4 (also referred to herein as PDL1×CD137×CTLA4 antibodies). In some embodiments, the multispecific antibody includes a CH3 domain or Fc region containing one or a combination of the manipulated disulfide bonds and / or salt bridges described herein. In some embodiments, the multispecific antibody does not include a CH3 domain or Fc region containing one or a combination of the manipulated 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 the variable domain of the immunoglobulin light chain. VH is a variable domain of the immunoglobulin heavy chain, scFv is a single-chain variable fragment, CL is the constant domain of the immunoglobulin light chain. CH1 is the constant domain 1 of the immunoglobulin heavy chain. CH2 is the constant domain 2 of the immunoglobulin heavy chain. The hinge is an immunoglobulin hinge region that connects the CH1 and CH2 domains. L1 is a binding or peptide linker. VL and VH associate to form Fv, which specifically binds to CD137, and scFv specifically binds to PDL1. In some embodiments, scFv includes VH, which includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 37, CDR-H2 containing the amino acid sequence of SEQ ID NO: 38, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 39; and / or VL, which includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 40, CDR-L2 containing the amino acid sequence of SEQ ID NO: 41, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 42. In some embodiments, scFv includes VH containing the amino acid sequence of SEQ ID NO: 43, and / or VL containing the amino acid sequence of SEQ ID NO: 44. In some embodiments, scFv includes VH-L1-VL from the N-terminus to the C-terminus, where L1 is a peptide linker. In some embodiments, L1 contains the amino acid sequence of SEQ ID NO: 82. In some embodiments, scFv includes a first cysteine ​​residue at position 44 of VH and a second cysteine ​​residue at position 100 of VL, where the first and second cysteine ​​residues form a disulfide bond, and the numbering is based on Kabat numbering. In some embodiments, scFv includes the amino acid sequence of SEQ ID NO: 77. In some embodiments, Fv includes VH including CDR-H1 containing the amino acid sequence of SEQ ID NO: 45, CDR-H2 containing the amino acid sequence of SEQ ID NO: 46, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 47; and / or VL including CDR-L1 containing the amino acid sequence of SEQ ID NO: 48, CDR-L2 containing the amino acid sequence of SEQ ID NO: 49, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 50. In some embodiments, Fv includes VH containing the amino acid sequence of SEQ ID NO: 51, and / or VL containing the amino acid sequence of SEQ ID NO: 52. 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 multispecific antibody includes 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., 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 containing the amino acid sequence of SEQ ID NO: 80 or 81.

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

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

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

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

[0159] In some embodiments, a triple-specific antibody is provided, which comprises a first polypeptide containing the amino acid sequence of SEQ ID NO: 118, a second polypeptide containing the amino acid sequence of SEQ ID NO: 119, and a third polypeptide containing the amino acid sequence of SEQ ID NO: 120. In some embodiments, a triple-specific antibody is provided, which comprises a first polypeptide containing the amino acid sequence of SEQ ID NO: 121, a second polypeptide containing the amino acid sequence of SEQ ID NO: 122, and a third polypeptide containing the amino acid sequence of SEQ ID NO: 123. In some embodiments, a triple-specific antibody is provided, which comprises a first polypeptide containing the amino acid sequence of SEQ ID NO: 124, a second polypeptide containing the amino acid sequence of SEQ ID NO: 125, and a third polypeptide containing 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 the variable domain of the immunoglobulin light chain. VH is a variable domain of the immunoglobulin heavy chain, scFv is a single-chain variable fragment, CL is the constant domain of the immunoglobulin light chain. CH1 is the constant domain 1 of the immunoglobulin heavy chain. CH2 is the constant domain 2 of the immunoglobulin heavy chain. The hinge is an immunoglobulin hinge region that connects the CH1 and CH2 domains. VL and VH associate to form Fv, which specifically binds to tumor antigens (e.g., HER2); scFv specifically binds to CD3. In some embodiments, scFv includes VH, which includes CDR-H1, which includes the amino acid sequence of SEQ ID NO: 61; CDR-H2, which includes the amino acid sequence of SEQ ID NO: 62; and CDR-H3, which includes the amino acid sequence of SEQ ID NO: 63; and / or VL, which includes CDR-L1, which includes the amino acid sequence of SEQ ID NO: 64; CDR-L2, which includes the amino acid sequence of SEQ ID NO: 65; and CDR-L3, which includes the amino acid sequence of SEQ ID NO: 66. In some embodiments, scFv includes VH, which includes the amino acid sequence of SEQ ID NO: 67; and / or VL, which includes the amino acid sequence of SEQ ID NO: 68. In some embodiments, scFv includes the amino acid sequence of SEQ ID NO: 79. In some embodiments, Fv specifically binds to HER2. In some embodiments, Fv includes VH, which includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 69, CDR-H2 containing the amino acid sequence of SEQ ID NO: 70, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 71; and / or VL, which includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 72, CDR-L2 containing the amino acid sequence of SEQ ID NO: 73, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 74. In some embodiments, Fv includes VH, which includes the amino acid sequence of SEQ ID NO: 75, and / or VL, which includes the amino acid sequence of SEQ ID NO: 76. In some embodiments, the first CH3 domain includes the D356K, E357K, S364K, and S400C substitutions, and the second CH3 domain includes the L351D, K370D, N390C, and K439D substitutions, or the first CH3 domain includes the L351D, K370D, N390C, and K439D substitutions, and the second CH3 domain includes the D356K, E357K, S364K, and S400C substitutions. In some embodiments, the multispecific antibody includes an IgG1 Fc region, e.g., IgG1 Fc having the N297A substitution. In some embodiments, a bispecific T cell engager molecule is provided, which includes a first polypeptide containing the amino acid sequence of SEQ ID NO: 112, a second polypeptide containing the amino acid sequence of SEQ ID NO: 113, and a third polypeptide containing the amino acid sequence of SEQ ID NO: 114.

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

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

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

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

[0165] In some embodiments, an activatable antibody is provided comprising a first polypeptide containing a first CH3 domain, a second polypeptide containing 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 a variable domain of the immunoglobulin heavy chain, scFv is a single-chain variable fragment, CL is the constant domain of the immunoglobulin light chain. CH1 is the constant domain 1 of the immunoglobulin heavy chain. CH2 is the constant domain 2 of the immunoglobulin heavy chain. The hinge is an 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; scFv specifically binds to a second target; if CM1 is not cleaved, MM1 inhibits the binding of scFv to the first target; if 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 contain any one or a combination of the manipulated disulfide bond or salt bridge described herein. In some embodiments, the first CH3 domain and the second CH3 domain include any one or a combination of the manipulated disulfide bond or salt bridge 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 the substitutions E357K, S364K, and S400C, and the second CH3 domain includes the substitutions L351D, K370D, and N390C, or the first CH3 domain includes the substitutions L351D, K370D, and N390C, and the second CH3 domain includes the substitutions E357K, S364K, and S400C. In some embodiments, the first CH3 domain includes the substitutions D356K, E357K, S364K, and N390C, and the second CH3 domain includes the substitutions L351D, K370D, K439D, and S400C, or the first CH3 domain includes the substitutions L351D, K370D, K439D, and S400C, and the second CH3 domain includes the substitutions D356K, E357K, S364K, and N390C. In some embodiments, the first CH3 domain includes the substitutions D356K, E357K, S364K, and S400C, and the second CH3 domain includes the substitutions L351D, K370D, N390C, and K439D, or the first CH3 domain includes the substitutions L351D, K370D, N390C, and K439D, and the second CH3 domain includes the substitutions D356K, E357K, S364K, and S400C.In some embodiments, the activatable antibody includes an IgG1 Fc region such as 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, an activatable antibody is provided comprising a first polypeptide containing a first CH3 domain, a second polypeptide containing 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 the variable domain of the immunoglobulin light chain. VH is a variable domain of the immunoglobulin heavy chain, scFv is a single-chain variable fragment, CL is the constant domain of the immunoglobulin light chain. CH1 is the constant domain 1 of the immunoglobulin heavy chain. CH2 is the constant domain 2 of the immunoglobulin heavy chain. The hinge is an 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; scFv specifically binds to a second target; if CM1 is not cleaved, MM1 inhibits the binding of the first Fv to the first target; if CM2 is not cleaved, MM2 inhibits the binding of scFv to the second target. In some embodiments, the first CH3 domain and the second CH3 domain do not contain any one or a combination of the manipulated disulfide bond or salt bridge described herein. In some embodiments, the first CH3 domain and the second CH3 domain include any one or a combination of the manipulated disulfide bond or salt bridge 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 the substitutions E357K, S364K, and S400C, and the second CH3 domain includes the substitutions L351D, K370D, and N390C, or the first CH3 domain includes the substitutions L351D, K370D, and N390C, and the second CH3 domain includes the substitutions E357K, S364K, and S400C. In some embodiments, the first CH3 domain includes the substitutions D356K, E357K, S364K, and N390C, and the second CH3 domain includes the substitutions L351D, K370D, K439D, and S400C, or the first CH3 domain includes the substitutions L351D, K370D, K439D, and S400C, and the second CH3 domain includes the substitutions D356K, E357K, S364K, and N390C. In some embodiments, the first CH3 domain includes the substitutions D356K, E357K, S364K, and S400C, and the second CH3 domain includes the substitutions L351D, K370D, N390C, and K439D, or the first CH3 domain includes the substitutions L351D, K370D, N390C, and K439D, and the second CH3 domain includes the substitutions D356K, E357K, S364K, and S400C.In some embodiments, the activatable antibody includes an IgG1 Fc region such as 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 comprising a first polypeptide containing a first CH3 domain, a second polypeptide containing 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 constant domain of the immunoglobulin light chain. CH1 is the constant domain 1 of the immunoglobulin heavy chain. CH2 is the constant domain 2 of the immunoglobulin heavy chain. The hinge is an 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, VL1 and VH1 associate to form a first Fv that specifically binds to a first target; VL2 and VH2 associate to form a second Fv that specifically binds to a second target; if CM1 is not cleaved, MM1 inhibits the binding of the first Fv to the first target; if 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 contain any one or a combination of the manipulated disulfide bond or salt bridge described herein. In some embodiments, the first CH3 domain and the second CH3 domain include any one or a combination of the manipulated disulfide bond or salt bridge 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 the substitutions E357K, S364K, and S400C, and the second CH3 domain includes the substitutions L351D, K370D, and N390C, or the first CH3 domain includes the substitutions L351D, K370D, and N390C, and the second CH3 domain includes the substitutions E357K, S364K, and S400C. In some embodiments, the first CH3 domain includes the substitutions D356K, E357K, S364K, and N390C, and the second CH3 domain includes the substitutions L351D, K370D, K439D, and S400C, or the first CH3 domain includes the substitutions L351D, K370D, K439D, and S400C, and the second CH3 domain includes the substitutions D356K, E357K, S364K, and N390C. In some embodiments, the first CH3 domain includes the substitutions D356K, E357K, S364K, and S400C, and the second CH3 domain includes the substitutions L351D, K370D, N390C, and K439D, or the first CH3 domain includes the substitutions L351D, K370D, N390C, and K439D, and the second CH3 domain includes the substitutions D356K, E357K, S364K, and S400C.In some embodiments, the activatable antibody includes an IgG1 Fc region such as 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, an activatable antibody is provided comprising a first polypeptide containing a first CH3 domain, a second polypeptide containing 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 constant domain of the immunoglobulin light chain. CH1 is the constant domain 1 of the immunoglobulin heavy chain. CH2 is the constant domain 2 of the immunoglobulin heavy chain. The hinge is an 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, VL1 and VH1 associate to form a first Fv that specifically binds to a first target; VL2 and VH2 associate to form a second Fv that specifically binds to a second target; if CM1 is not cleaved, MM1 inhibits the binding of the first Fv to the first target; if 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 contain any one or a combination of the manipulated disulfide bond or salt bridge described herein. In some embodiments, the first CH3 domain and the second CH3 domain include any one or a combination of the manipulated disulfide bond or salt bridge 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 the substitutions E357K, S364K, and S400C, and the second CH3 domain includes the substitutions L351D, K370D, and N390C, or the first CH3 domain includes the substitutions L351D, K370D, and N390C, and the second CH3 domain includes the substitutions E357K, S364K, and S400C. In some embodiments, the first CH3 domain includes the substitutions D356K, E357K, S364K, and N390C, and the second CH3 domain includes the substitutions L351D, K370D, K439D, and S400C, or the first CH3 domain includes the substitutions L351D, K370D, K439D, and S400C, and the second CH3 domain includes the substitutions D356K, E357K, S364K, and N390C. In some embodiments, the first CH3 domain includes the substitutions D356K, E357K, S364K, and S400C, and the second CH3 domain includes the substitutions L351D, K370D, N390C, and K439D, or the first CH3 domain includes the substitutions L351D, K370D, N390C, and K439D, and the second CH3 domain includes the substitutions D356K, E357K, S364K, and S400C.In some embodiments, the activatable antibody includes an IgG1 Fc region such as 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, an activatable antibody is provided comprising a first polypeptide containing a first CH3 domain, a second polypeptide containing 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 constant domain of the immunoglobulin light chain. CH1 is the constant domain 1 of the immunoglobulin heavy chain. CH2 is the constant domain 2 of the immunoglobulin heavy chain. The hinge is an 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, VL1 and VH1 associate to form a first Fv that specifically binds to a first target; VL2 and VH2 associate to form a second Fv that specifically binds to a second target; if CM1 is not cleaved, MM1 inhibits the binding of the first Fv to the first target; if 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 contain any one or a combination of the manipulated disulfide bond or salt bridge described herein. In some embodiments, the first CH3 domain and the second CH3 domain include any one or a combination of the manipulated disulfide bond or salt bridge 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 the substitutions E357K, S364K, and S400C, and the second CH3 domain includes the substitutions L351D, K370D, and N390C, or the first CH3 domain includes the substitutions L351D, K370D, and N390C, and the second CH3 domain includes the substitutions E357K, S364K, and S400C. In some embodiments, the first CH3 domain includes the substitutions D356K, E357K, S364K, and N390C, and the second CH3 domain includes the substitutions L351D, K370D, K439D, and S400C, or the first CH3 domain includes the substitutions L351D, K370D, K439D, and S400C, and the second CH3 domain includes the substitutions D356K, E357K, S364K, and N390C. In some embodiments, the first CH3 domain includes the substitutions D356K, E357K, S364K, and S400C, and the second CH3 domain includes the substitutions L351D, K370D, N390C, and K439D, or the first CH3 domain includes the substitutions L351D, K370D, N390C, and K439D, and the second CH3 domain includes the substitutions D356K, E357K, S364K, and S400C.In some embodiments, the activatable antibody includes an IgG1 Fc region such as 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).

[0170] In some embodiments, the activatable antibody is designed based on any one of the multispecific antibodies described herein by fusing a masking moiety (MM) to the target-binding moiety (TBM) of the multispecific antibody via a cleavable moiety (CM), for example, if 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, which is incorporated herein by reference in its entirety. The activatable antibody may include any one of the TBMs described in the subsection "Target-Binding Moiety (TBM)" of Section III "Multispecific Antibodies". The activatable antibody described herein may include, for example, one or more linkers described in the subsection "Linkers" of Section III "Multispecific Antibodies," located between the MM and CM of Fc, between the CM and TBM, or between the TBM and the hinge region.

[0171] MM refers to an amino acid sequence in which MM interferes with or inhibits the binding of TBM to the target, provided that the CM of the activatable antibody is intact (e.g., contains an uncleaved and / or unreduced cysteine-cysteine ​​disulfide bond by the corresponding enzyme). In some embodiments, MM interferes with or inhibits the binding of TBM to the target so efficiently that the binding of TBM to the target is very low and / or below the detection limit (e.g., the binding cannot be detected by ELISA or flow cytometry assays). The amino acid sequence of CM may overlap with or be contained within MM. It should be noted that, for convenience, “ABP” or “activatable antibody” is used herein to refer to both uncleaved (or “natural”) and cleaved ABP or activatable antibody. In some embodiments, cleaved ABP may lack MM due to cleavage of CM (e.g., by protease), and it will be apparent to those skilled in the art that at least MM is released (e.g., MM is not covalently bound to ABP (e.g., disulfide bonds between cysteine ​​residues)). Exemplary ABP is described in more detail below.

[0172] CMs generally contain cleavable amino acid sequences and function as substrates for enzymes and / or cysteine-cysteine ​​pairs capable of forming reducible disulfide bonds, for example. Therefore, when terms such as "cleavage," "cleavable," and "cleaved" are used in relation to CMs, the terms encompass cleavage of disulfide bonds between cysteine-cysteine ​​pairs via enzymatic cleavage by proteases and reduction of disulfide bonds that may occur upon exposure to reducing agents.

[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 (whether in active or inactive form) does not produce an ADCC effect of more than about 10% compared to the control (does not induce ADCC in more than about 10%, more than about 5%, more than about 1%, more than about 0.1%, or more than about 0.01%).

[0174] In some embodiments, activatable antibodies (e.g., BiTE molecules) can inhibit 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 in contact with activatable antibodies and T cells, compared to corresponding tumor cells not in contact with the activatable antibody (or compared to corresponding tumor cells in contact with isotype control antibodies and T cells). In some embodiments, activatable antibodies can reduce the tumor volume of a subject when the subject is administered the activatable antibody. In some embodiments, an activatable antibody (e.g., a BiTE molecule) can reduce 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 initial tumor volume of the subject (e.g., before administration of the activatable antibody; compared to the corresponding tumor in a subject administered with 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 remission 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 incorporated herein by reference in its entirety.

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

[0178] In some embodiments, a masking peptide (MM) interferes with, blocks, reduces, suppresses, inhibits, or competes with the target binding site (e.g., an "inactive activatable antibody") for binding to the target. In some embodiments, a masking peptide (MM) interferes with, blocks, reduces, suppresses, inhibits, or competes with the target binding site only if the antibody is not 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 protein ligand)). In some embodiments, activation induces cleavage of the cleavable site. In some embodiments, activation induces a conformational change in the polypeptide (e.g., displacement of the MM) to prevent the MM from inhibiting 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 site (CM) only if the cleavable site (CM) is not cleaved by one or more proteases cleaving within the cleavable site (CM). In some embodiments, MM has a masking efficiency of at least about 2.0 before activation (e.g., at least about 2.0, at least about 3.0, at least about 4.0, at least about 5.0, at least about 6.0, at least about 7.0, at least about 8.0, at least about 9.0, at least about 10, at least about 25, at least about 50, at least about 75, at least about 100, at least about 150, at least about 200, at least about 300, at least about 400, at least about 500, etc.). In some embodiments, the masking efficiency is measured as the difference in affinity of the activatable antibody containing MM (before activation) to the target compared to the affinity of the polypeptide lacking MM to the target (e.g., the difference in affinity of the activatable antibody containing MM (before activation) to the target antigen (e.g., CD3 or HER2) compared to the parent antibody lacking MM, or the difference in affinity of the activatable antibody containing MM (before activation) to the target antigen (e.g., CD3 or HER2) compared to the affinity of the activatable antibody (after activation) to the target antigen).In some embodiments, masking efficiency is measured by dividing the EC50 of the binding of the activatable antibody containing the MM (before activation) by the EC50 of the parent antibody (e.g., measuring EC50 by ELISA). In some embodiments, masking efficiency is measured as the difference in affinity of the activatable antibody containing the MM to the target before activation compared to the affinity of the activatable antibody containing the MM to the target after activation (e.g., the difference in affinity of the activatable antibody before activation to the target antigen (e.g., CD3 or HER2) compared to the activatable antibody after activation). In some embodiments, the MM binds to a 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) to the target. The dissociation constant can be measured 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 site (TBM) for binding to the target after the polypeptide has been activated (e.g., by treatment with one or more proteases that cleave within the cleavable portion (CM), by a change in pH (increase or decrease), by a change in temperature (increase or decrease), or by contact with a second molecule (e.g., an enzyme)). In some embodiments, the MM does not interfere with, block, reduce, suppress, inhibit, or compete with the target binding site (TBM) for binding to the target after the cleavable portion (CM) has been cleaved by one or more proteases that cleave within the cleavable portion (CM). In some embodiments, the MM has a masking efficiency (e.g., relative affinity of the activated activatable antibody compared to the affinity of the parent 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 (e.g., relative affinity of the activated activatable antibody compared to the affinity of the parent antibody).

[0180] In some embodiments, any of the MMs described herein may further include one or more additional amino acid sequences (e.g., one or more polypeptide tags). Suitable examples of additional amino acid sequences include, but are not limited to, purification tags (e.g., hist tags, flag tags, maltose-binding protein and glutathione-S transferase tags), detection tags (e.g., photometrically detectable tags (e.g., red or green fluorescent protein)), tags having detectable enzymatic activity (e.g., alkaline phosphatase), tags including secretion sequences, leader sequences and / or stabilization sequences, protease cleavage sites (e.g., furin cleavage sites, TEV cleavage sites, thrombin cleavage sites), and the like. In some embodiments, one or more additional amino acid sequences are located at the N-terminus of the MM.

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

[0182] In some embodiments, the CM includes 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 that is recognized and / or cleaved by any protease known in the art (e.g., a protease known to co-localize with a target of a polypeptide comprising the CM) may be used, for example, a protease cleavage site recognized and / or cleaved by a 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 HET virus (TEV) protease; Plasmin; Thrombin; PSA; PSMA; ADAM / ADAMTS (e.g., ADAM8, ADAM9, ADAMIO, ADAM12, ADAMIS, ADAM17 / TACE, ADAMDECI, ADAMTSI, ADAMTS4, and / or ADAMTS5); caspases (e.g., caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11, caspase-12, caspase-13, and / or caspase-14); aspartate proteases (e.g., RACE and / or renin); aspartate ca Thepsins (e.g., cathepsin D and / or cathepsin E); cysteine ​​cathepsins (e.g., cathepsin B, cathepsin C, cathepsin K, cathepsin L, cathepsin S, cathepsin V / L2, and / or cathepsin X / Z / P); cysteine ​​proteinases (e.g., cruzipain, regmain, and / or otsubine-2); KLK (e.g., KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, and / or KLK14);This includes 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; guanidinobenzoatase; HtrAl; human neutrophil elastase; lactoferrin; malapsin; NS3 / 4A; PACE4; tPA; tryptase; type II transmembrane serine proteases (TTSPs) (e.g., DESC1, DPP-4, FAP, hepsin, matryptase-2, MT-SP1 / matryptase, TMPRSS2, TMPRSS3, and / or TMPRSS4), etc. In some embodiments, the first protease cleavage site is a 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 protease cleavage includes 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 portion (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 portion (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 may be any preferred protease cleavage site 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 recognized and / or cleaved by the same protease. In some embodiments, the first (CS1) and second (CS2) cleavage sites are protease cleavage sites that 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.). In some embodiments, at least the second cleavage site (CS2) is C-terminal to the first linker (L1). In some embodiments, the cleavable portion (CM) includes a (CS1)-L1-(CS2) structure from the N-terminus to the C-terminus.

[0184] In some embodiments, the cleavable portion (CM) further includes 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 portion (CM) further includes a second linker (L2). The second linker (L2) may be any preferred linker 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 the C-terminus to the second cleavage site (CS2). In some embodiments, the cleavable portion (CM) includes a (CS1)-L1-(CS2)-L2 structure from the N-terminus to the C-terminus.

[0185] CD3-targeted activatable antibody This application provides a CD3-targeting activatable antibody, an activatable antibody fragment, and a polypeptide, all comprising a masking moiety (MM) containing the amino acid sequence of SEQ ID NO: 35.

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

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

[0188] In some embodiments, an activatable antibody targeting CD3 is provided, comprising a first polypeptide comprising 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 if CM is not cleaved, MM inhibits the binding of the activatable antibody to CD3; CM comprises at least a first cleavage site; TBM comprises VL, and the activatable antibody further comprises a second polypeptide comprising VH; if CM is cleaved, the activatable antibody binds to CD3 via VH and VL. In some embodiments, the activatable antibody comprises 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 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 VH, which contains the amino acid sequence of SEQ ID NO: 67, and / or VL, which contains the amino acid sequence of SEQ ID NO: 68.

[0189] In some embodiments, an activatable antibody targeting CD3 is provided, comprising a first polypeptide comprising 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 if CM is not cleaved, MM inhibits the binding of the activatable antibody to CD3; CM comprises at least a first cleavage site; TBM comprises VH, and the activatable antibody further comprises a second polypeptide comprising VL; if CM is cleaved, the activatable antibody binds to CD3 via VH and VL. In some embodiments, the activatable antibody comprises 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 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 VH, which contains the amino acid sequence of SEQ ID NO: 67, and / or VL, which contains the amino acid sequence of SEQ ID NO: 68.

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

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

[0192] In some embodiments, an activatable bispecific T cell engager molecule is provided, comprising a first polypeptide containing a first CH3 domain, a second polypeptide containing 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 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 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 contains the amino acid sequence of SEQ ID NO: 36. In some embodiments, VH contains CDR-H1 containing the amino acid sequence of SEQ ID NO: 69, CDR-H2 containing the amino acid sequence of SEQ ID NO: 70, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 71. In some embodiments, VL contains CDR-L1 containing the amino acid sequence of SEQ ID NO: 72, CDR-L2 containing the amino acid sequence of SEQ ID NO: 73, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 74. In some embodiments, VH contains the amino acid sequence of SEQ ID NO: 75. In some embodiments, VL contains the amino acid sequence of SEQ ID NO: 76.

[0194] In some embodiments, the activatable antibody (including the BiTE molecule) that targets CD3 as described herein comprises a first CH3 domain and a second CH3 domain that does not contain one or a combination of the manipulated disulfide bond or salt bridge described herein. In some embodiments, the activatable antibody comprises a first CH3 domain and a second CH3 domain that contains one or a combination of the manipulated disulfide bond or salt bridge 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 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 containing the amino acid sequence of SEQ ID NO: 115, a second polypeptide containing the amino acid sequence of SEQ ID NO: 116, and a third polypeptide containing the amino acid sequence of SEQ ID NO: 117.

[0196] Activatable antibodies targeting HER2 This application provides an activatable antibody targeting HER2, an activatable antibody fragment, and a polypeptide, all comprising a masking moiety (MM) containing the amino acid sequence of SEQ ID NO: 36.

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

[0198] In some embodiments, an antibody heavy chain is provided comprising a polypeptide including MM, CM, and TBM from the N-terminus to the C-terminus, where MM comprises the amino acid sequence of SEQ ID NO: 36; CM comprises at least a first cleavage site; and TBM comprises VH of the anti-HER2 antibody. In some embodiments, the anti-HER2 antibody comprises VH, which includes 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.

[0199] 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, wherein MM comprises the amino acid sequence of SEQ ID NO: 36, and if CM is not cleaved, MM inhibits the binding of the activatable antibody to HER2; CM comprises at least a first cleavage site; TBM comprises VL, and the activatable antibody further comprises a second polypeptide comprising VH; if CM is cleaved, the activatable antibody binds to HER2 via VH and VL. In some embodiments, the activatable antibody comprises 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 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 VH, which contains the amino acid sequence of SEQ ID NO: 75, and / or VL, which contains 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, wherein MM comprises the amino acid sequence of SEQ ID NO: 36, and if CM is not cleaved, MM inhibits the binding of the activatable antibody to HER2; CM comprises at least a first cleavage site; TBM comprises VH, and the activatable antibody further comprises a second polypeptide comprising VL; if CM is cleaved, the activatable antibody binds to HER2 via VH and VL. In some embodiments, the activatable antibody comprises 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 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 VH, which contains the amino acid sequence of SEQ ID NO: 75, and / or VL, which contains the amino acid sequence of SEQ ID NO: 76.

[0201] In some embodiments, an activatable antibody targeting HER2 is provided, comprising a first polypeptide comprising 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 if CM is not cleaved, MM inhibits the binding of the activatable antibody to HER2; CM comprises at least a first cleavage site; and if CM is cleaved, the activatable antibody binds to HER2 via scFv. In some embodiments, scFv comprises 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 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, scFv comprises VH, comprising the amino acid sequence of SEQ ID NO: 75, and / or VL, comprising the amino acid sequence of SEQ ID NO: 76. In some embodiments, scFv contains VL and VH from the N-terminus to the C-terminus. In some embodiments, scFv contains VH and VL from the N-terminus to the C-terminus. In some embodiments, scFv contains 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, the activatable antibody (including the BiTE molecule) that targets HER2 as described herein comprises a first CH3 domain and a second CH3 domain that do not contain one or a combination of the manipulated disulfide bond or salt bridge described herein. In some embodiments, the activatable antibody comprises a first CH3 domain and a second CH3 domain that contain one or a combination of the manipulated disulfide bond or salt bridge 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 IgG1 Fc having an N297A substitution.

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

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

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

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

[0208] Amino acid substitutions encompass 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 similarities in certain physicochemical properties of the residues involved, such as polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphiphilicity. For example, substitutions may typically be made within any of the following groups: (a) nonpolar (hydrophobic) amino acids, e.g., alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; (b) polar neutral amino acids, e.g., glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; (c) positively charged (basic) amino acids, e.g., arginine, lysine, and histidine; and (d) positively charged (acidic) amino acids, e.g., aspartic acid and glutamic acid.

[0209] Modifications may be made at any position in the amino acid sequence of the antibody, including the CDR, framework region, or constant region. In some embodiments, this application provides antibody derivatives that contain the VH and VL CDR sequences of the exemplary antibody described herein, but contain a framework sequence different from that of the exemplary antibody. Such framework sequences can be obtained from publicly available DNA databases or published references containing germline antibody gene sequences. For example, germline DNA sequences of human heavy chain 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, USD 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)). Framework sequences that may be used in constructing antibody derivatives include those structurally similar to the framework sequences used by the exemplary antibody of this application. For example, the CDR-H1, CDR-H2, and CDR-H3 sequences of the exemplary antibody, as well as the CDR-L1, CDR-L2, and CDR-L3 sequences, can be transplanted into framework regions having sequences identical to those found in the germline immunoglobulin genes from which the framework sequences originate, or the CDR sequences can be transplanted into framework regions containing one or more mutations compared to the germline sequences.

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

[0211] Another type of modification involves mutating amino acid residues within the CDR region 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 by in vitro or in vivo assays known in the art. Typically, conservative substitutions are introduced. The mutations may be additions and / or deletions of amino acids. Furthermore, typically, one, two, three, four, or five or fewer residues within the CDR region are altered. In some embodiments, the antibody derivative contains one, two, three, or four amino acid substitutions in the heavy chain CDR and / or light chain CDR. In another embodiment, the amino acid substitution is to change one or more cysteines in the antibody to another residue, such as alanine or serine, but not limited to these. The cysteine ​​may be standard or non-standard cysteine. In some embodiments, the antibody derivative has one, two, three, or four conservative amino acid substitutions in the heavy chain CDR region compared to the amino acid sequence of an exemplary antibody.

[0212] Modifications may also be made to framework residues within the VH and / or VL regions. Typically, such framework variants are created to reduce the immunogenicity of the antibody. One approach is to “reverse mutation” one or more framework residues to the corresponding germline sequence. Antibodies that have undergone somatic mutations may contain framework residues different from those in the germline sequence from which the antibody originates. Such residues can be identified by comparing the antibody framework sequence with the germline sequence from which the antibody originates. To restore the framework region sequence to its germline configuration, somatic mutations can be “reverse mutation” to the germline sequence, for example, by site-directed mutagenesis or PCR-mediated mutagenesis.

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

[0214] In some embodiments, the Fc region of the heterodimer protein or antibody described herein has at least one (e.g., at least one, two, or three or more) amino acid substitutions compared to the Fc region of wild-type IgG or wild-type antibody, in addition to the amino acid substitutions that form the manipulated 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 natural sequence Fc region and / or the Fc region of the parent polypeptide.

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

[0216] In some embodiments, the antibodies described herein (e.g., multispecific antibodies and activatable antibodies) may be 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) may be of the IgG class, such as the IgG1, IgG2, IgG3, or IgG4 subclass. The antibodies described herein can be converted from one class or subclass to another using methods known in the art. An exemplary method for generating an antibody of a desired class or subclass includes the steps of: isolating the nucleic acid encoding the heavy chain of the antibody described herein (e.g., a multispecific or activatable antibody) and the nucleic acid encoding the light chain of the antibody described herein (e.g., a multispecific 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 heavy chain construct in cells; and collecting the antibody.

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

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

[0219] In some embodiments, the heterodimer proteins or antibodies of this application are linked to pharmaceuticals. Examples of pharmaceuticals include cytotoxic agents or other cancer treatments, 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 their analogues or homologues. Therapeutic agents also include, for example, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechloretamine, thioepachlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclotosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum(II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomicin) 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 used for diagnostic or therapeutic purposes 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 linking therapeutic agents to antibodies, see, for example, Saito et al., Adv. Drug De / iv. Rev. 55:199-215 (2003); Trail, et al., Cancer Immunol. Immunother. 52:328-337 (2003); Payne, Cancer Cell 3:207-212 (2003); Allen, Nat. Rev. Cancer 2:750-763 (2002); Pastan and Kreitman, Curr. Opin. Investig. Drugs 3:1089-1091 (2002); Senter and Springer (2001) Adv. Drug De / iv. Rev. 53:247-264.

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

[0221] As used herein, a cytotoxic agent is a part that reduces the proliferative capacity of one or more cells. A cell's proliferative capacity is reduced when its potential for proliferation is diminished. This can be due, for example, to apoptosis or other causes of cell death, the inability of the cell cycle to progress and / or to divide, or the differentiation of cells. Non-limiting examples of cytotoxic agents include, but are not limited to, radioisotopes, toxins, and chemotherapeutic agents. Those skilled in the art can select suitable cytotoxicity according to their intended use.

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

[0223] VI. Preparation method In one embodiment, the present application provides a method for preparing heterodimer proteins, multispecific antibodies, or activatable antibodies as described herein. For example, a method is provided for preparing a heterodimer protein (e.g., a multispecific antibody) or activatable antibody, the method comprising culturing a host cell containing one or more nucleic acids (e.g., multispecific antibodies) or vectors encoding a heterodimer protein (e.g., a multispecific antibody) or activatable antibody polypeptide under conditions that enable the expression of nucleic acids or vectors, and recovering the heterodimer protein polypeptide or activatable antibody polypeptide from the host cell culture.

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

[0225] In the recombinant production of polypeptides according to this application (e.g., any of the heterodimer proteins, multispecific antibodies, or activatable antibodies described above), for example, nucleic acids encoding the polypeptide (e.g., the heterodimer proteins, multispecific antibodies, or activatable antibodies described above) are isolated for further cloning and / or expression in host cells 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 encoding the polypeptide(s)).

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

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

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

[0229] Plant cell cultures can also be used as hosts. See, for example, U.S. Patents 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe PLANTIBODIES® technology for antibody production in transgenic plants).

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

[0231] For certain secretory proteins to be expressed and secreted in large quantities, a leader sequence derived from a heterologous protein may be desirable. In some embodiments, using a heterologous leader sequence may be advantageous because the leader sequence is removed in the ER during the secretion process, so the resulting mature polypeptide may remain unchanged. The addition of a heterologous leader sequence may be necessary for the expression and secretion of certain proteins.

[0232] Specific exemplary leader sequences are listed, for example, in the online leader sequence database maintained by the Department of Biochemistry at the National University of Singapore. See Choo et al., BMC Bioinformatics, 6:249 (2005); and PCT Publication WO2006 / 081430.

[0233] VII. Compositions and Kits In some embodiments, this application provides a pharmaceutical composition comprising 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 formulations for the delivery of polypeptides (e.g., heterodimer proteins, multispecific antibodies, or activatable antibodies). Carriers may be antifouling agents, binders, coatings, disintegrants, fillers, or diluents, preservatives (e.g., antioxidants, antimicrobials, or antifungals), sweeteners, absorption retarders, wetting agents, emulsifiers, buffers, 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), saline, buffers, buffered saline, and isotonic agents, such as sugars, polyalcohols, sorbitol, and sodium chloride.

[0235] The composition may be in any preferred form, such as a liquid, semi-solid, or solid dosage form. Examples of liquid dosage forms include solutions (e.g., injectable and injectable solutions), microemulsions, liposomes, dispersions, or suspensions. Examples of solid dosage forms include tablets, pills, capsules, microcapsules, and powders. A particular form of composition suitable for the delivery of polypeptides (e.g., heterodimer proteins, multispecific antibodies, or activatable antibodies) is a sterile liquid, such as a solution, suspension, or dispersion for injection or infusion. A sterile solution can be prepared by incorporating the required amount of polypeptide (e.g., heterodimer proteins, multispecific antibodies, or activatable antibodies) into a suitable carrier, followed by sterile microfiltration. A dispersion may be prepared by incorporating the polypeptide into a sterile vehicle containing a basic dispersion medium and other carriers. In the case of a sterile powder for preparing a sterile liquid, the preparation method includes obtaining a powder of the active ingredient and any additional desired ingredients from the pre-sterile filtered solution by vacuum drying and freeze-drying (lyophilization). Various dosage forms of the composition can be prepared by conventional methods known in the art.

[0236] The relative amount of polypeptide (e.g., heterodimer protein, multispecific antibody, or activatable antibody) in a composition will vary depending on numerous 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., heterodimer protein, multispecific antibody, or activatable antibody) in a single dosage form is generally the amount that produces a therapeutic effect, but may be less. 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 of the total weight of the dosage form.

[0237] In addition to polypeptides (e.g., heterodimer proteins, multispecific antibodies, or activatable antibodies), one or more additional therapeutic agents may be included in the composition. The appropriate amount of additional therapeutic agents included in the composition can be easily selected by those skilled in the art and will vary depending on a number of factors, such as the specific drug and carrier used, the dosage form, and the desired release and pharmacodynamic properties. The amount of additional therapeutic agent included in a single dosage form is generally the amount of the drug that produces the therapeutic effect, but may be less.

[0238] Any polypeptide (e.g., heterodimer proteins, multispecific antibodies, or activatable antibodies) and / or composition (e.g., pharmaceutical compositions) described herein may be used in the preparation of drugs (e.g., drugs used in subjects requiring the treatment of cancer or the delay of cancer progression).

[0239] In some embodiments, kits comprising any one of the heterodimer proteins, multispecific antibodies, activatable antibodies, and / or compositions described herein are provided. In some embodiments, the kit further includes a package insert containing instructions for use of the heterodimer 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 relating to the use of the therapeutic product. In some embodiments, the kit further includes one or more buffers for, for example, storing, transporting, administering, or otherwise using the heterodimer protein, multispecific antibody, activatable antibody, and / or composition. In some embodiments, the kit further includes one or more containers (e.g., syringes) for storing or administering the heterodimer protein, multispecific antibody, activatable antibody, and / or composition. Manufacturing articles comprising any one of the heterodimer proteins, multispecific antibodies, activatable antibodies, and / or compositions described herein are also provided.

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

[0241] In some embodiments, methods are provided for treating a disease or condition in subjects requiring treatment of the disease or condition, the methods comprising administering an effective amount of a pharmaceutical composition comprising one of the heterodimer proteins, multispecific antibodies, or activatable antibodies (e.g., activatable BiTE molecules) described herein to the subject. In some embodiments, the disease or condition is cancer. Various cancers may be treated or prevented using the methods, uses, or pharmaceutical compositions provided herein.

[0242] In some embodiments, a method is provided for treating cancer in a subject requiring treatment of cancer, the method comprising administering an effective amount of a pharmaceutical composition to the subject containing one of the multispecific antibodies that target one or more immune checkpoint molecules described herein (e.g., one of 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-stage cancer.

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

[0244] In some embodiments, methods are provided for enhancing the immune response in mammals, which include administering to a mammal an effective amount of a pharmaceutical composition comprising one of the heterodimer proteins, multispecific antibodies, or activatable antibodies (e.g., activatable BiTE molecules) described herein. The term “enhancing the immune response” or its grammatical variations means stimulating, eliciting, increasing, improving, or amplifying the response of the immune system of interest. The immune response may be a cellular response (i.e., cell-mediated, e.g., cytotoxic T lymphocyte-mediated) or a humoral response (i.e., antibody-mediated), and may be a primary or secondary immune response. Examples of enhancing the immune response include activation of PBMCs and / or T cells (including increased secretion of one or more cytokines such as IL-2 and / or IFNγ). 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, tumor-bearing animal survival, antibody production, immune cell proliferation, cell surface marker expression, and cytotoxicity. Typically, the methods of this application enhance the immune response in mammals compared to the immune response in untreated mammals or mammals not treated using the enumerated methods.

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

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

[0247] The dosage, frequency of administration, and route of administration for the therapies described herein depend on a number of factors, such as the type and severity of the disorder to be treated, the specific heterodimer protein, multispecific antibody, or activatable antibody administered, the timing of administration, the duration of treatment, the specific additional therapy 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 field.

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

[0249] The following examples are intended purely to illustrate the invention and should therefore not be considered limiting. The following examples and detailed description are provided illustratively, not limitingly.

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

[0251] Example 2. Purity evaluation of heterodimers To test novel Fc mutations, TYM013 and a reference heterodimer were constructed from heterodimer TYM01. The corresponding novel Fc mutations are shown in Table 2. Fab-Fc / Fc1 arm constructs with mutations at the CH3 domain interface were designed (Figure 1A). To evaluate the effect of mutations on CH3 domain heterodimerization and homodimerization, cloning was performed in mammalian expression vectors so that the constructed CH3_A domain could be expressed as a light-heavy chain ("LC-HC") half-body, and the CH3_B domain could be expressed in Fc-only form. Plasmids encoding light chain ("LC"), heavy chain ("HC"), and Fc in a 2:1:1 molar ratio were co-transfected into HEK293 cells for transient expression. Excess LC relative to HC chain DNA was used to avoid LC restriction. Cell culture supernatant was filtered through a 0.45 μm sterile filter. Antibodies were purified by protein A affinity chromatography using a HiTrap MabSelect SuRe prepacked column (GE Healthcare), followed by buffer exchange. To assess heterodimer yield, the products were evaluated by SDS-PAGE and SEC-HPLC.

[0252] In the Fab-Fc / Fc1 arm construct system, the heterodimer and the 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"). The 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 heterodimers was also evaluated by SEC-HPLC. As shown in Figure 7, three peaks were detected in total. The first peak in the spectrum corresponds to the homodimer, the second peak at 46.6 corresponds to the heterodimer, and the third peak corresponds to the LC-HC half-body. Quantification of peak area using SEC-HPLC identified variant pairs that stabilize the heterodimer compared to the homodimer. Purity was calculated using the first and second peaks, and the results are shown in Table 2. [Table 7]

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

[0254] Example 4. Generation of heterodimers targeting CD137 and PDL1 Bispecific antibodies with Fc mutations were constructed using anti-CD137 and anti-PDL1 antibodies. The pharmacokinetics of monoclonal antibodies can be modulated by altering their interaction with the neonatal Fc receptor FcRn. Improving the affinity of the FcRn-IgG interaction can extend the half-life of 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 of endosomes (pH 6-6.5), FcRn exhibits low affinity (micromolar to nanomolar) for the Fc region of IgG. Therefore, FcRn binding properties may reflect how mutations affect the PK of the Fc region. Table 3 shows that the pH-dependent FcRn binding of the bispecific antibodies was unaffected at both acidic and physiological pH. [Table 8]

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

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

[0257] Example 5. Production and characterization of bispecific antibodies targeting CD137 and PDL1 A. Generation of bispecific antibodies The following example describes the development of a feasible and effective format for developing bispecific antibodies targeting CD137 and PDL1. The format was optimized based on the "Morrison format" (Figure 2). Expression plasmids were constructed in Fab or scFv form using DNA encoding anti-CD137Fv and anti-PDL1Fv. Since scFv has lower affinity compared to Fab, the orientation of the two Fv (i.e., CD137×PDL1 or PDL1×CD137 in the form of Fab×scFv) can affect the efficacy of the bispecific antibody.

[0258] IgG1 or IgG4(S228P) isotypes were used. The scFv was linked to the C-terminus of Fc with the SGGGS (SEQ ID NO: 80) or GGGGGGGS (SEQ ID NO: 81) linker, oriented from VH to VL. Of the scFv, the C-terminus of VH was linked to the N-terminus of VL with the (G4S)4 (SEQ ID NO: 82) linker. A disulfide bond from VH-44 to VL-100 was also incorporated into the scFv to stabilize the format. Novel manipulated N390C of the CH3 domain. CH3A -S400'C CH3B Disulfide bond pairs were also tested (SEQ ID NOs. 23-24). In addition, the N297A mutation was introduced to silence 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, first light chain, second heavy chain, and second light chain of exemplary CD137×PDL1 and PDL1×CD137 antibodies. [Table 9]

[0259] B. Evaluation of CMC characteristics of bispecific antibodies Plasmids encoding the heavy and light chains of a bispecific antibody were transiently transfected into mammalian cells. Seven days after transfection, the cell culture supernatant containing the bispecific antibody was collected by centrifugation at 14000 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 prepack column (GE Healthcare), followed by buffer exchange in 20 mM histidine (pH 5.5) buffer.

[0260] The agglutination ratio of the purified bispecific antibody was evaluated by analytical size exclusion chromatography (SEC). The analysis was performed as follows: SEC was performed using 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 pre-column (Tosoh Bioscience) was used. 10 μg of each sample was injected, and separation was performed at a flow rate of 0.5 mL / min. The elution buffer consisted of 200 mM sodium phosphate at pH 7.0. UV detection was performed at 214 nm. 100 μL of sample (1 mg / mL) was eluted at -80 o The freeze-thaw stability was tested by freezing at 1°C for 30 minutes, followed by thawing at room temperature for 60 minutes. 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, exhibiting low aggregation formation during expression and no tendency to aggregate during the freeze-thaw process. Both the replacement of the CH3 disulfide bond and the SGGGS (SEQ ID NO: 80) linker with the 9-amino acid linker GGGSGGGGS (SEQ ID NO: 81) improved 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 protein 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 tested proteins 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] Antibody affinity was also evaluated against human, monkey, and mouse CD137 or PDL1 transiently expressed on the surface of yeast or HEK293F cells. In summary, yeast or HEK293F cells were transfected with plasmids expressing human, monkey, or mouse CD137 or PDL1. After 48 hours, the transfected cells were harvested and then washed. The cells were then incubated with IgG (at 100 nM each) at 4°C for 1 hour, with shaking at 300 rpm in a shaking bed, protected from light. For simultaneous binding, cells were incubated with biotinylated human CD137 or PDL1 protein fused with human Fc fragments, and SA-PE (streptavidin, phycoerythrin conjugate). For cross-reactivity, cells were incubated with Alexa Fluor 647 conjugate mouse anti-human Fc antibody. The mixture was incubated at 4°C for 30 minutes with 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 simultaneously binds to human PDL1 and CD137. In addition, Figure 13 shows that the bispecific antibody maintained the cross-reactivity of the parental antibody with human, mouse, or monkey-derived PDL1 or CD137. [Table 12]

[0265] D. In vitro and in vivo efficacy The effects of PDL1xCD137 and CD137xPDL1 bispecific antibodies on in vitro reporter gene assays were tested (Figure 14). Anti-PDL1-based bispecific antibodies were evaluated in a PDL1 blockade bioassay. Briefly, Jurkat T cells expressing a luciferase reporter driven by human PD-1 and the NFAT response element (NFAT-RE) were used as PD-1 effector cells. CHO-K1 cells expressing human PDL1 and engineered cell surface proteins designed to activate the congeneral TCR in an antigen-independent manner were used as PDL1 aAPC / CHO-K1 cells. As shown in Figure 14, PD-1 effector cells were incubated with PDL1 aAPC / CHO-K1 cells in the absence or presence of anti-PDL1-based bispecific antibodies or PDL1 monomer 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 using an NFκB reporter assay. CD137-NFκB-293T stable cells were restored, cultured, and divided into 96-well plates (50 μL / well, density 6 x 10⁴). 5 After a 5.5-hour incubation, diluted test antibodies (shown in Figure 14) pre-mixed with a crosslinking agent in a 1:5 ratio were added. Luciferase levels were measured after 18 hours. Using the sea urchin luciferase activity, the relative luciferase amount (RLU) relative to the blank control (untreated with antibody) was normalized for transfection efficiency, and the results were expressed as the mean ± standard error of three trials.

[0266] As shown in the upper panel of Figure 14, the PDL1 reporter gene assay shows that the PDL1xCD137 bispecific antibody exhibited 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 exhibited stronger activity than the PDL1xCD137 bispecific antibody. These results indicate that Fab had superior activity compared to scFv. While we do not wish to be constrained by theory, this may be due to differences in affinity.

[0267] Since anti-CD137 and anti-PDL1 Fv cross-reacted with mouse and monkey antigens, the in vivo efficacy of bispecific antibodies was studied in a 3LL syngeneic mouse tumor model (Figures 15A-15C). 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, but far more effective than using 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. Production of a trispecific antibody The following examples provide a tripspecific antibody capable of binding to CD137, PDL1, and CTLA4 (Figure 3).

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

[0270] Since anti-CD137 and anti-PDL1 Fv cross-reacted with mouse and monkey antigens, the in vivo efficacy of the bispecific antibodies was studied in a 3LL syngeneic mouse tumor model. 2 × 10⁶ C57BL / 6 mice were subjected to this study. 6 3LL lung cancer cells were subcutaneously transplanted. When the tumor was established (70mm) 3 Treatment was initiated by intraperitoneal injection in up to six doses using isotype control IgG (n=6), PDL1xCD137 bispecificity (10 mg / kg, n=8), CD137xPDL1 bispecificity (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, but far more effective than one of the two parental antibodies alone. The PDL1xCD137 bispecific antibody was not as effective as the CD137xPDL1 antibody, indicating that the orientation of the two antigens in this bispecific format is important for efficacy.

[0271] Furthermore, to test in vivo efficacy using a 3LL syngeneic mouse tumor model, we selected the TYF02 trispecific antibody. 2 × 10⁶ C57BL / 6 mice were injected. 6 3LL lung cancer cells were subcutaneously transplanted. When the tumor was established (65 mm) 3Treatment was initiated by intraperitoneal injection with up to five doses using isotype control IgG (n=6), CD137xPDL1xCTLA4 trispecificity (10 mg / kg, n=8), CD137xPDL1xCTLA4 trispecificity (5 mg / kg, n=8), CD137xPDL1 bispecificity (10 mg / kg, n=6), CD137xCTLA4 bispecificity (10 mg / kg, n=6), CD137 monomer (7.5 mg / kg, n=6), PDL1 (3.75 mg / kg, n=6), CTLA4 (3.75 mg / kg, n=6), or CD137 monomer (7.5 mg / kg) + PDL1 (3.75 mg / kg) + CTLA4 (3.75 mg / kg) (n=6). Tumor growth was monitored every two days and reported as the mean tumor volume ± SEM over time. As shown in Figure 15C, this triplicate antibody exhibited superior tumor growth inhibition compared to the corresponding bispecific antibody or combination of three mono-IgG parental antibodies. [Table 13]

[0272] Example 7. Biophysical characterization of heterodimer HER2×CD3 T cell-inducing bispecific antibody A heterodimer bispecific scaffold was designed using the TYM13 Fc mutant. A bispecific antibody with the TYM13 mutation in the hetero-Fc domain was formed by combining light-chain / heavy-chain half-antibodies and scFv-Fc chains (Figure 4). This scaffold was used to construct a HER2xCD3 bispecific T-cell inducing antibody. For comparison, corresponding antibodies with knob-into-hole mutations Y394C, T366S, L368A, Y407V-S354'C T366'W, and the "Xencor mutation" E357Q, S364K-L368'D, K370'S were also constructed.

[0273] Mammalian cells were transiently transfected with plasmids encoding the heavy, light, and scFv-Fc chains of a bispecific antibody. Seven days after transfection, the cell culture supernatant containing the bispecific antibody was collected by centrifugation at 14000 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 prepack column (GE Healthcare) and subsequently buffer-exchanged in 20 mM histidine (pH 5.5) buffer.

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

[0275] When TY24051 was converted to the activatable antibody TY24052, several aggregates were formed (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 We constructed activatable HER2×CD3 bispecific antibodies (also referred to herein as "SAFE bodies" or "SAFE bispecificity") (Figure 5). The constructs are shown in Tables 10 and 11. [Table 15] [Table 16]

[0277] The affinity of bispecific antibodies (TY24051) and its SAFE body version (TY24052) was analyzed by enzyme-linked immunosorbent assay (ELISA). Human HER2 or CD3 (ε and δ chain heterodimers) fused to human Fc fragments were prepared at 2 μg / mL and used to coat ELISA plates overnight at 2–8°C. After washing and blocking, 50 μL of serially diluted IgG was added and incubated at 37°C for 1 hour. The 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 stopped, absorbance at 450 nm was measured. The data were analyzed using GraphPad Prism 6 with nonlinear fitting. As shown in Figures 18A–18B, TY24051 bound to both HER2 and CD3, while TY24052 showed significantly lower affinity than TY24051. After activation, the affinity of TY24052 was fully restored.

[0278] To compare the functional activity of TY24051 and TY24052, antibodies were expressed, purified, and evaluated for antigen-dependent bispecific antibody-mediated tumor cell killing activity (Figure 19). In an in vitro cytotoxic 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 bispecific antibody (target cells: 1x10⁻¹⁵). 4 Cells / well, E:T=10:1). As shown in Figure 19, dose-dependent killing was observed for TY24051 and TY24052, with TY24052 exhibiting higher EC levels compared to TY24051. 50 This showed an increase of approximately 800 times. No specific death was observed in the isotype control.

Claims

1. A heterodimeric protein comprising a first polypeptide comprising a first immunoglobulin heavy chain constant domain 3 (CH3 domain) and a second polypeptide comprising a second CH3 domain; the first polypeptide and the second polypeptide each comprise, from N-terminus to C-terminus, at least a portion of an immunoglobulin hinge region, an immunoglobulin heavy chain constant domain 2 (CH2 domain), and a CH3 domain, the CH2 domain and the CH3 domain forming an IgG F C region; 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, and the amino acid residue numbering is based on EU numbering; Heterodimeric protein.

2. The heterodimeric protein of 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.

3. 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), The amino acid residue numbering is based on EU numbering. The heterodimeric protein according to claim 1.

4. 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. The heterodimeric 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 heterodimeric protein according to claim 4.

6. i) the first CH3 domain comprises an E357K and a T411K substitution and the second CH3 domain comprises an L351D and a K370D substitution, or the first CH3 domain comprises an L351D and a K370D substitution and the second CH3 domain comprises an E357K and a T411K substitution; or ii) the first CH3 domain comprises an E357K and an S364K substitution and the second CH3 domain comprises an L351D and a K370D substitution, or the first CH3 domain comprises an L351D and a K370D substitution and the second CH3 domain comprises an E357K and an S364K substitution; 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; The heterodimeric protein according to claim 5 .

7. i) the first CH3 domain further comprises a K392D and a K409D substitution and the second CH3 domain further comprises a D356K and a D399K substitution, or the first CH3 domain further comprises a D356K and a D399K substitution and the second CH3 domain further comprises a K392D and a K409D substitution; or ii) the first CH3 domain further comprises an L368D and a K370S substitution and the second CH3 domain further comprises an E357Q and a S364K substitution, or the first CH3 domain further comprises an E357Q and a S364K substitution and the second CH3 domain further comprises an L368D and a K370S substitution; or iii) the first CH3 domain further comprises a L351K and a T366K substitution and the second CH3 domain further comprises a L351D and a L368E substitution, or the first CH3 domain further comprises a L351D and a L368E substitution and the second CH3 domain further comprises a L351K and a T366K substitution; or (iv) the first CH3 domain further comprises P395K, P396K, and V397K substitutions and the second CH3 domain further comprises T394D, P395D, and P396D substitutions; or the first CH3 domain further comprises T394D, P395D, and P396D substitutions and the second CH3 domain further comprises P395K, P396K, and V397K substitutions; or (v) the first CH3 domain further comprises F405E, Y407E, and K409E substitutions and the second CH3 domain further comprises F405K and Y407K substitutions; or the first CH3 domain further comprises F405K and Y407K substitutions and the second CH3 domain further comprises F405E, Y407E, and K409E substitutions. The heterodimeric protein according to any one of claims 1 to 3.

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 a knob-into-hole residue.

10. i) the first CH3 domain comprises a T366S, L368A, and Y407V substitution and the second CH3 domain comprises a T366W substitution, or the first CH3 domain comprises a T366W substitution and the second CH3 domain comprises a T366S, L368A, and Y407V substitution; or ii) the first CH3 domain comprises a L368V and a Y407V substitution and the second CH3 domain comprises a T366W substitution, or the first CH3 domain comprises a T366W substitution and the second CH3 domain comprises a L368V and a Y407V substitution; The heterodimeric protein according to claim 9.

11. A heterodimeric protein described in any one of claims 1 to 3, wherein the first CH3 domain and the second CH3 domain are human CH3 domains.

12. A heterodimeric protein described in any one of claims 1 to 3, wherein the Fc region is of the human IgG1 subclass.

13. A heterodimeric protein described in any one of claims 1 to 3, wherein the Fc region is of the human IgG4 subclass.

14. The heterodimeric protein described in Claim 13, wherein the Fc region further contains an S228P substitution.

15. The heterodimeric protein described in claim 12, wherein the Fc region further contains an N297A substitution.

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.

17. The heterodimeric protein of claim 16, wherein the heterodimeric protein is a multispecific antibody.

18. further comprising a third polypeptide and a fourth polypeptide; (i) the first polypeptide comprises a structure represented by the following formula: VH1-CH1-hinge-CH2-first CH3-L1-scFv1(Ia), (ii) the second polypeptide comprises a structure represented by the following formula: VH2-CH1-hinge-CH2-2nd CH3-L2-scFv2(IIa), (iii) the third polypeptide comprises a structure represented by the following formula: VL1-CL(Ib), (iv) the fourth polypeptide comprises a structure represented by the following formula: VL2-CL (IIb), VL1 is the first immunoglobulin light chain variable domain, VH1 is the first immunoglobulin heavy chain variable domain, VL2 is a second immunoglobulin light chain variable domain, VH2 is a second immunoglobulin heavy chain variable domain, scFv1 is the first single chain variable fragment, scFv2 is a second single chain variable fragment, CL is an immunoglobulin light chain constant domain, CH1 is immunoglobulin heavy chain constant domain 1; CH2 is immunoglobulin heavy chain constant domain 2; the hinge is an immunoglobulin hinge region connecting the CH1 and CH2 domains; L1 and L2 are each independently a bond or a peptide linker; VL1 and VH1 associate to form a first Fv that specifically binds to a first target; VL2 and VH2 associate to form a second Fv that specifically binds to a second target; scFv1 specifically binds to a third target; The scFv2 specifically binds to a fourth target; The heterodimeric protein according to claim 17.

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

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

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

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

25. The heterodimeric protein of claim 24, wherein the first target is a tumor antigen and the second target is CD3.

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

35.

27. 26. The heterodimeric protein of claim 25, wherein the first target is HER2.

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

36.

29. The heterodimeric protein described in claim 25, wherein the scFv which 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.

30. The heterodimeric protein described in 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.

31. 25. The heterodimeric protein of claim 24, wherein the activatable antibody comprises an Fc region comprising a first CH3 domain and a second CH3 domain, wherein the first CH3 domain comprises D356K, E357K, S364K, and S400C substitutions, and the second CH3 domain comprises L351D, K370D, N390C, and K439D substitutions, or wherein the first CH3 domain comprises L351D, K370D, N390C, and K439D substitutions, and the second CH3 domain comprises D356K, E357K, S364K, and S400C substitutions.

32. One or more nucleic acids (multiple are possible) encoding the heterodimeric protein described in any one of claims 1 to 3.

33. 33. A vector comprising one or more nucleic acid(s) according to claim 32.

34. 33. A host cell comprising one or more nucleic acid(s) of claim 32.

35. A host cell comprising the vector of claim 33.

36. 1. A method for preparing a heterodimeric protein, comprising: (a) culturing the host cell of claim 35 under conditions allowing expression of the one or more nucleic acid(s) or vectors; and (b) recovering the heterodimeric protein from the culture of the host cells; A method comprising:

37. A pharmaceutical composition comprising the heterodimeric protein according to any one of claims 1 to 3 and a pharma- ceutically acceptable carrier.

38. A pharmaceutical for treating a disease or condition, comprising a heterodimeric protein described in any one of claims 1 to 3 or a pharmaceutical composition described in claim 37.

39. The pharmaceutical of claim 38, wherein the disease or condition is cancer.

40. The pharmaceutical composition of claim 39, wherein the cancer is lung cancer.

41. The pharmaceutical composition of claim 39, wherein the cancer is ovarian cancer.

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