Bispecific antibody fusion molecules and methods of use thereof

Abstract

Described herein are compositions and methods for the efficient production of heteromultimeric antibodies, such as bispecific antibodies. The compositions and methods improve the assembly of heteromultimeric antibodies at higher yields and efficiencies than conventional methods. The antibody is optionally fused to a cytokine or co-stimulatory peptide or a portion thereof. Also described herein are monoclonal antibodies and antigen-binding fragments, variants, multimeric forms or bispecific antibodies thereof that specifically bind CD3. Described herein are methods of making anti-CD3 antibodies and antigen-binding fragments thereof, and methods of using the anti-CD3 antibodies and antigen-binding fragments thereof in a variety of therapeutic, diagnostic, and prophylactic indications.

Description

[0001] Related applications

[0002] This application claims priority to and the benefit of U.S. Provisional Application No. 63 / 368,852, filed on July 19, 2022, U.S. Provisional Application No. 63 / 330,250, filed on April 12, 2022, and U.S. Provisional Application No. 63 / 321,563, filed on March 18, 2022, the contents of each of which are incorporated herein by reference in their entirety.

[0003] Incorporation of Sequence Listing

[0004] The electronic sequence listing, created on March 20, 2023, is named "EVIM_001_001WO_SeqList_ST26.xml," is 883,743 bytes in size, and is incorporated herein by reference in its entirety. Technical Field

[0005] This article describes compositions and methods for efficiently producing antibodies or antigen-binding fragments thereof. Antibodies may be able to specifically bind to more than one target molecule or different epitopes on a single target molecule. Compositions and methods improve antibody assembly and production, resulting in higher efficiency and yield compared to traditional methods. Background Art

[0006] IgG monoclonal antibodies contain two identical antigen-binding arms and a constant domain (Fc). Antibodies with different specificities in their binding arms do not typically occur in nature and must therefore be produced using chemical engineering (e.g., chemical cross-linking), recombinant DNA, and / or cell fusion techniques.

[0007] Bispecific antibodies can bind to two different antigens simultaneously. This property enables the development of therapeutic strategies that are not feasible with traditional monoclonal antibodies. Another type of multispecific molecule is recombinant fusion proteins. Recombinant fusion proteins composed of the extracellular domain of an immunomodulatory protein and the constant region (Fc) domain of an immunoglobulin (Ig) represent a growing class of human therapeutic agents.

[0008] For antibodies, especially multispecific antibodies, the production of clinical-grade materials remains challenging. There are many approaches to produce molecules with mixed binding arms (i.e., the binding arms are different from each other). However, each of these methods has significant disadvantages.

[0009] Chemical cross-linking is labor-intensive, as related substances may still need to be purified from homodimers and other unwanted byproducts, necessitating additional steps to separate undesirable products from the desired product. Furthermore, the chemical modification step can alter the integrity of the protein, leading to poor stability. Consequently, this approach is often inefficient and can result in loss of antibody activity.

[0010] Cell fusion technology (e.g., hybridomas) express two heavy chains and two light chains. When two cells, each expressing a separate antibody, fuse, these two heavy chains and two light chains randomly assemble, resulting in 10 antibody combinations. The desired heteromultimeric antibodies only account for a small fraction of the antibodies thus produced. Purification of the desired heteromultimeric proteins significantly reduces production yields and increases manufacturing costs.

[0011] Recombinant DNA technology has been used to generate various heteromultimeric formats, such as single-chain Fvs (scFvs) and diabodies, which lack an Fc domain. The main disadvantage of these antibody molecules is the lack of an Fc domain, and therefore the ability to trigger effector functions and extend serum half-life (e.g., complement activation, Fc receptor binding, etc.). Therefore, there is a need for bispecific antibodies that contain a functional Fc domain.

[0012] Recombinant DNA technology has also been used to generate “knobs-into-holes” bispecific antibodies. A limitation of this strategy is that the light chains of the two parent antibodies must be identical to prevent mispairing and the formation of unwanted and / or inactive molecules when expressed in the same cell.

[0013] Furthermore, one of the limiting factors during annealing and purification is redox efficiency. BioAnalyzer and MS-TOF analysis of intact mass after oxidation indicates that heterodimers oxidized after this step typically only account for 70-80% of the protein after reduction. The remaining 20-30% of the antibody is dimerized and lacks covalent bonds (SEC-MALS). This can be removed but will severely impact overall yield.

[0014] Therefore, there is still a need to improve the total output of antibody production, especially heterodimeric antibodies, such as bispecific antibodies. This paper describes a method for improving the total output of bispecific antibodies, heterodimers, etc. Based on the description of the invention provided herein, these and other aspects and advantages of the present invention will be apparent. Summary of the Invention

[0015] The present disclosure provides an antibody comprising the following structures: a. a first heavy chain polypeptide (H1) comprising a variable region (VH1) and a constant region (CH1), wherein the constant region (CH1) has a constant region 1 domain (CH1 H1 ), hinge region (H1H), constant region 2 domain (CH1 H2 ) and constant region 3 domain (CH1 H3 ); and a first light chain polypeptide (L1) comprising a variable region (VL1) and a constant region (CL1), b. a second heavy chain polypeptide (H2) comprising a variable region (VH2) and a constant region (CH2), wherein the constant region (CH2) has a constant region 1 domain (CH2 H1), hinge region (H2H), constant region 2 domain (CH2 H2 ) and constant region 3 domain (CH2 H3 ); and a second light chain polypeptide (L2) comprising a variable region (VL2) and a constant region (CL2), wherein i. the amino acid at position 39 (Kabat numbering) of said VH1 and VH2 is a charged or polar amino acid residue, and the amino acid at position 38 (Kabat numbering) of said VL1 and VL2 is an amino acid residue with an opposite charge or polarity compared to the amino acid at position 39 of said VH1 and VH2; or the amino acid at position 100 (Kabat numbering) of said VH1 and VH2 is a charged or polar amino acid residue, and the amino acid at position 44 (Kabat numbering) of said VL1 and VL2 is an amino acid residue with an opposite charge or polarity compared to the amino acid at position 100 of said VH1 and VH2; ii. CH1 H1 and CH1 H2 The amino acid at position 147 (EU numbering) of CL1 or CL2 is a charged or polar amino acid residue, and one of the amino acids at position 131, 179, or 180 (EU numbering) of CL1 or CL2 is aligned with the amino acid residue at position 147 (EU numbering) of CH1. H1 and CH1 H2 iii. CH1 H1 and CH1 H2 The amino acid at position 185 (EU numbering) of CL1 and CL2 is a charged or polar amino acid residue, and the amino acid at position 137 (EU numbering) of CL1 and CL2 is aligned with the amino acid at position 137 (EU numbering) of CH1. H1 and CH1 H2 an amino acid residue with an opposite charge or polarity compared to the amino acid at position 185 (EU numbering) of CH1; or H1 and CH1 H2 The amino acid at position 187 (EU numbering) of CL1 and CL2 is a charged or polar amino acid residue, and one of the amino acids at position 137 or 138 (EU numbering) of CL1 and CL2 is aligned with the amino acid residue at position 137 or 138 (EU numbering) of CH1. H1 and CH1 H2 iv. CH1 is an amino acid residue with an opposite charge or polarity compared to the amino acid at position 187 (EU numbering); and iv. H1 and CH1 H2 The amino acid at position 145 (EU numbering) of CL1 and CL2 is a charged or polar amino acid residue, and the amino acid at position 131 (EU numbering) of CL1 and CL2 is aligned with the amino acid at position 131 of CH1. H1 and CH1 H2 The amino acid residue at position 145 (EU numbering) is an oppositely charged or polar amino acid residue compared to the amino acid at position 145 (EU numbering).

[0016] In some embodiments, the charged amino acid residue is a naturally occurring amino acid or a non-naturally occurring amino acid. In some embodiments, the naturally occurring charged amino acid residue is arginine, lysine, histidine, glutamic acid or aspartic acid.

[0017] In some embodiments, the H1 amino acid at position 39, 100, 147, 185, 187, or 145 is positively charged and the L1 amino acid at position 38, 44, 131, 179, 180, 137, or 138 is negatively charged; and the H2 amino acid at position 39, 100, 147, 185, 187, or 145 is negatively charged and the L2 amino acid at position 38, 44, 131, 179, 180, 137, or 138 is positively charged. In some embodiments, the H1 amino acid at position 39, 100, 147, 185, 187, and 145 is negatively charged, while the L1 amino acid at position 38, 44, 131, 179, 180, 137, or 138 is positively charged; and the H2 amino acid at position 39, 100, 147, 185, 187, or 145 is positively charged, while the L2 amino acid at position 38, 44, 131, 179, 180, 137, or 138 is negatively charged.

[0018] In some embodiments, L1 and L2 are lambda or kappa light chains. In some embodiments, L1 is a lambda light chain and L2 is a kappa light chain. In some embodiments, L1 is a kappa light chain and L2 is a lambda light chain.

[0019] In some embodiments, the antibody is IgG1, IgG 2, In some embodiments, the antibody can be a chimeric antibody, a human antibody, or a humanized antibody. In some embodiments, the antibody is a bispecific antibody.

[0020] In some embodiments, the bispecific antibody comprises: i) a first antigen-binding domain that binds to a cell-surface antigen, wherein the cell-surface antigen is expressed on T cells, NK cells, neutrophils, B cells, or dendritic cell engagers; and ii) a second antigen-binding domain that binds to a disease-associated antigen (DAA). In some embodiments, the cell-surface antigen is expressed on T cells.

[0021] In some embodiments, the cell surface antigen expressed on T cells is CD3. In some embodiments, the cell surface antigen expressed on T cells is CD3ε.

[0022] In some embodiments, the DAA is UL16 binding protein 2 (ULBP2). In some embodiments, the DAA is UL16 binding protein 5 (ULBP5). In some embodiments, the DAA is UL16 binding protein 6 (ULBP6).

[0023] In some embodiments, the polypeptide is fused to the N-terminus or C-terminus of H1 and / or H2. In some embodiments, the polypeptide is fused to the N-terminus of H1. In some embodiments, the polypeptide is fused to the N-terminus of H2. In some embodiments, the polypeptide is fused to the C-terminus of H1. In some embodiments, the polypeptide is fused to the C-terminus of H2. In some embodiments, the polypeptide is CD58, IL-7, or a fragment thereof. In some embodiments, the polypeptide is the CD58 extracellular domain (CD58-ECD). In some embodiments, the polypeptide is the CD58 variable domain (CD58v*).

[0024] In some embodiments, the polypeptides are fused via a linker peptide. In some embodiments, the linker peptide is at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 amino acid residues in length. In some embodiments, the linker peptide comprises the amino acid sequence of SEQ ID NOs: 52-54.

[0025] In some embodiments, the antibody comprises: i) amino acid at position 87 (Kabat numbering) of VH1 and / or VH2 is G; and ii) amino acid at position 45 (Kabat numbering) of VL1 and / or VL2 is W.

[0026] In some embodiments, the antibody comprises: i) CH1 H3 C at position 349, S at position 366, A at position 368, and V at position 407 (EU numbering); and CH2 H3 C at position 354 and W at position 366 (EU numbering); ii) CH2 H3 C at position 349, S at position 366, A at position 368, and V at position 407 (EU numbering); and CH1 H3 C at position 354 and W at position 366 (EU numbering); iii) CH1 H3 C at position 354, S at position 366, A at position 368, and V at position 407 (EU numbering); and CH2 H3 C at position 349 and W at position 366 (EU numbering); or iv) CH2 H3C at position 354, S at position 366, A at position 368, and V at position 407 (EU numbering); and CH1 H3 It has a C at position 349 and a W at position 366 (EU numbering).

[0027] In some embodiments, CH1 H3 and / or CH2 H3 The amino acid at position 447 (EU numbering) is deleted.

[0028] In some embodiments, i) H1H and / or H2H have A at positions 234 and 235 (EU numbering); ii) H1H and / or H2H have A at positions 234, 235, and 237 (EU numbering); or iii) H1H and / or H2H have A at positions 234 and 235 and a G at position 329 (EU numbering).

[0029] In some embodiments, the antibody comprises: i) CH1 H3 and / or CH2 H3 A at position 297 (EU numbering); ii) CH1 H3 and / or CH2 H3 G at position 297 (EU numbering); or iii) CH1 H3 and / or CH2 H3 At position 297 (EU numbering) there is an S. In some embodiments, CH1 H3 and / or CH2 H3 There is an S at position 331 (EU numbering).

[0030] In some embodiments, the antibody comprises: i) CH1 H2 and / or CH2 H2 C at position 370 (Kabat numbering); and ii) CH1 H2 and / or CH2 H2 There is a C at position 375 (Kabat numbering).

[0031] In some embodiments, a) H1 and L1 comprise the following: i. the amino acid at position 39 (Kabat numbering) of VH1 is K, and the amino acid at position 38 (Kabat numbering) of VL1 is D; ii. CH1 H1 The amino acid at position 185 (EU numbering) of CL1 is K, and the amino acid at position 137 (EU numbering) of CL1 is D; iii. CH1 H1the amino acid at position 128 (EU numbering) of VH2 is C, and the amino acid at position 118 (EU numbering) of CL1 is C; and iv. the amino acid at position 220 (EU numbering) of H1H is S, and the amino acid at position 214 (EU numbering) of CL1 is S; and b) H2 and L2 include the following: i. the amino acid at position 39 (Kabat numbering) of VH2 is D, and the amino acid at position 38 (Kabat numbering) of VL2 is K; and ii. CH2 H1 The 147th amino acid (EU numbering) of is D, and the 180th amino acid (EU numbering) of CL2 is R.

[0032] In some embodiments, a) H1 and L1 comprise the following: i. the amino acid at position 39 (Kabat numbering) of VH1 is K, and the amino acid at position 38 (Kabat numbering) of VL1 is D; and ii. CH1 H1 the amino acid at position 185 (EU numbering) of VH2 is K, and the amino acid at position 137 (EU numbering) of CL1 is D; and b) H2 and L2 comprise the following: i. the amino acid at position 39 (Kabat numbering) of VH2 is D, and the amino acid at position 38 (Kabat numbering) of VL2 is K; ii. CH2 H1 The amino acid at position 147 (EU numbering) of CL2 is D, and the amino acid at position 180 (EU numbering) of CL2 is R; iii. CH2 H1 The amino acid at position 134 (EU numbering) of is C, and the amino acid at position 116 (EU numbering) of CL2 is C; and the amino acid at position 220 (EU numbering) of iv.H2H is S, and the amino acid at position 214 (EU numbering) of CL2 is S.

[0033] In some embodiments, a) H1 and L1 comprise the following: i. the amino acid at position 39 (Kabat numbering) of VH1 is K, and the amino acid at position 38 (Kabat numbering) of VL1 is D; ii. CH1 H1 the amino acid at position 185 (EU numbering) of VH2 is K, and the amino acid at position 137 (EU numbering) of CL1 is D; and b) H2 and L2 comprise the following: i. the amino acid at position 39 (Kabat numbering) of VH2 is D, and the amino acid at position 38 (Kabat numbering) of VL2 is K; ii. CH2 H1 The amino acid at position 147 (EU numbering) of CL2 is D, and the amino acid at position 180 (EU numbering) of CL2 is R; iii. CH2 H1The amino acid at position 136 (EU numbering) of iv.H2H is C, and the amino acid at position 114 (EU numbering) of CL2 is C; and the amino acid at position 220 (EU numbering) of iv.H2H is S, and the amino acid at position 214 (EU numbering) of CL2 is S.

[0034] In some embodiments, a) H1 and L1 comprise the following: i. the amino acid at position 39 (Kabat numbering) of VH1 is K, and the amino acid at position 38 (Kabat numbering) of VL1 is D; ii. CH1 H1 The amino acid at position 147 (EU numbering) of CL1 is K, and the amino acid at position 131 (EU numbering) of CL1 is D; iii. CH1 H1 the amino acid at position 173 (EU numbering) of VH2 is C, and the amino acid at position 162 (EU numbering) of CL1 is C; iv. the amino acid at position 220 (EU numbering) of H1H is S, and the amino acid at position 214 (EU numbering) of CL1 is S; and b) H2 and L2 include the following: i. the amino acid at position 39 (Kabat numbering) of VH2 is D, and the amino acid at position 38 (Kabat numbering) of VL2 is K; and ii. CH2 H1 The 147th amino acid (EU numbering) of is D, and the 180th amino acid (EU numbering) of CL2 is R.

[0035] In some embodiments, a) H1 and L1 comprise the following: i. the amino acid at position 39 (Kabat numbering) of VH1 is K, and the amino acid at position 38 (Kabat numbering) of VL1 is D; ii. CH1 H1 The amino acid at position 185 (EU numbering) of CL1 is K, and the amino acid at position 137 (EU numbering) of CL1 is D; iii. CH1 H1 the amino acid at position 173 (EU numbering) of VH2 is C, and the amino acid at position 162 (EU numbering) of CL1 is C; and iv. the amino acid at position 220 (EU numbering) of H1H is S, and the amino acid at position 214 (EU numbering) of CL1 is S; and b) H2 and L2 include the following: i. the amino acid at position 39 (Kabat numbering) of VH2 is D, and the amino acid at position 38 (Kabat numbering) of VL2 is K; and ii. CH2 H1 The 147th amino acid (EU numbering) of is D, and the 180th amino acid (EU numbering) of CL2 is R.

[0036] In some embodiments, a) H1 and L1 comprise the following: i. the amino acid at position 39 (Kabat numbering) of VH1 is K, and the amino acid at position 38 (Kabat numbering) of VL1 is D; ii. CH1H1 the amino acid at position 185 (EU numbering) of VH2 is K, and the amino acid at position 137 (EU numbering) of CL1 is D; and b) H2 and L2 comprise the following: i. the amino acid at position 39 (Kabat numbering) of VH2 is D, and the amino acid at position 38 (Kabat numbering) of VL2 is K; ii. CH2 H1 The amino acid at position 147 (EU numbering) of CL2 is D, and the amino acid at position 180 (EU numbering) of CL2 is R; iii. CH2 H1 The amino acid at position 131 (EU numbering) of is C, and the amino acid at position 114 (EU numbering) of CL2 is C; and the amino acid at position 220 (EU numbering) of iv.H2H is S, and the amino acid at position 214 (EU numbering) of CL2 is S.

[0037] In some embodiments, a) H1 and L1 comprise the following: i. the amino acid at position 39 (Kabat numbering) of VH1 is K, and the amino acid at position 38 (Kabat numbering) of VL1 is D; ii. CH1 H1 the amino acid at position 185 (EU numbering) of VH2 is K, and the amino acid at position 137 (EU numbering) of CL1 is D; and b) H2 and L2 comprise the following: i. the amino acid at position 39 (Kabat numbering) of VH2 is D, and the amino acid at position 38 (Kabat numbering) of VL2 is K; ii. CH2 H1 The amino acid at position 187 (EU numbering) of CL2 is D, and the amino acid at position 138 (EU numbering) of CL2 is K; iii. CH2 H1 The amino acid at position 170 (EU numbering) of iv.H2H is C, and the amino acid at position 162 (EU numbering) of CL2 is C; and the amino acid at position 220 (EU numbering) of iv.H2H is S, and the amino acid at position 214 (EU numbering) of CL2 is S.

[0038] In some embodiments, a) H1 and L1 comprise the following: i. the amino acid at position 39 (Kabat numbering) of VH1 is K, and the amino acid at position 38 (Kabat numbering) of VL1 is D; ii. CH1 H1 the amino acid at position 185 (EU numbering) of VH2 is K, the amino acid at position 137 (EU numbering) of CL1 is K; and iii. the amino acid at position 179 (EU numbering) of CL1 is E; and b) H2 and L2 comprise the following: i. the amino acid at position 39 (Kabat numbering) of VH2 is D, and the amino acid at position 38 (Kabat numbering) of VL2 is K; ii. CH2 H1The amino acid at position 187 (EU numbering) of CL2 is D, and the amino acid at position 138 (EU numbering) of CL2 is K; iii. CH2 H1 The amino acid at position 171 (EU numbering) of iv.H2H is C, and the amino acid at position 162 (EU numbering) of CL2 is C; and the amino acid at position 220 (EU numbering) of iv.H2H is S, and the amino acid at position 214 (EU numbering) of CL2 is S.

[0039] In some embodiments, a) H1 and L1 comprise the following: i. the amino acid at position 39 (Kabat numbering) of VH1 is K, and the amino acid at position 38 (Kabat numbering) of VL1 is D; ii. CH1 H1 the amino acid at position 185 (EU numbering) of VH2 is K, and the amino acid at position 137 (EU numbering) of CL1 is D; and b) H2 and L2 comprise the following: i. the amino acid at position 39 (Kabat numbering) of VH2 is D, and the amino acid at position 38 (Kabat numbering) of VL2 is K; ii. CH2 H1 The amino acid at position 187 (EU numbering) of CL2 is D, and the amino acid at position 138 (EU numbering) of CL2 is K; iii. CH2 H1 The amino acid at position 171 (EU numbering) of iv.H2H is C, and the amino acid at position 162 (EU numbering) of CL2 is C; and the amino acid at position 220 (EU numbering) of iv.H2H is S, and the amino acid at position 214 (EU numbering) of CL2 is S.

[0040] In some embodiments, a) H1 and L1 comprise the following: i. the amino acid at position 39 (Kabat numbering) of VH1 is K, and the amino acid at position 38 (Kabat numbering) of VL1 is D; ii. CH1 H1 the amino acid at position 185 (EU numbering) of VH2 is E, and the amino acid at position 137 (EU numbering) of CL1 is K; and iii. the amino acid at position 179 (EU numbering) of CL1 is E; and b) H2 and L2 include the following: i. the amino acid at position 39 (Kabat numbering) of VH2 is D, and the amino acid at position 38 (Kabat numbering) of VL2 is K; ii. CH2 H1 The amino acid at position 147 (EU numbering) of CL2 is D, and the amino acid at position 180 (EU numbering) of CL2 is R; iii. CH2 H1 The amino acid at position 171 (EU numbering) of iv.H2H is C, and the amino acid at position 162 (EU numbering) of CL2 is C; and the amino acid at position 220 (EU numbering) of iv.H2H is S, and the amino acid at position 214 (EU numbering) of CL2 is S.

[0041] In some embodiments, a) H1 and L1 comprise the following: i. the amino acid at position 39 (Kabat numbering) of VH1 is K, and the amino acid at position 38 (Kabat numbering) of VL1 is D; ii. CH1 H1 the amino acid at position 185 (EU numbering) of VH2 is K, and the amino acid at position 137 (EU numbering) of CL1 is D; and b) H2 and L2 comprise the following: i. the amino acid at position 39 (Kabat numbering) of VH2 is D, and the amino acid at position 38 (Kabat numbering) of VL2 is K; ii. CH2 H1 The amino acid at position 147 (EU numbering) of CL2 is D, and the amino acid at position 180 (EU numbering) of CL2 is R; iii. CH2 H1 The amino acid at position 171 (EU numbering) of iv.H2H is C, and the amino acid at position 162 (EU numbering) of CL2 is C; and the amino acid at position 220 (EU numbering) of iv.H2H is S, and the amino acid at position 214 (EU numbering) of CL2 is S.

[0042] In some embodiments, a) H1 and L1 comprise the following: amino acid at position 39 (Kabat numbering) of VH1 is K, and amino acid at position 38 (Kabat numbering) of VL1 is D; CH1 H1 The amino acid at position 147 (EU numbering) of CL1 is K, and the amino acid at position 131 (EU numbering) of CL1 is D; and CH1 H1 the amino acid at position 185 (EU numbering) of VH2 is K, the amino acid at position 137 (EU numbering) of CL1 is K; and b) H2 and L2 comprise the following: the amino acid at position 39 (Kabat numbering) of VH2 is D, and the amino acid at position 38 (Kabat numbering) of VL2 is K; CH2 H1 The amino acid at position 147 (EU numbering) of CL2 is D, and the amino acid at position 180 (EU numbering) of CL2 is R; CH2 H1 The amino acid at position 171 (EU numbering) of H2H is C, and the amino acid at position 162 (EU numbering) of CL2 is C; and the amino acid at position 220 (EU numbering) of H2H is S, and the amino acid at position 214 (EU numbering) of CL2 is S.

[0043] In some embodiments, a) H1 and L1 comprise the following: i. the amino acid at position 39 (Kabat numbering) of VH1 is D, and the amino acid at position 38 (Kabat numbering) of VL1 is K; and ii. CH1 H1the amino acid at position 185 (EU numbering) of VH2 is E, and the amino acid at position 137 (EU numbering) of CL1 is K; and b) H2 and L2 comprise the following: i. the amino acid at position 39 (Kabat numbering) of VH2 is K, and the amino acid at position 38 (Kabat numbering) of VL2 is D; ii. CH2 H1 The amino acid at position 187 (EU numbering) of CL2 is D, and the amino acid at position 138 (EU numbering) of CL2 is K; iii. CH2 H1 The amino acid at position 136 (EU numbering) of is C, and the amino acid at position 114 (EU numbering) of CL2 is C; and the amino acid at position 220 (EU numbering) of iv.H2H is S, and the amino acid at position 214 (EU numbering) of CL2 is S.

[0044] In some embodiments, a) H1 and L1 comprise the following: i. the amino acid at position 39 (Kabat numbering) of VH1 is D, and the amino acid at position 38 (Kabat numbering) of VL1 is K; and ii. CH1 H1 the amino acid at position 185 (EU numbering) of VH2 is E, and the amino acid at position 137 (EU numbering) of CL1 is K; and b) H2 and L2 comprise the following: i. the amino acid at position 39 (Kabat numbering) of VH2 is K, and the amino acid at position 38 (Kabat numbering) of VL2 is D; ii. CH2 H1 The amino acid at position 187 (EU numbering) of CL2 is D, and the amino acid at position 138 (EU numbering) of CL2 is K; iii. CH2 H1 The amino acid at position 171 (EU numbering) of iv.H2H is C, and the amino acid at position 162 (EU numbering) of CL2 is C; and the amino acid at position 220 (EU numbering) of iv.H2H is S, and the amino acid at position 214 (EU numbering) of CL2 is S.

[0045] In some embodiments, a) H1 and L1 comprise the following: i. the amino acid at position 39 (Kabat numbering) of VH1 is D, and the amino acid at position 38 (Kabat numbering) of VL1 is K; and ii. CH1 H1 the amino acid at position 185 (EU numbering) of VH2 is K, and the amino acid at position 137 (EU numbering) of CL1 is D; and b) H2 and L2 comprise the following: i. the amino acid at position 39 (Kabat numbering) of VH2 is K, and the amino acid at position 38 (Kabat numbering) of VL2 is D; ii. CH2 H1 The amino acid at position 187 (EU numbering) of CL2 is D, and the amino acid at position 138 (EU numbering) of CL2 is K; iii. CH2 H1The amino acid at position 171 (EU numbering) of iv.H2H is C, and the amino acid at position 162 (EU numbering) of CL2 is C; and the amino acid at position 220 (EU numbering) of iv.H2H is S, and the amino acid at position 214 (EU numbering) of CL2 is S.

[0046] In some embodiments, a) H1 and L1 comprise the following: i. the amino acid at position 39 (Kabat numbering) of VH1 is D, and the amino acid at position 38 (Kabat numbering) of VL1 is K; ii. CH1 H1 The amino acid at position 147 (EU numbering) of CL1 is K, and the amino acid at position 131 (EU numbering) of CL1 is D; iii. CH1 H1 The amino acid at position 185 (EU numbering) of CL1 is E, and the amino acid at position 137 (EU numbering) of CL1 is K; and iv. CH1 H1 the amino acid at position 145 (EU numbering) of VH2 is S, and the amino acid at position 180 (EU numbering) of CL1 is E; and b) H2 and L2 comprise the following: i. the amino acid at position 39 (Kabat numbering) of VH2 is K, and the amino acid at position 38 (Kabat numbering) of VL2 is D; ii. CH2 H1 the amino acid at position 187 (EU numbering) of VH2 is D, and the amino acid at position 138 (EU numbering) of CL2 is K; iii. the amino acid at position 170 (EU numbering) of VH2 is C, and the amino acid at position 162 (EU numbering) of VL2 is C; and iv. the amino acid at position 220 (EU numbering) of H2H is S, and the amino acid at position 214 (EU numbering) of CL2 is S.

[0047] In some embodiments, a) H1 and L1 comprise the following: i. the amino acid at position 39 (Kabat numbering) of VH1 is K, and the amino acid at position 38 (Kabat numbering) of VL1 is D; ii. CH1 H1 The amino acid at position 147 (EU numbering) of CL1 is D, and the amino acid at position 131 (EU numbering) of CL1 is K; and iii. CH1 H1 the amino acid at position 185 (EU numbering) of VH2 is E, and the amino acid at position 137 (EU numbering) of CL1 is D; and b) H2 and L2 comprise the following: i. the amino acid at position 39 (Kabat numbering) of VH2 is D, and the amino acid at position 38 (Kabat numbering) of VL2 is K; ii. CH2 H1 The amino acid at position 187 (EU numbering) of CL2 is D, and the amino acid at position 137 (EU numbering) of CL2 is K; iii. the amino acid at position 138 (EU numbering) of CL2 is R; iv. CH2 H1The amino acid at position 170 (EU numbering) of v.H2H is C, and the amino acid at position 162 (EU numbering) of CL2 is C; and the amino acid at position 220 (EU numbering) of v.H2H is S, and the amino acid at position 214 (EU numbering) of CL2 is S.

[0048] In some embodiments, a) H1 and L1 comprise the following: i. the amino acid at position 39 (Kabat numbering) of VH1 is K, and the amino acid at position 38 (Kabat numbering) of VL1 is D; ii. CH1 H1 The amino acid at position 147 (EU numbering) of CL1 is D, and the amino acid at position 131 (EU numbering) of CL1 is K; and iii. CH1 H1 the amino acid at position 145 (EU numbering) of VH2 is S; and b) H2 and L2 comprise the following: i. the amino acid at position 39 (Kabat numbering) of VH2 is D, and the amino acid at position 38 (Kabat numbering) of VL2 is K; ii. CH2 H1 The amino acid at position 187 (EU numbering) of CL2 is D, and the amino acid at position 138 (EU numbering) of CL2 is K; iii. CH2 H1 The amino acid at position 170 (EU numbering) of iv.H2H is C, and the amino acid at position 162 (EU numbering) of CL2 is C; and the amino acid at position 220 (EU numbering) of iv.H2H is S, and the amino acid at position 214 (EU numbering) of CL2 is S.

[0049] In some embodiments, a) H1 and L1 comprise the following: i. the amino acid at position 39 (Kabat numbering) of VH1 is D, and the amino acid at position 38 (Kabat numbering) of VL1 is K; ii. CH1 H1 The amino acid at position 147 (EU numbering) of CL1 is K, and the amino acid at position 131 (EU numbering) of CL1 is D; and iii. CH1 H1 the amino acid at position 185 (EU numbering) of VH2 is E, and the amino acid at position 137 (EU numbering) of CL1 is K; and b) H2 and L2 comprise the following: i. the amino acid at position 39 (Kabat numbering) of VH2 is D, and the amino acid at position 38 (Kabat numbering) of VL2 is K; ii. CH2 H1 The amino acid at position 147 (EU numbering) of CL2 is D, and the amino acid at position 180 (EU numbering) of CL2 is R; iii. CH2 H1 The amino acid at position 136 (EU numbering) of is C, and the amino acid at position 114 (EU numbering) of CL2 is C; and the amino acid at position 220 (EU numbering) of iv.H2H is S, and the amino acid at position 214 (EU numbering) of CL2 is S.

[0050] In some embodiments, a) H1 and L1 comprise the following: i. the amino acid at position 39 (Kabat numbering) of VH1 is K, and the amino acid at position 38 (Kabat numbering) of VL1 is E; ii. CH1 H1 The amino acid at position 147 (EU numbering) of CL1 is K, and the amino acid at position 131 (EU numbering) of CL1 is D; and iii. CH1 H1 the amino acid at position 185 (EU numbering) of VH2 is E, and the amino acid at position 137 (EU numbering) of CL1 is K; and b) H2 and L2 comprise the following: i. the amino acid at position 39 (Kabat numbering) of VH2 is K, and the amino acid at position 38 (Kabat numbering) of VL2 is K; ii. CH2 H1 The amino acid at position 187 (EU numbering) of CL2 is D, and the amino acid at position 138 (EU numbering) of CL2 is K; iii. CH2 H1 The amino acid at position 136 (EU numbering) of is C, and the amino acid at position 114 (EU numbering) of CL2 is C; and the amino acid at position 220 (EU numbering) of iv.H2H is S, and the amino acid at position 214 (EU numbering) of CL2 is S.

[0051] In some embodiments, a) H1 and L1 comprise the following: i. the amino acid at position 39 (Kabat numbering) of VH1 is K, and the amino acid at position 38 (Kabat numbering) of VL1 is D; ii. CH1 H1 The amino acid at position 147 (EU numbering) of CL1 is D, and the amino acid at position 131 (EU numbering) of CL1 is K; and iii. CH1 H1 the amino acid at position 185 (EU numbering) of VH2 is D, and the amino acid at position 137 (EU numbering) of CL1 is K; and b) H2 and L2 comprise the following: i. the amino acid at position 39 (Kabat numbering) of VH2 is K, and the amino acid at position 38 (Kabat numbering) of VL2 is K; ii. CH2 H1 The amino acid at position 187 (EU numbering) of CL2 is D, and the amino acid at position 138 (EU numbering) of CL2 is K; iii. CH2 H1 The amino acid at position 171 (EU numbering) of iv.H2H is C, and the amino acid at position 162 (EU numbering) of CL2 is C; and the amino acid at position 220 (EU numbering) of iv.H2H is S, and the amino acid at position 214 (EU numbering) of CL2 is S.

[0052] In some embodiments, i) the amino acid at position 87 (Kabat numbering) of VH1 and / or VH2 is G; and ii) the amino acid at position 45 (Kabat numbering) of VL1 and / or VL2 is W.

[0053] In some embodiments, i) CH1 H3 C at position 349, S at position 366, A at position 368, and V at position 407 (EU numbering); and CH2 H3 C at position 354 and W at position 366 (EU numbering); ii) CH2 H3 C at position 349, S at position 366, A at position 368, and V at position 407 (EU numbering); and CH1 H3 C at position 354 and W at position 366 (EU numbering); iii) CH1 H3 C at position 354, S at position 366, A at position 368, and V at position 407 (EU numbering); and CH2 H3 C at position 349 and W at position 366 (EU numbering); or iv) CH2 H3 C at position 354, S at position 366, A at position 368, and V at position 407 (EU numbering); and CH1 H3 There is a C at position 349 and a W at position 366 (EU numbering). In some embodiments, CH1 H3 and / or CH2 H3 The amino acid at position 447 (EU numbering) is deleted.

[0054] In some embodiments, i) H1H and / or H2H have A at positions 234 and 235 (EU numbering); ii) H1H and / or H2H have A at positions 234, 235, and 237 (EU numbering); or iii) H1H and / or H2H have A at positions 234 and 235 and a G at position 329 (EU numbering).

[0055] In some embodiments, i) CH1 H3 and / or CH2 H3 A at position 297 (EU numbering); ii) CH1 H3 and / or CH2 H3 G at position 297 (EU numbering); or iii) CH1 H3 and / or CH2 H3 At position 297 (EU numbering) there is an S. In some embodiments, CH1 H3 and / or CH2 H3There is an S at position 331 (EU numbering).

[0056] In some embodiments, the antibody comprises: i) CH1 H2 and / or CH2 H2 C at position 370 (Kabat numbering); and ii) CH1 H2 and / or CH2 H2 There is a C at position 375 (Kabat numbering).

[0057] In some embodiments, a) VH1 comprises the amino acid sequence of SEQ ID NO: 13; and b) VL1 comprises the amino acid sequence of SEQ ID NO: 22.

[0058] In some embodiments, a) VH1 comprises the amino acid sequence of EVQLLESGGGLVQPGGS LKLSCAASGFTFNX1YAMX2WVRX3APGKGLEWVAX4IRSKYNNYAT YYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGX5FGNX6X7YVSWFAYWGQGTLVTVSS (SEQ ID NO: 440), wherein X1 of SEQ ID NO: 440 is D or T, wherein X2 of SEQ ID NO: 440 is D or N, wherein X3 of SEQ ID NO: 440 is D or K, wherein X4 of SEQ ID NO: 440 is K or R, wherein X5 of SEQ ID NO: 440 is N or T, wherein X6 of SEQ ID NO: 440 is D or N, and wherein X7 of SEQ ID NO: 440 is D or N. NO:440 X7 is D or S; and b) VL1 comprises the amino acid sequence of ELVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQX1KPGQAP RGLIGX2TNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCAL WX3SX4LWVFGGGTKLTVL (SEQ ID NO:441), wherein X1 of SEQ ID NO:441 is an amino acid sequence of E or D, wherein X2 of SEQ ID NO:441 is D or G, wherein X3 of SEQ ID NO:441 is D or Y, and wherein X4 of SEQ ID NO:441 is D, N or Q.

[0059] In some embodiments, a) VH2 comprises: a complementarity determining region 1 (VH2) comprising the amino acid sequence of SEQ ID NO: 428 CDR1 ); Complementarity determining region 2 (VH2) comprising the amino acid sequence of SEQ ID NO: 430 CDR2); and a complementarity determining region 3 (VH2 comprising the amino acid sequence SEQ ID NO: 432 CDR3 ); and b) VL2 comprises: a complementarity determining region 1 (VL2 comprising the amino acid sequence of SEQ ID NO: 433 CDR1 ); Complementarity determining region 2 (VL2) comprising the amino acid sequence of SEQ ID NO: 434 CDR2 ); and a complementarity determining region 3 (VL2 comprising the amino acid sequence of SEQ ID NO: 435 CDR3 ).

[0060] In some embodiments, a) VH2 comprises: a complementarity determining region 1 (VH2) comprising the amino acid sequence of SEQ ID NO: 5 CDR1 ); Complementarity determining region 2 (VH2) comprising the amino acid sequence of SEQ ID NO: 7 CDR2 ); and a complementarity determining region 3 (VH2 comprising the amino acid sequence SEQ ID NO: 9 CDR3 ); and b) VL2 comprises: a complementary determining region 1 (VL2) comprising the amino acid sequence SEQ ID NO: 10 CDR1 ); Complementarity determining region 2 (VL2) comprising the amino acid sequence SEQ ID NO: 11 CDR2 ); and a complementary determining region 3 (VL2 comprising the amino acid sequence SEQ ID NO: 12 CDR3 ).

[0061] In some embodiments, CD58 comprises the amino acid sequence of SEQ ID NOs: 49-50. In some embodiments, IL-7 comprises the amino acid sequence of SEQ ID NO: 51.

[0062] The present disclosure provides polynucleotides comprising a nucleic acid sequence encoding any one of the antibodies disclosed herein. The present disclosure also provides vectors comprising the polynucleotides disclosed herein.

[0063] The present disclosure also provides a pharmaceutical composition comprising any one of the antibodies, polynucleotides or vectors of the present invention and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition is packaged in liposomes or lipid nanoparticles.

[0064] The present disclosure provides a method for treating cancer in a subject in need thereof, comprising administering a therapeutically effective amount of any pharmaceutical composition of the present disclosure. The present disclosure also provides a method for retargeting T cells in a subject in need thereof, comprising administering a therapeutically effective amount of any pharmaceutical composition of the present disclosure. The present disclosure also provides a method for activating T cells in a subject in need thereof, comprising administering a therapeutically effective amount of any pharmaceutical composition of the present disclosure.

[0065] In some embodiments, the subject has cancer. In some embodiments, the cancer is a ULBP2-positive cancer. In some embodiments, the cancer is a primary tumor, a metastatic cancer, a multidrug-resistant cancer, an advanced tumor, or a recurrent cancer. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is urothelial cancer, lung cancer, brain cancer, head and neck cancer, breast cancer, skin cancer, melanoma, liver cancer, pancreatic cancer, stomach cancer, colon cancer, rectal cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer, testicular cancer, skin cancer, or esophageal cancer.

[0066] In some embodiments, the method further comprises administering a therapeutically effective amount of a ligand or cytokine or an agnostic antibody that binds to a receptor of the ligand or cytokine. In some embodiments, the ligand is CD48, CD58, CD86, TNFSF9, OX40L, 4-1BBL, GITL, CD70, CD80, MR1, TNFSF4, ICOSL, ICOSLG, MICA, MICB, ULBP1, ULBP2, ULBP3, RAET1G, and RAET1L. In some embodiments, the cytokine is IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, or IL-21. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] This patent or application file contains at least one drawing printed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the U.S. Patent and Trademark Office upon request and payment of the necessary fee.

[0068] Figure 1 AD depicts schematic diagrams of antibodies with charged pair mutations, disulfide bond relocation, and knob-into-hole mutations. The gray shaded area represents the first heavy chain polypeptide (H1), which has a heavy chain variable region (VH1), a constant region 1 domain (CH1 H1 ), hinge region (H1H), constant region 2 domain (CH1 H2 ) and constant region 3 domain (CH1 H3 ); and a first light chain polypeptide (L1), which comprises a variable region (VL1) and a constant region (CL1). The white shaded area represents a second heavy chain polypeptide (H2), which comprises a variable region (VH2) and a constant region (CH2), wherein the constant region (CH2) has a constant region 1 domain (CH2 H1 ), hinge region (H2H), constant region 2 domain (CH2 H2 ) and constant region 3 domain (CH2 H3 ); and a second light chain polypeptide (L2) comprising a variable region (VL2) and a constant region (CL2). The + and - symbols between the antigen binding domains represent charged pair mutations. CH1 H1and CL1 domain and CH2 H1 The lines between the CH1 and CL2 domains represent disulfide bonds, where solid lines represent endogenous disulfide bonds and dashed lines represent relocated disulfide bonds. H3 and CH2 H3 The protrusions and depressions between the domains represent knob-into-hole mutations. Charged pair mutations, disulfide bond repositioning, and knob-into-hole mutations provide increased heterodimerization of the heavy and light chains, which facilitates the production and purification of the bispecific antibodies of the present disclosure.

[0069] Figures 2A-2B are two graphs depicting the biophysical characteristics of light chain paired bispecific antibodies EIP0187 (light chain pairing C), EIP0205 (light chain pairing D), EIP0356 (light chain pairing O), and EIP0377 (light chain pairing P) compared to the EIP0112 Crossmab control antibody. Figure 2A Size exclusion chromatogram obtained from tandem protein A and size exclusion chromatography purification of light chain paired bispecific antibodies. Figure 2B Depicted are differential scanning calorimetry analyses of light chain paired bispecific antibodies.

[0070] Figures 3A-3B yes Figures 2A-2B NuPAGE gel analysis of representative variants of the light chain paired bispecific antibodies depicted in . Figure 3A This is non-reducing NuPAGE analysis. Figure 3B is a reducing NuPAGE analysis showing the intact bispecific antibody and the expected protein masses of the heavy and light chains together.

[0071] Figures 4A-4B Yes Display Figures 2A-2B A series of line graphs showing antigen binding of the light chain-paired bispecific antibodies depicted in , compared to isotype and bispecific antibody controls. Figure 4A is a line graph depicting antigen binding of light chain paired bispecific antibody variants as determined by sandwich ELISA, where the antibodies were captured on plates coated with CD3ε. Figure 4B is a line graph depicting antigen binding of light chain-paired bispecific antibody variants as determined by sandwich ELISA, in which the antibodies were captured on plates coated with recombinant ULBP2.

[0072] Figure 5A is a mass spectrometric analysis of EIP0205 showing the intact mass after PNGase F deglycosylation under non-reducing conditions and chromatographic separation using a reversed-phase C4 column.

[0073] Figure 5BFigure 2 is a mass spectrometry analysis of EIP0205 showing a decrease in heavy chain mass after rapid PNGase F deglycosylation under reducing conditions and chromatographic separation using a reversed-phase C4 column.

[0074] Figure 5C Figure 2 is a mass spectrometry analysis of EIP0205 showing a reduction in light chain mass after rapid PNGase F deglycosylation under reducing conditions and chromatographic separation using a reversed-phase C4 column.

[0075] Figure 5D Figure 5 is a mass spectrometric analysis of EIP0187 showing the intact mass after PNGase F deglycosylation under non-reducing conditions and chromatographic separation using a reversed-phase C4 column.

[0076] Figure 5E Figure 2 is a mass spectrometry analysis of EIP0187 showing a decrease in heavy chain mass after rapid PNGase F deglycosylation under reducing conditions and chromatography using a reversed-phase C4 column.

[0077] Figure 5F Figure 2 is a mass spectrometry analysis of EIP0187 showing a reduction in light chain mass following rapid PNGase F deglycosylation under reducing conditions and chromatography using a reversed-phase C4 column.

[0078] Figure 6 It is a depiction Figures 2A-2B Line graph of functional evaluation (cytotoxicity) of the light chain paired bispecific antibodies depicted in FIG5 compared to a bispecific control antibody (EIP0112) in a tumor cell and T cell co-culture assay.

[0079] Figure 7A is a chromatogram of the bispecific antibody variant obtained by tandem purification.

[0080] Figure 7B is a non-reducing NuPAGE analysis showing the protein mass of the intact bispecific antibody variants.

[0081] Figure 7C is a reducing NuPAGE analysis showing the protein masses of the heavy and light chains of the bispecific antibody variants.

[0082] Figure 7D is a line graph depicting antigen binding of light chain paired bispecific antibody variants as determined by sandwich ELISA, where the antibodies were captured on plates coated with CD3ε.

[0083] Figure 7E is a line graph depicting antigen binding of light chain paired bispecific antibody variants as determined by sandwich ELISA, where the antibodies were captured on plates coated with antigen.

[0084] Figure 8Ais a line graph depicting the binding of αULBP2-αCD3 bispecific antibody variants to human CD3ε as determined by ELISA.

[0085] Figure 8B is a line graph depicting the binding of αULBP2-αCD3 bispecific variants to cynomolgus monkey CD3ε as determined by ELISA.

[0086] Figures 9A-9B is a series of line graphs depicting luciferase activity in co-culture of tumor cells and Jurkat NFAT luciferase reporter cells in the presence of αULBP2-αCD3 bispecific antibody variants. Figure 9A Co-culture of SiHa tumor cells is depicted.

[0087] Figure 9B Depicted is the co-culture of HCT116 tumor cells.

[0088] Figures 10A-10C It depicts Figures 9A-9B A series of line graphs showing T cell-mediated cytotoxicity against three tumor cell lines in the presence of αULBP2-αCD3 bispecific antibody variants. Figure 10A Cytotoxicity of HCT116 tumor cells is depicted. Figure 10B The cytotoxicity of MDA-MB-231GFP tumor cells is depicted.

[0089] Figure 10C Depicted are the cytotoxicity of SiHa tumor cells in the presence of αULBP2-αCD3 bispecific antibody variants.

[0090] Figures 11A-11C It depicts Figures 9A-9B A series of line graphs showing cytokine secretion by activated T cells in the presence of αULBP2-αCD3 bispecific affinity variants when co-cultured with SiHa tumor cells. Figure 11A IFNγ secretion is depicted.

[0091] Figure 11B IL-2 secretion is depicted.

[0092] Figure 11C Secretion of TNFα is depicted.

[0093] Figure 12A Depicted are bispecific antibody variants without CD58 fusion.

[0094] Figure 12B Depicts CD58 and CH1 H3 Bispecific antibody variants with carboxyl-terminal fusion.

[0095] Figure 12CDepicts CD58 and CH2 H3 Bispecific antibody variants with carboxyl-terminal fusion.

[0096] Figure 12D Depicts CD58 and CH1 H3 The carboxyl terminus of CD58 is fused to CH2 H3 Carboxyl-terminally fused bispecific antibody variants.

[0097] Figure 12E Depicted are bispecific antibody variants with CD58 fused amino-terminally to VL2.

[0098] Figure 12F Depicted are bispecific antibody variants with CD58 fused amino-terminally to VH2.

[0099] Figure 12G Depicted are bispecific antibody variants with CD58 fused amino-terminally to VL1.

[0100] Figure 12H Depicted are bispecific antibody variants with CD58 fused amino-terminally to VH1.

[0101] Figure 12L depicts bispecific antibody variants with CD58 fused to the carboxy terminus of CL2.

[0102] Figure 12J Depicted are bispecific antibody variants in which CD58 is fused to the carboxy terminus of CL1.

[0103] Figure 12K Depicted are bispecific antibody variants with CD58 fused to the carboxyl termini of CL1 and CL2.

[0104] Figure 13 Chromatograms obtained from tandem purification of costimulatory ligand or cytokine fusion bispecific variants (EIP0205, EIP0359, EIP0360, EIP0363) engineered using light chain pairing technology.

[0105] Figure 14 Depicted are differential scanning calorimetry analyses of co-stimulatory ligand or cytokine fusion αULBP2-αCD3 bispecific variants (EIP0205, EIP0359, EIP0363).

[0106] Figure 15A is a line graph depicting antigen binding of co-stimulatory ligand or cytokine fusion bispecific antibody variants to plates coated with recombinant CD3ε as determined by sandwich ELISA.

[0107] Figure 15Bis a line graph depicting antigen binding of co-stimulatory ligand or cytokine fusion bispecific antibody variants to plates coated with recombinant ULBP2 protein as determined by sandwich ELISA.

[0108] Figure 16A Depicted are cytolysis of MDA-MB-231GFP tumor cells after 7 days of incubation with naive T cells in the presence of the αULBP2-αCD3 bispecific variant.

[0109] Figure 16B Representative brightfield and fluorescence microscopy images showing naive T cell activation and MDA-MB-231 cell death.

[0110] Figure 16C Depicted are cytolysis of ULBP2-deficient MDA-MB-231GFP tumor cells after 7 days of incubation with naive T cells in the presence of αULBP2-αCD3 bispecific antibody variants.

[0111] Figures 17A-17B It is depicted in Figures 16A-16C A series of line graphs showing IFNγ secretion from naive T cells after 24 h of incubation with MDA-MB-231GFP tumor cells and ULBP2-deficient MDA-MB-231GFP tumor cells in the presence of the bispecific antibody variants. Figure 17A MDA-MB-231GFP tumor cells are described.

[0112] Figure 17B ULBP2-deficient MDA-MB-231GFP tumor cells are depicted.

[0113] Figures 18A-18B It is depicted in Figures 16A-16C A series of line graphs showing IL-2 secretion from naive T cells after 24 hours of incubation with MDA-MB-231GFP tumor cells and ULBP2-deficient MDA-MB-231GFP tumor cells in the presence of bispecific antibody variants. Figure 18A MDA-MB-231GFP tumor cells are depicted.

[0114] Figure 18B ULBP2-deficient MDA-MB-231GFP tumor cells are depicted.

[0115] Figures 19A-19B It is depicted in Figures 16A-16C A series of line graphs showing IFNγ secretion from naive T cells after 24 h of incubation with MDA-MB-231GFP tumor cells and ULBP2-deficient MDA-MB-231GFP tumor cells in the presence of the bispecific antibody variants. Figure 19AMDA-MB-231GFP tumor cells are depicted.

[0116] Figure 19B ULBP2-deficient MDA-MB-231GFP tumor cells are depicted.

[0117] Figure 20A Depicted are cytolysis of SiHa cells following 48 h incubation with activated T cells in the presence of αULBP2-αCD3 bispecific variants and αULBP2-αCD3-CD58 bispecific variants.

[0118] Figure 20B Depicted are cytolysis of SiHa cells following 48 h incubation with activated T cells in the presence of αULBP2-αCD3 bispecific variants and αULBP2-αCD3-CD58 bispecific variants.

[0119] Figure 21A Depicted are cytolysis of SiHa cells following 48 h incubation with activated T cells in the presence of αULBP2-αCD3 bispecific variants and αULBP2-αCD3-CD58 bispecific variants.

[0120] Figure 21B Depicted are cytolysis of SiHa cells following 48 h incubation with activated T cells in the presence of αULBP2-αCD3 bispecific variants and αULBP2-αCD3-CD58 bispecific variants.

[0121] Figure 22A is a line graph depicting IFNγ secretion by activated T cells after co-culture with SiHa cells for 48 hours in the presence of αULBP2-αCD3 bispecific antibody variants and αULBP2-αCD3-CD58 bispecific antibody variants.

[0122] Figure 22B is a line graph depicting IFNγ secretion by activated T cells after co-culture with SiHa cells for 48 hours in the presence of αULBP2-αCD3 bispecific antibody variants and αULBP2-αCD3-CD58 bispecific antibody variants.

[0123] Figure 23A is a line graph depicting IFNγ secretion by activated T cells after co-culture with SiHa cells for 48 hours in the presence of αULBP2-αCD3 bispecific antibody variants and αULBP2-αCD3-CD58 bispecific antibody variants.

[0124] Figure 23Bis a line graph depicting IFNγ secretion by activated T cells after co-culture with SiHa cells for 48 hours in the presence of αULBP2-αCD3 bispecific antibody variants and αULBP2-αCD3-CD58 bispecific antibody variants.

[0125] Figure 24A is a line graph depicting IL-2 secretion by activated T cells after co-culture with SiHa cells for 48 hours in the presence of αULBP2-αCD3 bispecific antibody variants and αULBP2-αCD3-CD58 bispecific antibody variants.

[0126] Figure 24B is a line graph depicting IL-2 secretion by activated T cells after co-culture with SiHa cells for 48 hours in the presence of αULBP2-αCD3 bispecific antibody variants and αULBP2-αCD3-CD58 bispecific antibody variants.

[0127] Figure 25A is a line graph depicting IL-2 secretion by activated T cells after co-culture with SiHa cells for 48 hours in the presence of αULBP2-αCD3 bispecific antibody variants and αULBP2-αCD3-CD58 bispecific antibody variants.

[0128] Figure 25B is a line graph depicting IL-2 secretion by activated T cells after co-culture with SiHa cells for 48 hours in the presence of αULBP2-αCD3 bispecific antibody variants and αULBP2-αCD3-CD58 bispecific antibody variants.

[0129] Figure 26A is a line graph depicting TNFα secretion by activated T cells after co-culture with SiHa cells for 48 hours in the presence of αULBP2-αCD3 bispecific antibody variants and αULBP2-αCD3-CD58 bispecific antibody variants.

[0130] Figure 26B is a line graph depicting TNFα secretion by activated T cells after co-culture with SiHa cells for 48 hours in the presence of αULBP2-αCD3 bispecific antibody variants and αULBP2-αCD3-CD58 bispecific antibody variants.

[0131] Figure 27A is a line graph depicting TNFα secretion by activated T cells after co-culture with SiHa cells for 48 hours in the presence of αULBP2-αCD3 bispecific antibody variants and αULBP2-αCD3-CD58 bispecific antibody variants.

[0132] Figure 27Bis a line graph depicting TNFα secretion by activated T cells after co-culture with SiHa cells for 48 hours in the presence of αULBP2-αCD3 bispecific antibody variants and αULBP2-αCD3-CD58 bispecific antibody variants.

[0133] Figure 28 is a line graph depicting cytolysis of MDA-MB-231GFP cells after 7 days of incubation with PBMCs in the presence of αULBP2-αCD3, αULBP2-αCD3-CD58 bispecific αULBP2-αCD3-CD58 variable domain fusion bispecific antibody variants.

[0134] Figure 29A is a line graph depicting cytolysis of MDA-MB-231 cells in the presence of bispecific antibody variants.

[0135] Figure 29B is a line graph depicting IFNγ secretion from MDA-MB-231 cells in the presence of bispecific antibody variants.

[0136] Figure 30 Representative microscopy images of cell lysis of MDA-MB-231-GFP cells after 48 h of incubation with naive, 3rd, and 5th round depleted T cells in the presence of bispecific antibody variants.

[0137] Figure 31 Radar graph depicting normalized levels of T cell markers IL2, IFNγ, CD25, CD69, GZMB, CD2, PD-1, CD38, and TIM3 present after 72 hours of incubation of PBMCs with MDA-MB-231 cells in the presence of bispecific antibody variants.

[0138] Figures 32A-32D is a series of graphs depicting improved tumor growth inhibition, pharmacokinetics, and survival of humanized mice following treatment with bispecific antibody variants. Figure 32A is a line graph depicting inhibition of SiHa tumor growth over time following adoptive transfer of human T cells and dosing with bispecific antibody variants EIP0542, EIP0205, and EIP0359. Figure 32B is adoptively transferred human T cells and Figure 32A Survival curves after administration of bispecific antibody variants. Figure 32C It is a depiction Figure 32A Line graph of the pharmacokinetics of bispecific antibody variants. Figure 32D is a graph depicting the pharmacokinetics of EIP0561 following administration of a 10 mg / kg dose to humanized mice.

[0139] Figures 33A-33Care a series of histograms from flow cytometric analysis of tumor-infiltrating lymphocytes in SiHa tumors treated with bispecific antibody variants 3 days after transplantation. Figure 33A An analysis of granzyme B showing that the bispecific antibody variants increased tumor cell lysis. Figure 33B An analysis of CD25 showing that the bispecific antibody variants increased T cell activation. Figure 33C An analysis of CD38 showing that the bispecific antibody variants reduced T cell exhaustion.

[0140] Figures 34A-34C This is a series of structural renderings simulating the binding of A06 and E12 to ULBP2. Figure 34A is a homology model of the ULBP2-NKG2D complex. Figure 34B This is the docking model of ULBP2 and A06 antibody. Figure 34C The docking model of ULBP2 and E12 antibodies. Residue R106 is shown as a stick model in all figures. ULBP2 is shown in dark grey, while NKG2D, A06, and E12 are shown in light grey.

[0141] Figure 35 is a line graph depicting the growth inhibition of CORL-105 tumors over time following co-transplantation of human T cells and administration of bispecific antibodies with various CD3 affinities and bispecific CD58 fusions.

[0142] Figures 36A-36F is a series of graphs depicting cytolysis of tumor cells following 48 hours of incubation with activated T cells in the presence of a bispecific CD58 fusion. Figure 36A Cytolysis of HCT116 cells is shown. Figure 36B Cytolysis of U266B1 cells is shown. Figure 36C Cytolysis of JeKo-1 cells is shown. Figure 36D Cytolysis of PSMA-low LNCAP prostate cancer cells (LNCAP-vL) is shown. Figure 36E Cytolysis of MM1s cells is shown. Figure 36F Cytolysis of Raji cells is shown.

[0143] Figure 37 is a graph depicting chromatograms obtained from tandem purification of exemplary antibodies with and without exemplary disulfide stabilizing mutations.

[0144] Figures 38A-38Dare two graphs and microscopy images depicting cytolysis of MDA-MB-231GFP tumor cells and ULBP2-deficient MDA-MB-231GFP tumor cells in the presence of αULBP2-αCD3 bispecific antibody variants after incubation with naive T cells at a 1:10 biI ratio for 5 days. Figure 38A Tumor cells incubated with different concentrations of EIP0205 are shown. Figure 38B Representative brightfield and fluorescence microscopy images of naive T cell activation and cell death in MDA-MB-231 cells incubated with EIP0205 are shown. Figure 38C Shown are tumor cells incubated with different concentrations of EIP0359. Figure 38D Representative brightfield and fluorescence microscopy images of naive T cell activation and cell death in MDA-MB-231 cells incubated with EIP0359 are shown.

[0145] Figure 39 Graph depicting cytolysis of MDA-MB-231GFP tumor cells in the presence of αULBP2-αCD3 bispecific antibody variants after incubation with naive T cells at a 1:10 ratio for 5 days.

[0146] Figures 40A-40C is a series of line graphs depicting cytolysis and cytokine secretion of tumor cells after 5 days of incubation with naive T cells at an E:T ratio of 10:1 in the presence of αULBP2-αCD3 bispecific variants and αULBP2-αCD3-CD58 bispecific variants. Figure 40A Cytolysis of tumor cells is depicted. Figure 40B Depicted is the secretion of IFNγ by activated T cells after 48 hours of coculture with tumor cells. Figure 40C Depicted is the secretion of IL-2 by activated T cells after 48 hours of coculture with tumor cells.

[0147] Figure 41 is a graph depicting killing of MDA-MB-231 tumor cells in the presence of activated T cells following incubation with activated T cells for 48 hours in the presence of αULBP2-αCD3-CD58 bispecific variants at an E:T ratio of 5:1.

[0148] Figures 42A-42F is a series of graphs depicting cytolysis of tumor cells after 2 days in the presence of activated T cells and αCD3 bispecific antibody variants comprising light chain pairings of the present disclosure compared to controls. Figure 42A JeKo-1 tumor cells with an effector to target (E:T) ratio of 5:1 are shown. Figure 42B Ramos cells with an effector to target (E:T) ratio of 10:1 are shown. Figure 42C Raji cells with an effector to target (E:T) ratio of 10:1 are shown. Figure 42D SUDHL10 cells with an effector to target (E:T) ratio of 10:1 are shown. Figure 42E MV411 cells with an effector to target (E:T) ratio of 5:1 are shown. Figure 42F OCI-AML2 cells showing an effector to target (E:T) ratio of 5:1.

[0149] Figures 43A-43B is a series of graphs depicting lysis of tumor cells after z3 days in the presence of αCD3 bispecific antibody variants comprising light chain pairings (with and without CD58 fusion molecules of the present disclosure) at an effector to target cell (E:T) ratio of 7.5:1 in the presence of naive T cells compared to controls. Figure 43A Tumor lysis of JeKo-1 cells is shown. Figure 43B Tumor lysis of MV411 cells is shown.

[0150] Figures 44A-44B is a series of graphs depicting cytolysis of tumor cells after 48 hours of incubation in the presence of activated T cells in the presence of αBCMA-αCD3 and αBCMA-αCD3-CD58 bispecific variants compared to a no antibody control. Figure 43A NCI929 tumor cells with an effector to target (E:T) ratio of 2:1 are shown. Figure 43B U266B1 cells showing an effector to target (E:T) ratio of 1.6:1. DETAILED DESCRIPTION

[0151] The production of heteromultimeric antibodies (e.g., bispecific antibodies) using conventional techniques faces many challenges, including the production of mismatched antibodies, reduced yields, and reduced / eliminated effector functions. In addition, aggregation and precipitation often occur during the preparation and assembly of heteromultimeric antibodies. Mismatching, aggregation, and precipitation of heavy and light chains can greatly reduce the yield of the desired heteromultimeric antibodies. Therefore, the compositions and methods herein are needed to produce heteromultimeric antibodies more efficiently and at higher levels.

[0152] Disclosed herein are compositions and efficient production processes / methods for economically producing heteromultimeric antibodies (e.g., bispecific antibodies) by using or modulating one or more of the following (including but not limited to): a pair of oppositely charged mutations on the light and heavy chains, a disulfide bond repositioning mutation, or a knob-into-hole mutation. The methods of the invention described herein reduce mispairing of heavy and light chains, reduce losses from protein precipitation and / or aggregation, and increase the overall yield of heteromultimeric antibody production (e.g., bispecific antibody production).

[0153] T cell retargeting (or T cell redirecting) bispecific antibodies are a new class of therapeutics that can recruit T cells to tumor cells and induce tumor-specific (but MHC-independent) T cell effector activity. Typically, T cell retargeting bispecific antibodies contain an antigen-binding domain that targets the CD3 portion of the T cell receptor for T cell recruitment and an antigen-binding domain that targets a disease-associated antigen (DAA). This targeting design promotes the recruitment of T cells and positions them in close contact with target tumor cells, leading to the formation of immune synapses, local T cell activation, and subsequent destruction of the target cells by perforins and granzymes released by T cell cytotoxic granules.

[0154] Because the CD3 binding affinity of T cell-retargeting bispecific antibodies is crucial for T cell recruitment, the present invention also contemplates the generation of a panel of antibodies that bind to human CD3 and cross-react with cynomolgus monkey CD3. Cross-reactivity with cynomolgus monkey CD3 is an important characteristic for facilitating preclinical development of T cell-retargeting bispecific antibodies incorporating the anti-CD3ε antibodies described herein. Furthermore, these anti-CD3ε antibodies exhibit varying binding affinities. The affinity of the CD3 arm of a bispecific antibody can significantly alter the functional activity of the bispecific antibody. Therefore, anti-CD3 antibodies with varying affinities are desirable and advantageous.

[0155] In addition, bispecific antibodies disclosed herein can have cytokines or costimulatory molecule fusion peptides, which are used as antagonists to suppress or block harmful interactions, or as agonists to simulate or enhance physiological responses. Physiological responses include but are not limited to T cell activation, T cell proliferation and prevention of T cell exhaustion. These characteristics are superior to traditional CD3 bispecific antibodies or tumor-targeted costimulatory receptor agonists because they cannot optimally activate T cells and induce (or promote) T cell dysfunction. Therefore, cytokines and / or costimulatory fusion peptides are conducive to enhancing the therapeutic potential of bispecific antibodies.

[0156] In summary, these features (such as pairs of oppositely charged mutations on the light and heavy chains, disulfide bond repositioning mutations, knob-into-hole mutations, CD3 affinity variants, cytokine or co-stimulatory molecule fusion peptides) have considerable advantages from a development perspective. For example, it provides T cell co-stimulation and prevents T cell exhaustion. In addition, bispecific T cell retargeting agents have drug-like properties of human monoclonal antibodies. In addition, T cell retargeting bispecific antibodies are superior to other existing therapies (such as CAR-T therapy) because they provide off-the-shelf products with a high safety profile (such as alleviating cytokine release syndrome and reducing the level of tonic signaling that causes T cell dysfunction) as well as the possibility of dose titration and escalation.

[0157] Antibody composition and structure

[0158] The present disclosure provides antibodies comprising the following domain structures: a) a first heavy chain polypeptide (H1) comprising a variable region (VH1) and a constant region (CH1) having a constant region 1 domain (CH1) H1 ), hinge region (H1H), constant region 2 domain (CH1 H2 ) and constant region 3 domain (CH1 H3 ); and a first light chain polypeptide (L1) comprising a variable region (VL1) and a constant region (CL1), and b) a second heavy chain polypeptide (H2) comprising a variable region (VH2) and a constant region (CH2) having a constant region 1 domain (CH2 H1 ), hinge region (H2H), constant region 2 domain (CH2 H2 ) and constant region 3 domain (CH2 H3 ); and a second light chain polypeptide (L2), which comprises a variable region (VL2) and a constant region (CL2). The schematic diagram of the antibody structure of the present disclosure is as follows Figure 1 As shown in A-1D.

[0159] As used herein, the term "antibody" refers to immunoglobulin (Ig) molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen. "Specifically binds" or "immunoreacts with" or "directed against" means that the antibody reacts with one or more antigenic determinants of the desired antigen and does not react with other polypeptides or with much lower affinity (K). d >10 -6 ) binding. Antibodies include, but are not limited to, polyclonal antibodies, monoclonal antibodies, and chimeric antibodies. Antibodies can be derived from recombinant sources and / or produced in transgenic animals.

[0160] The basic structural unit of known antibodies is a tetramer. Each tetramer is composed of two pairs of identical polypeptide chains, each pair of polypeptide chains having a "light" chain (about 25kDa) and a "heavy" chain (about 50-70kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids that is primarily responsible for antigen recognition. The carboxyl-terminal portion of each chain defines a constant region that is primarily responsible for effector function.

[0161] Generally speaking, antibody molecules obtained from humans belong to any of the IgG, IgM, IgA, IgE, and IgD classes, which differ from each other by the nature of the heavy chains present in the molecule. Certain classes also have subclasses, such as IgG1, IgG2, IgG4, etc. Furthermore, in humans, light chains can be either kappa chains or lambda chains. Thus, in one embodiment, the antibodies disclosed herein are IgG antibodies.

[0162] Antibodies can be purified by well-known techniques, such as affinity chromatography using protein A or protein G, which primarily provide the IgG portion of the immune serum. Subsequently or alternatively, the specific antigen or epitope thereof that is the target of the immunoglobulin being sought can be immobilized on a column to purify the immunospecific antibodies by immunoaffinity chromatography. For example, D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia PA, Vol. 14, No. 8 (April 17, 2000), pp. 25-28) discusses the purification of immunoglobulins.

[0163] The term "antibody fragment" as used herein is intended to include, but is not limited to, Fv, Fab, Fab', F(ab')2, scFv, dsFv, ds-scFv, dimers, minibodies, diabodies and multimers thereof, multispecific antibody fragments and domain antibodies. Antibody fragmentation can be performed using conventional techniques. For example, F(ab')2 fragments can be produced by treating antibodies with pepsin. The resulting F(ab')2 fragments can be treated to reduce disulfide bridges to produce Fab' fragments. Papain digestion can result in the formation of Fab fragments. Fab, Fab' and F(ab')2, scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments and other fragments can also be synthesized by recombinant techniques.

[0164] The technology can be applied to produce single-chain antibodies specific for the antigenic proteins of the present disclosure (see, e.g., U.S. Patent No. 4,946,778). In addition, the method can be applied to construct Fab expression libraries (see, e.g., Huse et al., 1989 Science 246: 1275-1281) to allow rapid and efficient identification of monoclonal Fab fragments with desired specificity for proteins or their derivatives, fragments, analogs or homologs.

[0165] As used herein, the term "epitope" refers to a site on an antigen that is recognized by the antibodies and fragments disclosed herein. The term "epitope" includes any protein determinant capable of specific binding to an immunoglobulin. Epitope determinants are typically composed of chemically active surface groups or sugar side chains of molecules (such as amino acids) and have specific three-dimensional structural characteristics and specific charge characteristics. An antibody is said to specifically bind to an antigen when the dissociation constant is <1 micromolar, e.g., <100 nM, preferably <10 nM, more preferably <1 nM.

[0166] Bispecific antibodies are monoclonal antibodies that have binding specificities for at least two different antigens. The present disclosure provides bispecific antibodies having a first antigen-binding region that binds to a first antigen (eg, CD3) and a second antigen-binding region that binds to a second antigen (eg, a disease-associated antigen).

[0167] Antibodies with more than two valencies are also contemplated. For example, trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147:60 (1991).

[0168] Antibody variants

[0169] In certain embodiments, it is contemplated that the amino acid sequence variants of the antibodies provided herein. For example, it may be necessary to improve the heavy chain heterodimerization, light chain heterodimerization, binding affinity and / or other biological properties of the antibody. The amino acid sequence variants of the antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions and / or insertions and / or substitutions of residues in the amino acid sequence of the antibody. Any combination of deletions, insertions and substitutions can be performed to obtain the final construct, with the precursor condition being that the final construct has the desired tez (e.g., light chain heterodimerization, heavy chain heterodimerization, antigen binding).

[0170] Based on common side chain properties, amino acids can be grouped as follows:

[0171] (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;

[0172] (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

[0173] (3) Acidic (negatively charged): Asp, Glu;

[0174] (4) Basic (positively charged): His, Lys, Arg;

[0175] (5) Residues that affect chain orientation: Gly, Pro;

[0176] (6) Aromatic: Trp, Tyr, Phe.

[0177] The present disclosure also encompasses functional variants of the antibodies or antigen-binding fragments described herein. As used herein, the term "functional variant" includes modifications or chemical equivalents of the amino acid and nucleic acid sequences disclosed herein that perform substantially the same function as the polypeptides or nucleic acid molecules disclosed herein in substantially the same manner. For example, functional variants of the polypeptides disclosed herein include, but are not limited to, conservative amino acid substitutions.

[0178] As used herein, "conservative amino acid substitution" refers to a substitution of one amino acid residue by another amino acid residue, thereby changing an amino acid to a different amino acid with similar biochemical properties (e.g., charge, hydrophobicity, and size). Variants of polypeptides also include additions and deletions to the polypeptide sequences disclosed herein. In addition, variant nucleotide sequences include analogs and derivatives thereof. Variants of binding proteins disclosed herein include proteins that bind to the same antigen or epitope as the binding protein.

[0179] In some embodiments, the charged amino acid residue is a naturally occurring amino acid or a non-naturally occurring amino acid. In some embodiments, the naturally occurring charged amino acid residue is arginine, lysine, histidine, glutamic acid or aspartic acid.

[0180] Light and heavy chain substitution variants

[0181] In order to generate a substantially homogeneous population of bispecific antibodies with correct pairing of heavy and light chains (i.e., homologous pairing or heterodimerization of the light chain with the heavy chain is required to form the variable domain or antigen-binding domain of the original antibody), the first heavy chain polypeptide (H1) has a strong preference for binding to the first light chain polypeptide (L1) relative to the second light chain polypeptide (L2); and the second heavy chain polypeptide (H2) has a strong preference for binding to the second light chain polypeptide (L2) relative to the first light chain polypeptide (L1). In addition, the first heavy chain polypeptide (H1) and the second heavy chain polypeptide (H2) have a stronger preference for heterodimerization than for homodimerization (i.e., heavy chain heterodimerization).

[0182] Provided herein are antibody variants having one or more amino acid substitutions. Exemplary substitution mutagenesis sites include the charged substitution pairs shown in Tables 1.1-1.3 and 2-6.

[0183] For the bispecific antibodies of the present disclosure, it is advantageous to use a Fab heterodimerization strategy to allow the correct association of Fab domains belonging to the same arm and to minimize abnormal pairing of Fab domains belonging to different arms. Exemplary Fab heterodimerization strategies include, but are not limited to, those shown in Table 1.1 and Table 1.2. The antibody domains listed in Table 1.1 and Table 1.2 correspond to Figure 1 The domains of the present disclosure depicted in .

[0184] Table 1.1-Fab heterodimerization strategy

[0185]

[0186]

[0187] Table 1.2-Fc heterodimerization strategy

[0188]

[0189]

[0190]

[0191]

[0192]

[0193]

[0194]

[0195]

[0196]

[0197]

[0198]

[0199] Table 1.3. Kappa Light Chain and Heavy Chain-Constant Domain Mutation Pairs

[0200] Mutation type Heavy chain constant domain Light chain constant domain Charged pair K147D S131K Charged pair K147D T180R Charged pair K147(WT) T180E Charged pair T187D N137K / N138R Charged pair T187D N138R Charged pair T187D N138K Charged pair K147(WT) S131D Charged pair L145S S131K

[0201] All location information is reported using the EU numbering scheme

[0202] Wild type (WT) indicates the natural amino acid at the indicated position

[0203] The charge pairs with negative and positive residues can be reversed between the heavy and light chains, where D or E (negative charge) is replaced by K or R (positive charge), and K or R (positive charge) is replaced by D or E (negative charge) in the homologous chain.

[0204] Table 2. Kappa light chain and heavy chain variable domain mutation pairs

[0205] Mutation type Heavy chain variable domain Light chain variable domain Charged pair Q39K Q38D Charged pair Q39K Q38E Charged pair Q39D Q38K Charged pair Q39E Q38K Charged pair G100K G44D Knob entry hole Y87G L45W

[0206] All position information is reported using the Kabat numbering scheme.

[0207] Wild type (WT) indicates the natural amino acid at the indicated position

[0208] The charge pairs of negatively and positively charged residues can be reversed between the heavy and light chains, where D or E (negatively charged) is replaced by K or R (positively charged), while in the homologous chain K or R (positively charged) is replaced by D or E (negatively charged).

[0209] Table 3. Lambda light chain and heavy chain-constant domain mutation pairs

[0210] Mutation type Heavy chain constant domain Light chain constant domain Charged pair V185K S137D Charged pair V185E S137K Charged pair K147D T131R Charged pair K147D S179R Charged pair K147D S179K Charged pair K147D T131K Charged pair L145S T131K Charged pair K147(WT) T131D Charged pair K147(WT) S179E

[0211] All location information is reported using the EU numbering scheme

[0212] The charge pairs with negative and positive residues can be reversed between the heavy and light chains, where D or E (negative charge) is replaced by K or R (positive charge), and K or R (positive charge) is replaced by D or E (negative charge) in the homologous chain.

[0213] Table 4. Lambda light chain and heavy chain variable domain mutation pairs

[0214] Mutation type Heavy chain variable domain Light chain variable domain Charged pair Q39K Q38D Charged pair Q39K Q38E Charged pair Q39D Q38K Charged pair Q39E Q38K Charged pair G100D G44K Knob entry hole Y87G L45W

[0215] All position information is reported using the Kabat numbering scheme.

[0216] The charge pairs with negative and positive residues can be reversed between the heavy and light chains, where D or E (negative charge) is replaced by K or R (positive charge), and K or R (positive charge) is replaced by D or E (negative charge) in the homologous chain.

[0217] Table 5. Kappa constant chain cysteine ​​mutation pairs

[0218] Mutation type Heavy chain constant domain Light chain constant domain Cys-Cys S131C S114C Cys-Cys S136C S114C Cys-Cys S134C F116C Cys-Cys F170C S162C Cys-Cys P171C S162C

[0219] All location information is reported using the EU numbering scheme

[0220] Table 6. Lambda constant chain cysteine ​​mutation pairs

[0221] Mutation type Heavy chain constant domain Light chain constant domain Cys-Cys V173C T162C Cys-Cys L128C S118C Cys-Cys S134C T116C

[0222] All location information is reported using the EU numbering scheme

[0223] In certain embodiments, the antibody variant comprises the following substitutions: i) the amino acid at position 39 (Kabat numbering) of VH1 and VH2 is a charged or polar amino acid residue, and the amino acid at position 38 (Kabat numbering) of VL1 and VL2 is an amino acid residue with an opposite charge or polarity compared to the amino acid at position 39 of VH1 and VH2; or the amino acid at position 100 (Kabat numbering) of VH1 and VH2 is a charged or polar amino acid residue, and the amino acid at position 44 (Kabat numbering) of VL1 and VL2 is an amino acid residue with an opposite charge or polarity compared to the amino acid at position 100 of VH1 and VH2; ii) CH1 H1 and CH1 H2The amino acid at position 147 (EU numbering) of CL1 or CL2 is a charged or polar amino acid residue, and one of the amino acids at position 131, 179, or 180 (EU numbering) of CL1 or CL2 is a charged or polar amino acid residue. H1 and CH1 H2 iii) an amino acid residue with an opposite charge or polarity compared to the amino acid at position 147 of CH1; H1 and CH1 H2 The amino acid at position 185 (EU numbering) of CL1 and CL2 is a charged or polar amino acid residue, and the amino acid at position 137 (EU numbering) of CL1 and CL2 is a charged or polar amino acid residue. H1 and CH1 H2 an amino acid residue with an opposite charge or polarity compared to the amino acid at position 185 of the CH1 H1 and CH1 H2 The amino acid at position 187 (EU numbering) of CL1 and CL2 is a charged or polar amino acid residue, and one of the amino acids at position 137 or 138 (EU numbering) of CL1 and CL2 is aligned with CH1. H1 and CH1 H2 iv) an amino acid residue with an opposite charge or polarity compared to the amino acid at position 187 (EU numbering) of CH1; and H1 and CH1 H2 The amino acid at position 145 (EU numbering) of CL1 and CL2 is a charged or polar amino acid residue, and the amino acid at position 131 (EU numbering) of CL1 and CL2 is aligned with the amino acid at position 131 of CH1. H1 and CH1 H2 The amino acid residue at position 145 is an oppositely charged or polar amino acid residue compared to the amino acid at position 145.

[0224] In certain embodiments, the antibody variant comprises the following substitutions: the H1 amino acid at position 39, 100, 147, 185, 187 or 145 is positively charged and the L1 amino acid at position 38, 44, 131, 179, 180, 137 or 138 is negatively charged; and the H2 amino acid at position 39, 100, 147, 185, 187 or 145 is negatively charged and the L2 amino acid at position 38, 44, 131, 179, 180, 137 or 138 is positively charged.

[0225] In certain embodiments, the antibody variant comprises the following substitutions: the H1 amino acid at position 39, 100, 147, 185, 187, and 145 is negatively charged, while the L1 amino acid at position 38, 44, 131, 179, 180, 137, or 138 is positively charged; and the H2 amino acid at position 39, 100, 147, 185, 187, or 145 is positively charged, while the L2 amino acid at position 38, 44, 131, 179, 180, 137, or 138 is negatively charged.

[0226] In certain embodiments, the antibody variant comprises a "light chain pairing mutation set A" comprising the following substitutions: a) H1 and L1 comprise the following: i) amino acid position 39 (Kabat numbering) of VH1 is K, and amino acid position 38 (Kabat numbering) of VL1 is D; ii) CH1 H1 The amino acid at position 185 (EU numbering) of CL1 is K, and the amino acid at position 137 (EU numbering) of CL1 is D; iii) CH1 H1 wherein the amino acid at position 128 (EU numbering) of H1H is C, and the amino acid at position 118 (EU numbering) of CL1 is C; and iv) the amino acid at position 220 (EU numbering) of H1H is S, and the amino acid at position 214 (EU numbering) of CL1 is S; and b) H2 and L2 include the following: i) the amino acid at position 39 (Kabat numbering) of VH2 is D, and the amino acid at position 38 (Kabat numbering) of VL2 is K; and ii) CH2 H1 The 147th amino acid (EU numbering) of is D, and the 180th amino acid (EU numbering) of CL2 is R.

[0227] In certain embodiments, the antibody variant comprises a "light chain pairing mutation set B" comprising the following substitutions: a) H1 and L1 comprise the following: i) amino acid position 39 (Kabat numbering) of VH1 is K, and amino acid position 38 (Kabat numbering) of VL1 is D; and ii) CH1 H1 the amino acid at position 185 (EU numbering) of VH2 is K, and the amino acid at position 137 (EU numbering) of CL1 is D; and b) H2 and L2 include the following: i) the amino acid at position 39 (Kabat numbering) of VH2 is D, and the amino acid at position 38 (Kabat numbering) of VL2 is K; ii) CH2 H1 The amino acid at position 147 (EU numbering) of CL2 is D, and the amino acid at position 180 (EU numbering) of CL2 is R; iii) CH2 H1 The amino acid at position 134 (EU numbering) of H2H is C, and the amino acid at position 116 (EU numbering) of CL2 is C; and iv) the amino acid at position 220 (EU numbering) of H2H is S, and the amino acid at position 214 (EU numbering) of CL2 is S.

[0228] In certain embodiments, the antibody variant comprises a "light chain paired mutation set C" comprising the following substitutions: a) H1 and L1 comprise the following: i) amino acid position 39 (Kabat numbering) of VH1 is K, and amino acid position 38 (Kabat numbering) of VL1 is D; and ii) CH1 H1the amino acid at position 185 (EU numbering) of VH2 is K, and the amino acid at position 137 (EU numbering) of CL1 is D; and b) H2 and L2 include the following: i) the amino acid at position 39 (Kabat numbering) of VH2 is D, and the amino acid at position 38 (Kabat numbering) of VL2 is K; ii) CH2 H1 The amino acid at position 147 (EU numbering) of CL2 is D, and the amino acid at position 180 (EU numbering) of CL2 is R; iii) CH2 H1 The amino acid at position 136 (EU numbering) of H2H is C, and the amino acid at position 114 (EU numbering) of CL2 is C; and iv) the amino acid at position 220 (EU numbering) of H2H is S, and the amino acid at position 214 (EU numbering) of CL2 is S.

[0229] In certain embodiments, the antibody variant comprises a "light chain paired mutation set D" comprising the following substitutions: a) H1 and L1 comprise the following: i) amino acid position 39 (Kabat numbering) of VH1 is K, and amino acid position 38 (Kabat numbering) of VL1 is D; ii) CH1 H1 The amino acid at position 147 (EU numbering) of CL1 is K, and the amino acid at position 131 (EU numbering) of CL1 is D; iii) CH1 H1 wherein the amino acid at position 173 (EU numbering) of H1H is C, and the amino acid at position 162 (EU numbering) of CL1 is C; iv) the amino acid at position 220 (EU numbering) of H1H is S, and the amino acid at position 214 (EU numbering) of CL1 is S; and b) H2 and L2 include the following: i) the amino acid at position 39 (Kabat numbering) of VH2 is D, and the amino acid at position 38 (Kabat numbering) of VL2 is K; and ii) CH2 H1 The 147th amino acid (EU numbering) of is D, and the 180th amino acid (EU numbering) of CL2 is R.

[0230] In certain embodiments, the antibody variant comprises a "light chain paired mutation set E" comprising the following substitutions: a) H1 and L1 comprise the following: i) amino acid position 39 (Kabat numbering) of VH1 is K, and amino acid position 38 (Kabat numbering) of VL1 is D; ii) CH1 H1 The amino acid at position 185 (EU numbering) of CL1 is K, and the amino acid at position 137 (EU numbering) of CL1 is D; iii) CH1 H1wherein the amino acid at position 173 (EU numbering) of H1H is C, and the amino acid at position 162 (EU numbering) of CL1 is C; and iv) the amino acid at position 220 (EU numbering) of H1H is S, and the amino acid at position 214 (EU numbering) of CL1 is S; and b) H2 and L2 include the following: i) the amino acid at position 39 (Kabat numbering) of VH2 is D, and the amino acid at position 38 (Kabat numbering) of VL2 is K; and ii) CH2 H1 The 147th amino acid (EU numbering) of is D, and the 180th amino acid (EU numbering) of CL2 is R.

[0231] In certain embodiments, the antibody variant comprises a "light chain pairing mutation set F" comprising the following substitutions: a) H1 and L1 comprise the following: i) amino acid position 39 (Kabat numbering) of VH1 is K, and amino acid position 38 (Kabat numbering) of VL1 is D; and ii) CH1 H1 the amino acid at position 185 (EU numbering) of VH2 is K, and the amino acid at position 137 (EU numbering) of CL1 is D; and b) H2 and L2 include the following: i) the amino acid at position 39 (Kabat numbering) of VH2 is D, and the amino acid at position 38 (Kabat numbering) of VL2 is K; ii) CH2 H1 The amino acid at position 147 (EU numbering) of CL2 is D, and the amino acid at position 180 (EU numbering) of CL2 is R; iii) CH2 H1 The amino acid at position 131 (EU numbering) of H2H is C, and the amino acid at position 114 (EU numbering) of CL2 is C; and iv) the amino acid at position 220 (EU numbering) of H2H is S, and the amino acid at position 214 (EU numbering) of CL2 is S.

[0232] In certain embodiments, the antibody variant comprises a "light chain pairing mutation set G" comprising the following substitutions: a) H1 and L1 comprise the following: i) amino acid position 39 (Kabat numbering) of VH1 is K, and amino acid position 38 (Kabat numbering) of VL1 is D; and ii) CH1 H1 the amino acid at position 185 (EU numbering) of VH2 is K, and the amino acid at position 137 (EU numbering) of CL1 is D; and b) H2 and L2 include the following: i) the amino acid at position 39 (Kabat numbering) of VH2 is D, and the amino acid at position 38 (Kabat numbering) of VL2 is K; ii) CH2 H1 The amino acid at position 187 (EU numbering) of CL2 is D, and the amino acid at position 138 (EU numbering) of CL2 is K; iii) CH2 H1the amino acid at position 170 (EU numbering) of H2H is C, and the amino acid at position 162 (EU numbering) of CL2 is C; and iv) the amino acid at position 220 (EU numbering) of H2H is S, and the amino acid at position 214 (EU numbering) of CL2 is S.

[0233] In certain embodiments, the antibody variant comprises a "light chain paired mutation set H" comprising the following substitutions: a) H1 and L1 comprise the following: i) amino acid position 39 (Kabat numbering) of VH1 is K, and amino acid position 38 (Kabat numbering) of VL1 is D; ii) CH1 H1 the amino acid at position 185 (EU numbering) of VH2 is E, and the amino acid at position 137 (EU numbering) of CL1 is K; and iii) the amino acid at position 179 (EU numbering) of CL1 is E; and b) H2 and L2 include the following: i) the amino acid at position 39 (Kabat numbering) of VH2 is D, and the amino acid at position 38 (Kabat numbering) of VL2 is K; ii) CH2 H1 The amino acid at position 187 (EU numbering) of CL2 is D, and the amino acid at position 138 (EU numbering) of CL2 is K; iii) CH2 H1 the amino acid at position 171 (EU numbering) of H2H is C, and the amino acid at position 162 (EU numbering) of CL2 is C; and iv) the amino acid at position 220 (EU numbering) of H2H is S, and the amino acid at position 214 (EU numbering) of CL2 is S.

[0234] In certain embodiments, the antibody variant comprises a "light chain paired mutation set 1" comprising the following substitutions: a) H1 and L1 comprise the following: i) amino acid position 39 (Kabat numbering) of VH1 is K, and amino acid position 38 (Kabat numbering) of VL1 is D; and ii) CH1 H1 the amino acid at position 185 (EU numbering) of VH2 is K, and the amino acid at position 137 (EU numbering) of CL1 is D; and b) H2 and L2 include the following: i) the amino acid at position 39 (Kabat numbering) of VH2 is D, and the amino acid at position 38 (Kabat numbering) of VL2 is K; ii) CH2 H1 The amino acid at position 187 (EU numbering) of CL2 is D, and the amino acid at position 138 (EU numbering) of CL2 is K; iii) CH2 H1 the amino acid at position 171 (EU numbering) of H2H is C, and the amino acid at position 162 (EU numbering) of CL2 is C; and iv) the amino acid at position 220 (EU numbering) of H2H is S, and the amino acid at position 214 (EU numbering) of CL2 is S.

[0235] In certain embodiments, the antibody variant comprises a "light chain paired mutation set J" comprising the following substitutions: a) H1 and L1 comprise the following: i) amino acid position 39 (Kabat numbering) of VH1 is K, and amino acid position 38 (Kabat numbering) of VL1 is D; ii) CH1 H1 wherein the amino acid at position 185 (EU numbering) of VH2 is E, and the amino acid at position 137 (EU numbering) of CL1 is K; and iii) the amino acid at position 179 (EU numbering) of CL1 is E; and b) H2 and L2 comprise the following: i) the amino acid at position 39 (Kabat numbering) of VH2 is D, and the amino acid at position 38 (Kabat numbering) of VL2 is K; ii) CH2 H1 The amino acid at position 147 (EU numbering) of CL2 is D, and the amino acid at position 180 (EU numbering) of CL2 is R; iii) CH2 H1 the amino acid at position 171 (EU numbering) of H2H is C, and the amino acid at position 162 (EU numbering) of CL2 is C; and iv) the amino acid at position 220 (EU numbering) of H2H is S, and the amino acid at position 214 (EU numbering) of CL2 is S.

[0236] In certain embodiments, the antibody variant comprises a "light chain pairing mutation set K" comprising the following substitutions: a) H1 and L1 comprise the following: i) amino acid position 39 (Kabat numbering) of VH1 is K, and amino acid position 38 (Kabat numbering) of VL1 is D; and ii) CH1 H1 the amino acid at position 185 (EU numbering) of VH2 is K, and the amino acid at position 137 (EU numbering) of CL1 is D; and b) H2 and L2 include the following: i) the amino acid at position 39 (Kabat numbering) of VH2 is D, and the amino acid at position 38 (Kabat numbering) of VL2 is K; ii) CH2 H1 The amino acid at position 147 (EU numbering) of CL2 is D, and the amino acid at position 180 (EU numbering) of CL2 is R; iii) CH2 H1 the amino acid at position 171 (EU numbering) of H2H is C, and the amino acid at position 162 (EU numbering) of CL2 is C; and iv) the amino acid at position 220 (EU numbering) of H2H is S, and the amino acid at position 214 (EU numbering) of CL2 is S.

[0237] In certain embodiments, the antibody variant comprises a "light chain paired mutation set L" comprising the following substitutions: a) H1 and L1 comprise the following: i) amino acid position 39 (Kabat numbering) of VH1 is K, and amino acid position 38 (Kabat numbering) of VL1 is D; ii) CH1 H1The amino acid at position 147 (EU numbering) of CL1 is K, and the amino acid at position 131 (EU numbering) of CL1 is D; iii) CH1 H1 the amino acid at position 185 (EU numbering) of VH2 is K, and the amino acid at position 137 (EU numbering) of CL1 is D; and b) H2 and L2 include the following: i) the amino acid at position 39 (Kabat numbering) of VH2 is D, and the amino acid at position 38 (Kabat numbering) of VL2 is K; ii) CH2 H1 The amino acid at position 147 (EU numbering) of CL2 is D, and the amino acid at position 180 (EU numbering) of CL2 is R; iii) CH2 H1 The amino acid at position 171 (EU numbering) of H2H is C, and the amino acid at position 162 (EU numbering) of CL2 is C; and the amino acid at position 220 (EU numbering) of H2H is S, and the amino acid at position 214 (EU numbering) of CL2 is S.

[0238] In certain embodiments, the antibody variant comprises a "light chain pairing mutation set 0340" comprising the following substitutions: a) H1 and L1 comprise the following: i) amino acid position 39 (Kabat numbering) of VH1 is D, and amino acid position 38 (Kabat numbering) of VL1 is K; and ii) CH1 H1 the amino acid at position 185 (EU numbering) of VH2 is E, and the amino acid at position 137 (EU numbering) of CL1 is K; and b) H2 and L2 include the following: i) the amino acid at position 39 (Kabat numbering) of VH2 is K, and the amino acid at position 38 (Kabat numbering) of VL2 is D; ii) CH2 H1 The amino acid at position 187 (EU numbering) of CL2 is D, and the amino acid at position 138 (EU numbering) of CL2 is K; iii) CH2 H1 The amino acid at position 136 (EU numbering) of H2H is C, and the amino acid at position 114 (EU numbering) of CL2 is C; and iv) the amino acid at position 220 (EU numbering) of H2H is S, and the amino acid at position 214 (EU numbering) of CL2 is S.

[0239] In certain embodiments, the antibody variant comprises a "light chain paired mutation set M" comprising the following substitutions: a) H1 and L1 comprise the following: i) amino acid position 39 (Kabat numbering) of VH1 is D, and amino acid position 38 (Kabat numbering) of VL1 is K; and ii) CH1 H1the amino acid at position 185 (EU numbering) of VH2 is E, and the amino acid at position 137 (EU numbering) of CL1 is K; and b) H2 and L2 include the following: i) the amino acid at position 39 (Kabat numbering) of VH2 is K, and the amino acid at position 38 (Kabat numbering) of VL2 is D; ii) CH2 H1 The amino acid at position 187 (EU numbering) of CL2 is D, and the amino acid at position 138 (EU numbering) of CL2 is K; iii) CH2 H1 the amino acid at position 171 (EU numbering) of H2H is C, and the amino acid at position 162 (EU numbering) of CL2 is C; and iv) the amino acid at position 220 (EU numbering) of H2H is S, and the amino acid at position 214 (EU numbering) of CL2 is S.

[0240] In certain embodiments, the antibody variant comprises a "light chain paired mutation set N" comprising the following substitutions: a) H1 and L1 comprise the following: i) amino acid position 39 (Kabat numbering) of VH1 is D, and amino acid position 38 (Kabat numbering) of VL1 is K; and ii) CH1 H1 the amino acid at position 185 (EU numbering) of VH2 is K, and the amino acid at position 137 (EU numbering) of CL1 is D; and b) H2 and L2 include the following: i) the amino acid at position 39 (Kabat numbering) of VH2 is K, and the amino acid at position 38 (Kabat numbering) of VL2 is D; ii) CH2 H1 The amino acid at position 187 (EU numbering) of CL2 is D, and the amino acid at position 138 (EU numbering) of CL2 is K; iii) CH2 H1 the amino acid at position 171 (EU numbering) of H2H is C, and the amino acid at position 162 (EU numbering) of CL2 is C; and iv) the amino acid at position 220 (EU numbering) of H2H is S, and the amino acid at position 214 (EU numbering) of CL2 is S.

[0241] In certain embodiments, the antibody variant comprises a "light chain pairing mutation set O" comprising the following substitutions: a) H1 and L1 comprise the following: i) amino acid position 39 (Kabat numbering) of VH1 is D, and amino acid position 38 (Kabat numbering) of VL1 is K; ii) CH1 H1 The amino acid at position 147 (EU numbering) of CL1 is K, and the amino acid at position 131 (EU numbering) of CL1 is D; iii) CH1 H1 The amino acid at position 185 (EU numbering) of CL1 is E, and the amino acid at position 137 (EU numbering) of CL1 is K; and iv) CH1 H1the amino acid at position 145 (EU numbering) of VH2 is S, and the amino acid at position 180 (EU numbering) of CL1 is E; and b) H2 and L2 include the following: i) the amino acid at position 39 (Kabat numbering) of VH2 is K, and the amino acid at position 38 (Kabat numbering) of VL2 is D; ii) CH2 H1 the amino acid at position 187 (EU numbering) of H2H is D, and the amino acid at position 138 (EU numbering) of CL2 is K; iii) the amino acid at position 170 (EU numbering) of VH2 is C, and the amino acid at position 162 (EU numbering) of VL2 is C; and iv) the amino acid at position 220 (EU numbering) of H2H is S, and the amino acid at position 214 (EU numbering) of CL2 is S.

[0242] In certain embodiments, the antibody variant comprises a "light chain paired mutation set 367" comprising the following substitutions: a) H1 and L1 comprise the following: i) amino acid position 39 (Kabat numbering) of VH1 is K, and amino acid position 38 (Kabat numbering) of VL1 is D; ii) CH1 H1 The amino acid at position 147 (EU numbering) of CL1 is D, and the amino acid at position 131 (EU numbering) of CL2 is K; and iii) CH1 H1 the amino acid at position 185 (EU numbering) of VH2 is E, the amino acid at position 137 (EU numbering) of CL1 is D; and b) H2 and L2 comprise the following: i) the amino acid at position 39 (Kabat numbering) of VH2 is D, and the amino acid at position 38 (Kabat numbering) of VL2 is K; ii) CH2 H1 The amino acid at position 187 (EU numbering) of CL2 is D, and the amino acid at position 137 (EU numbering) of CL2 is K; iii) the amino acid at position 138 (EU numbering) of CL2 is R; iv) CH2 H1 The amino acid at position 170 (EU numbering) in H2H is C, and the amino acid at position 162 (EU numbering) in CL2 is C; and v) the amino acid at position 220 (EU numbering) in H2H is S, and the amino acid at position 214 (EU numbering) in CL2 is S.

[0243] In certain embodiments, the antibody variant comprises a "light chain pairing mutation set P" comprising the following substitutions: a) H1 and L1 comprise the following: i) amino acid position 39 (Kabat numbering) of VH1 is K, and amino acid position 38 (Kabat numbering) of VL1 is D; ii) CH1 H1 The amino acid at position 147 (EU numbering) of CL1 is D, and the amino acid at position 131 (EU numbering) of CL1 is K; and iii) CH1 H1and b) H2 and L2 comprise the following: i) the amino acid at position 39 (Kabat numbering) of VH2 is D, and the amino acid at position 38 (Kabat numbering) of VL2 is K; ii) CH2 H1 The amino acid at position 187 (EU numbering) of CL2 is D, and the amino acid at position 138 (EU numbering) of CL2 is K; iii) CH2 H1 the amino acid at position 170 (EU numbering) of H2H is C, and the amino acid at position 162 (EU numbering) of CL2 is C; and iv) the amino acid at position 220 (EU numbering) of H2H is S, and the amino acid at position 214 (EU numbering) of CL2 is S.

[0244] In certain embodiments, the antibody variant comprises a "light chain paired mutation set 404" comprising the following substitutions: a) H1 and L1 comprise the following: i) amino acid position 39 (Kabat numbering) of VH1 is D, and amino acid position 38 (Kabat numbering) of VL1 is K; ii) CH1 H1 The amino acid at position 147 (EU numbering) of CL1 is K, and the amino acid at position 131 (EU numbering) of CL1 is D; and iii) CH1 H1 the amino acid at position 185 (EU numbering) of VH2 is E, and the amino acid at position 137 (EU numbering) of CL1 is K; and b) H2 and L2 include the following: i) the amino acid at position 39 (Kabat numbering) of VH2 is K, and the amino acid at position 38 (Kabat numbering) of VL2 is D; ii) CH2 H1 The amino acid at position 147 (EU numbering) of CL2 is D, and the amino acid at position 180 (EU numbering) of CL2 is R; iii) CH2 H1 The amino acid at position 136 (EU numbering) of H2H is C, and the amino acid at position 114 (EU numbering) of CL2 is C; and iv) the amino acid at position 220 (EU numbering) of H2H is S, and the amino acid at position 214 (EU numbering) of CL2 is S.

[0245] In certain embodiments, the antibody variant comprises a "light chain paired mutation set 406" comprising the following substitutions: a) H1 and L1 comprise the following: i) amino acid position 39 (Kabat numbering) of VH1 is K, and amino acid position 38 (Kabat numbering) of VL1 is E; ii) CH1 H1 The amino acid at position 147 (EU numbering) of CL1 is K, and the amino acid at position 131 (EU numbering) of CL1 is D; and iii) CH1 H1the amino acid at position 185 (EU numbering) of VH2 is E, and the amino acid at position 137 (EU numbering) of CL1 is K; and b) H2 and L2 include the following: i) the amino acid at position 39 (Kabat numbering) of VH2 is D, and the amino acid at position 38 (Kabat numbering) of VL2 is K; ii) CH2 H1 The amino acid at position 187 (EU numbering) of CL2 is D, and the amino acid at position 138 (EU numbering) of CL2 is K; iii) CH2 H1 The amino acid at position 136 (EU numbering) of H2H is C, and the amino acid at position 114 (EU numbering) of CL2 is C; and iv) the amino acid at position 220 (EU numbering) of H2H is S, and the amino acid at position 214 (EU numbering) of CL2 is S.

[0246] In certain embodiments, the antibody variant comprises a "light chain paired mutation set 473" comprising the following substitutions: a) H1 and L1 comprise the following: i) amino acid position 39 (Kabat numbering) of VH1 is K, and amino acid position 38 (Kabat numbering) of VL1 is D; ii) CH1 H1 The amino acid at position 147 (EU numbering) of CL1 is D, and the amino acid at position 131 (EU numbering) of CL1 is K; and iii) CH1 H1 the amino acid at position 185 (EU numbering) of VH2 is D, and the amino acid at position 137 (EU numbering) of CL1 is K; and b) H2 and L2 include the following: i) the amino acid at position 39 (Kabat numbering) of VH2 is D, and the amino acid at position 38 (Kabat numbering) of VL2 is K; ii) CH2 H1 The amino acid at position 187 (EU numbering) of CL2 is D, and the amino acid at position 138 (EU numbering) of CL2 is K; iii) CH2 H1 the amino acid at position 171 (EU numbering) of H2H is C, and the amino acid at position 162 (EU numbering) of CL2 is C; and iv) the amino acid at position 220 (EU numbering) of H2H is S, and the amino acid at position 214 (EU numbering) of CL2 is S.

[0247] In certain embodiments, the antibody variant comprises a "light chain pairing mutation set Q" comprising the following substitutions: a) H1 and L1 comprise the following: i) amino acid position 39 (Kabat numbering) of VH1 is K, and amino acid position 38 (Kabat numbering) of VL1 is D; ii) CH1 H1 The amino acid at position 170 (EU numbering) of CL1 is S, and the amino acid at position 131 (EU numbering) of CL1 is D; iii) CH1 H1wherein the amino acid at position 173 (EU numbering) of H1H is C, and the amino acid at position 162 (EU numbering) of CL1 is C; iv) the amino acid at position 220 (EU numbering) of H1H is S, and the amino acid at position 214 (EU numbering) of CL1 is S; and b) H2 and L2 include the following: i) the amino acid at position 39 (Kabat numbering) of VH2 is D, and the amino acid at position 38 (Kabat numbering) of VL2 is K; and ii) CH2 H1 The 147th amino acid (EU numbering) of is D, and the 180th amino acid (EU numbering) of CL2 is R.

[0248] It may be desirable to modify the antibodies disclosed herein with respect to effector functions in order to enhance, for example, the effectiveness of the antibodies in treating diseases and disorders. For example, cysteine ​​residues can be introduced into the Fc region, thereby allowing interchain disulfide bonds to form in this region. The resulting homodimeric antibodies can have improved internalization capacity and / or enhanced complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). (See Caron et al., J Exp Med., 176: 1191-1195 (1992) and Shopes, J. Immunol., 148: 2918-2922. (1992)). Alternatively, antibodies can be engineered to have dual Fc regions, thereby having enhanced complement lysis and ADCC capabilities. (See Stevenson et al., Anti-Cancer Drug Design, 3: 219-230 (1989)).

[0249] Certain antibody variants with improved or decreased binding to FcRs are described. (See, e.g., U.S. Patent No. 6,737,056; WO 2004 / 056312 and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001).)

[0250] In certain embodiments, the antibody variant comprises an Fc region with one or more amino acid substitutions that improve ADCC, such as substitutions at positions 298, 333, and / or 334 of the Fc region (EU numbering of residues).

[0251] In some embodiments, alterations are made to the Fc region that result in altered (i.e., improved or reduced) C1q binding and / or complement dependent cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

[0252] Antibodies with extended half-life and improved binding to the neonatal Fc receptor (FcRn) are described in US 2005 / 0014934A1 (Hinton et al.), which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)). These antibodies comprise an Fc region with one or more substitutions that improve binding of the Fc region to FcRn. Such Fc variants include those having substitutions at one or more of Fc region residues 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434, such as substitutions at Fc region residue 434 ( U.S. Patent No. 7,371,826 ). See also Duncan and Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94 / 29351 for other examples of Fc region variants.

[0253] In some embodiments, the antibody may comprise a substitution mutation in the Fc region that reduces effector function. In some embodiments, the substitution mutation is a non-glycosylation site mutation. In some embodiments, the non-glycosylation site mutation is located at amino acid residue 297, and the amino acid substitution is located at residues 234, 235, 265, and 331 (EU numbering) to disrupt the Fc receptor binding interface. In some embodiments, the non-glycosylation site mutation reduces the effector function of the antibody.

[0254] In some embodiments, i) CH1 H3 and / or CH2 H3 A at position 297 (EU numbering); ii) CH1 H3 and / or CH2 H3 G at position 297 (EU numbering); or iii) CH1 H3 and / or CH2 H3 At position 297 (EU numbering) there is an S. In some embodiments, CH1 H3 and / or CH2 H3 There is an S at position 331 (EU numbering).

[0255] In some embodiments, i) H1H and / or H2H have A at positions 234 and 235 (EU numbering); or ii) H1H and / or H2H have A at positions 234, 235, and 237 (EU numbering); iii) H1H and / or H2H have A at positions 234 and 235 and a G at position 329 (EU numbering).

[0256] In some embodiments, the antibody may comprise a substitution mutation in the Fc region, which may improve expression titer and increase homogeneity after antibody purification. In some embodiments, the antibody comprises a variant human IgG4 Fc domain. In some embodiments, i) CH1 H2 and / or CH2 H2 C at position 370 (Kabat numbering); and ii) CH1 H2 and / or CH2 H2 There is a C at position 375 (Kabat numbering).

[0257] The use of knobs-in-holes as a method for producing multispecific antibodies is well known in the art. See U.S. Patent No. 5,731,168, issued on March 24, 1998 and assigned to Genentech, PCT Publication No. WO2009089004, issued on July 16, 2009 and assigned to Amgen, and U.S. Patent Publication No. 20090182127, issued on July 16, 2009 and assigned to Novo Nordisk A / S. See also Marvin and Zhu, Acta Pharmacologica Sincia (2005) 26(6):649-658 and Kontermann (2005) Acta Pharacol. Sin., 26:1-9.

[0258] "Protrusion" refers to at least one amino acid side chain that protrudes from the interface of a first polypeptide and can therefore be located in a compensatory cavity of an adjacent interface (i.e., the interface of a second polypeptide) so as to stabilize the heteromultimeric antibody and thereby, for example, favor the formation of heteromultimeric antibodies rather than the formation of homomultimeric antibodies. The protrusion may be present in the original interface, or may be introduced synthetically (e.g., by changing the nucleic acid encoding the interface). Typically, the nucleic acid encoding the interface of the first polypeptide is changed to encode the protrusion. To achieve this, the nucleic acid encoding at least one "original" amino acid residue in the interface of the first polypeptide is replaced with a nucleic acid encoding at least one "input" amino acid residue having a side chain volume larger than that of the original amino acid residue. It should be understood that there may be more than one original and corresponding input residue. The upper limit of the number of original residues replaced is the total number of residues in the interface of the first polypeptide.

[0259] Preferred input residues for forming the protrusions are generally naturally occurring amino acid residues and are preferably selected from arginine (R), phenylalanine (F), tyrosine (Y) and tryptophan (W). Most preferred are tryptophan and tyrosine. In one embodiment, the original residue forming the protrusion has a smaller side chain volume, such as alanine, asparagine, aspartic acid, glycine, serine, threonine or valine. CH1 H3 or CH2 H3 Exemplary amino acid substitutions in the domain that form a protuberance include, but are not limited to, a T366W substitution.

[0260] "Cavity" refers to at least one amino acid side chain that is recessed from the interface of the second polypeptide, and thus accommodates the corresponding protrusion on the adjacent interface of the first polypeptide. The cavity may be present in the original interface, or may be introduced synthetically (e.g., by changing the nucleic acid encoding the interface). Typically, the nucleic acid encoding the interface of the second polypeptide is changed to encode the cavity. To achieve this, the nucleic acid encoding at least one "original" amino acid residue in the interface of the second polypeptide is replaced with DNA encoding at least one "input" amino acid residue, the "input" amino acid residue having a side chain volume smaller than the original amino acid residue. It should be understood that there may be more than one original and corresponding input residue. The upper limit of the number of original residues replaced is the total number of residues in the second polypeptide interface. The side chain volumes of various amino residues are shown in Table 3 above. The preferred input residues for forming the cavity are typically naturally occurring amino acid residues and are preferably selected from alanine (A), serine (S), threonine (T) and valine (V). Most preferably, serine, alanine or threonine. In one embodiment, the original residue forming the cavity has a large side chain volume, such as tyrosine, arginine, phenylalanine or tryptophan. CH1 for generating the cavity H3 or CH2 H3 Exemplary amino acid substitutions in the domains include, but are not limited to, T366S, L368A, Y407A, Y407T, and Y407V substitutions. In certain embodiments, the knob half-antibody comprises a T366W substitution, while the hole half-antibody comprises a T366S / L368A / Y407V substitution.

[0261] In certain embodiments, the antibody variant comprises the following substitutions: CH1 H3 C at position 349, S at position 366, A at position 368, and V at position 407 (EU numbering); and CH2 H3 It has a C at position 354 and a W at position 366 (EU numbering).

[0262] In certain embodiments, the antibody variant comprises the following substitutions: CH2 H3C at position 349, S at position 366, A at position 368, and V at position 407 (EU numbering); and CH1 H3 It has a C at position 354 and a W at position 366 (EU numbering).

[0263] In certain embodiments, the antibody variant comprises the following substitutions: CH1 H3 C at position 354, S at position 366, A at position 368, and V at position 407 (EU numbering); and CH2 H3 C at position 349 and W at position 366 (EU numbering);

[0264] In certain embodiments, the antibody variant comprises the following substitutions: CH2 H3 C at position 354, S at position 366, A at position 368, and V at position 407 (EU numbering); and CH1 H3 It has a C at position 349 and a W at position 366 (EU numbering).

[0265] T cell surface antigens

[0266] The present disclosure provides an antibody comprising a first antigen binding domain that binds to a cell surface antigen expressed on a T cell, NK cell, neutrophil, B cell or dendritic cell engagement cell, and a second antigen binding domain that binds to a disease-associated antigen (DAA). In some embodiments, the cell surface antigen is expressed on a T cell. Exemplary T cell surface antigens include but are not limited to CD3. In some embodiments, the T cell surface antigen is CD3. In some embodiments, the T cell surface antigen is CD3ε.

[0267] Cluster of differentiation 3 (CD3)

[0268] In some embodiments, the present invention is based in part on anti-CD3 antibodies. In certain embodiments, the anti-CD3 antibodies are multispecific (e.g., bispecific) and, in addition to binding to CD3 or a fragment thereof, also bind to a second biomolecule (e.g., a cell surface antigen, e.g., a disease-associated antigen). The antibodies of the present invention can be used, for example, to treat or slow the progression of cell proliferative disorders (e.g., cancer) or autoimmune disorders, or to enhance immune function in subjects suffering from such disorders.

[0269] Unless otherwise indicated, the term "cluster of differentiation 3" or "CD3" as used herein refers to any native CD3 from any vertebrate source, including mammals, such as primates (such as humans, cynomolgus monkeys) and rodents (such as mice and rats), including, for example, CD3ε, CD3γ, CD3α and CD3β chains. CD3 is a cell surface complex expressed on T cells that associates with the T cell receptor. The CD3 complex is required for the activation of CD8+ and CD4+ T lymphocytes. It is formed by three different but highly related chains: one CD3γ chain, one CD3δ chain and two CD3ε chains, which associate with each other to form CD3ε / γ heterodimers and CD3ε / δ heterodimers. The two CD3 heterodimers form the T cell receptor complex together with the T cell receptor (TCR) and the signal transduction ζ chain homodimer.

[0270] The term includes "full-length" unprocessed CD3 (e.g., unprocessed or unmodified CD3ε or CD3γ), as well as any form of CD3 produced by processing in cells. The term also encompasses naturally occurring variants of CD3, including, for example, splice variants or allelic variants. CD3 includes, for example, human CD3ε protein (NCBI RefSeq No. NP_000724), which is 207 amino acids in length.

[0271] In some embodiments, the present invention provides isolated antibodies that bind to CD3. In some embodiments, the present invention provides isolated antibodies that bind to CD3ε. In some cases, the anti-CD3ε antibody binds to a human CD3ε polypeptide or a cynomolgus monkey (cyno) CD3ε polypeptide. In some cases, the human CD3 polypeptide or cynoCD3 polypeptide is a human CD3ε polypeptide (SEQ ID NO: 419) or a cynoCD3ε polypeptide (SEQ ID NO: 420), respectively. In some cases, the anti-CD3 antibody binds to an epitope within a CD3ε (e.g., human CD3ε) fragment consisting of amino acid residues 1-26 or amino acid residues 1-27 of human CD3ε (SEQ ID NO: 419).

[0272] A method for identifying residues or regions that can be used as mutagenesis targets in antibodies is called "alanine scanning mutagenesis," as described by Cunningham and Wells (1989) Science, 244: 1081-1085. In this method, groups of residues or target residues are identified (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) and substituted with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with the antigen is affected. Further substitutions can be introduced at amino acid positions that show functional sensitivity to the initial substitution. Alternatively or in addition, the contact points between the antibody and the antigen can be identified using the crystal structure of the antigen-antibody complex. Such contact residues and adjacent residues can be targeted or eliminated as candidates for substitution. Variants can be screened to determine whether they contain desired properties.

[0273] anti-CD3ε antibody

[0274] Anti-CD3ε antibodies are provided herein.In some embodiments, alanine scanning mutagenesis is performed on the "SP34" anti-CD3ε antibody to generate affinity-modulated anti-CD3ε antibodies of the present invention.

[0275] In some embodiments, an anti-CD3ε antibody of the present disclosure comprises any one of the VH and VL sequences listed in Table 7. In Table 7, the underlined sequences are CDR sequences according to Kabat and the bolded sequences are CDR sequences according to Chothia.

[0276] In some embodiments, the anti-CD3 antibodies of the present disclosure comprise: a) a heavy chain variable region (VH) comprising a VH complementarity determining region 1 (VH CDR1 ), VH complementarity determining region 2 (VH CDR2 ) and VH complementarity determining region 3 (VH CDR3 ); and b) a light chain variable region (VL), which comprises a VL complementarity determining region 1 (VL CDR1 ), VL complementarity determining region 2 (VL CDR2 ) and VL complementarity determining region 3 (VL CDR3 Tables 8 and 9 provide examples of CDR sequences of the anti-CD3 antibodies provided herein.

[0277] Table 7. Anti-CD3 variable heavy chain and variable light chain domains

[0278]

[0279]

[0280]

[0281]

[0282]

[0283] Table 8. Anti-CD3 Heavy Chain CDRs

[0284]

[0285]

[0286] Table 9. Anti-CD3 light chain CDRs

[0287]

[0288]

[0289] In some embodiments, the present disclosure provides antibodies (e.g., including antibody fragments, such as single-chain variable fragments (scFv) that specifically bind to CD3ε, wherein the antibody comprises a) a heavy chain variable region (VH) comprising i) a VH complementarity determining region 1 (VH) comprising the amino acid sequence of SEQ ID NO: 29, 30, 31, 32, or 33; CDR1 ), ii) a VH complementarity determining region 2 (VH) comprising the amino acid sequence of SEQ ID NO: 34, 35 or 36 CDR2 ), iii) a VH complementarity determining region 3 (VH) comprising the amino acid sequence of SEQ ID NO: 37, 38, 39, 40 or 41 CDR3 ); and b) a light chain variable region (VL) comprising ai) i) a VL complementarity determining region 1 (VL) comprising the amino acid sequence of SEQ ID NO: 42 CDR1 ), ii) a VL complementary determining region 2 (VL CDR2 ), iii) a VL complementary determining region 3 (VL CDR3 ).

[0290] Exemplary anti-CD3 antibodies of the invention include CD3-A1, CD3-A2, CD3-A3, CD3-A4, CD3-A5, CD3-A6, CD3-A7, CD3-A8, CD3-A9, CD3-A10, CD3-A11, CD3-A12, and CD3-A13.

[0291] In some embodiments, the anti-CD3 antibody CD3-A1 comprises a VH region comprising a VH comprising the amino acid sequence of SEQ ID NO: 29. CDR1 , VH comprising the amino acid sequence of SEQ ID NO: 34 CDR2and a VH comprising the amino acid sequence of SEQ ID NO: 37 CDR3 ; and a VL region comprising a VL comprising the amino acid sequence of SEQ ID NO: 42 CDR1 , a VL comprising the amino acid sequence of SEQ ID NO: 43 CDR2 and a VL comprising the amino acid sequence of SEQ ID NO: 46 CDR3 .

[0292] In some embodiments, the anti-CD3 antibody CD3-A1 comprises a VH region comprising the amino acid sequence of SEQ ID NO:13 and a VL region comprising the amino acid sequence of SEQ ID NO:25.

[0293] In some embodiments, the anti-CD3 antibody CD3-A2 comprises a VH region comprising a VH comprising the amino acid sequence of SEQ ID NO: 29. CDR1 , VH comprising the amino acid sequence of SEQ ID NO: 34 CDR2 and a VH comprising the amino acid sequence of SEQ ID NO: 37 CDR3 ; and a VL region comprising a VL comprising the amino acid sequence of SEQ ID NO: 42 CDR1 , a VL comprising the amino acid sequence of SEQ ID NO: 44 CDR2 and a VL comprising the amino acid sequence of SEQ ID NO: 45 CDR3 .

[0294] In some embodiments, the anti-CD3 antibody CD3-A2 comprises a VH region comprising the amino acid sequence set forth in SEQ ID NO:13 and a VL region comprising the amino acid sequence set forth in SEQ ID NO:27.

[0295] In some embodiments, the anti-CD3 antibody CD3-A3 comprises a VH region comprising a VH comprising the amino acid sequence of SEQ ID NO: 29. CDR1 , VH comprising the amino acid sequence of SEQ ID NO: 34 CDR2 and a VH comprising the amino acid sequence of SEQ ID NO: 37 CDR3 ; and a VL region comprising a VL comprising the amino acid sequence of SEQ ID NO: 42 CDR1 , a VL comprising the amino acid sequence of SEQ ID NO: 43 CDR2 and a VL comprising the amino acid sequence of SEQ ID NO: 45 CDR3 .

[0296] In some embodiments, the anti-CD3 antibody CD3-A3 comprises a VH region comprising the amino acid sequence set forth in SEQ ID NO:14 and a VL region comprising the amino acid sequence set forth in SEQ ID NO:23.

[0297] In some embodiments, the anti-CD3 antibody CD3-A4 comprises a VH region comprising a VH comprising the amino acid sequence of SEQ ID NO: 29. CDR1 , VH comprising the amino acid sequence of SEQ ID NO: 34 CDR2 and a VH comprising the amino acid sequence of SEQ ID NO: 38 CDR3 ; and a VL region comprising a VL comprising the amino acid sequence of SEQ ID NO: 42 CDR1 , a VL comprising the amino acid sequence of SEQ ID NO: 43 CDR2 and a VL comprising the amino acid sequence of SEQ ID NO: 47 CDR3 .

[0298] In some embodiments, the anti-CD3 antibody CD3-A4 comprises a VH region comprising the amino acid sequence set forth in SEQ ID NO:15 and a VL region comprising the amino acid sequence set forth in SEQ ID NO:26.

[0299] In some embodiments, the anti-CD3 antibody CD3-A5 comprises a VH region comprising a VH comprising the amino acid sequence of SEQ ID NO: 29. CDR1 , VH comprising the amino acid sequence of SEQ ID NO: 34 CDR2 and a VH comprising the amino acid sequence of SEQ ID NO: 39 CDR3 ; and a VL region comprising a VL comprising the amino acid sequence of SEQ ID NO: 42 CDR1 , a VL comprising the amino acid sequence of SEQ ID NO: 43 CDR2 and a VL comprising the amino acid sequence of SEQ ID NO: 47 CDR3 .

[0300] In some embodiments, the anti-CD3 antibody CD3-A5 comprises a VH region comprising the amino acid sequence set forth in SEQ ID NO:16 and a VL region comprising the amino acid sequence set forth in SEQ ID NO:26.

[0301] In some embodiments, the anti-CD3 antibody CD3-A6 comprises a VH region comprising a VH comprising the amino acid sequence of SEQ ID NO: 30. CDR1 , VH comprising the amino acid sequence of SEQ ID NO: 34 CDR2and a VH comprising the amino acid sequence of SEQ ID NO: 37 CDR3 ; and a VL region comprising a VL comprising the amino acid sequence of SEQ ID NO: 42 CDR1 , a VL comprising the amino acid sequence of SEQ ID NO: 43 CDR2 and a VL comprising the amino acid sequence of SEQ ID NO: 45 CDR3 .

[0302] In some embodiments, the anti-CD3 antibody CD3-A6 comprises a VH region comprising the amino acid sequence set forth in SEQ ID NO: 17 and a VL region comprising the amino acid sequence set forth in SEQ ID NO: 22.

[0303] In some embodiments, the anti-CD3 antibody CD3-A7 comprises a VH region comprising a VH comprising the amino acid sequence of SEQ ID NO: 29. CDR1 , VH comprising the amino acid sequence of SEQ ID NO: 35 CDR2 and a VH comprising the amino acid sequence of SEQ ID NO: 38 CDR3 ; and a VL region comprising a VL comprising the amino acid sequence of SEQ ID NO: 42 CDR1 , a VL comprising the amino acid sequence of SEQ ID NO: 43 CDR2 and a VL comprising the amino acid sequence of SEQ ID NO: 45 CDR3 .

[0304] In some embodiments, the anti-CD3 antibody CD3-A7 comprises a VH region comprising the amino acid sequence set forth in SEQ ID NO:18 and a VL region comprising the amino acid sequence set forth in SEQ ID NO:22.

[0305] In some embodiments, the anti-CD3 antibody CD3-A8 comprises a VH region comprising a VH comprising the amino acid sequence of SEQ ID NO: 29. CDR1 , VH comprising the amino acid sequence of SEQ ID NO: 35 CDR2 and a VH comprising the amino acid sequence of SEQ ID NO: 38 CDR3 ; and a VL region comprising a VL comprising the amino acid sequence of SEQ ID NO: 42 CDR1 , a VL comprising the amino acid sequence of SEQ ID NO: 43 CDR2 and a VL comprising the amino acid sequence of SEQ ID NO: 47 CDR3 .

[0306] In some embodiments, the anti-CD3 antibody CD3-A8 comprises a VH region comprising the amino acid sequence set forth in SEQ ID NO:18 and a VL region comprising the amino acid sequence set forth in SEQ ID NO:26.

[0307] In some embodiments, the anti-CD3 antibody CD3-A9 comprises a VH region comprising a VH comprising the amino acid sequence of SEQ ID NO: 29. CDR1 , VH comprising the amino acid sequence of SEQ ID NO: 34 CDR2 and a VH comprising the amino acid sequence of SEQ ID NO: 40 CDR3 ; and a VL region comprising a VL comprising the amino acid sequence of SEQ ID NO: 42 CDR1 , a VL comprising the amino acid sequence of SEQ ID NO: 43 CDR2 and a VL comprising the amino acid sequence of SEQ ID NO: 47 CDR3 .

[0308] In some embodiments, the anti-CD3 antibody CD3-A9 comprises a VH region comprising the amino acid sequence set forth in SEQ ID NO: 19 and a VL region comprising the amino acid sequence set forth in SEQ ID NO: 26.

[0309] In some embodiments, the anti-CD3 antibody CD3-A10 comprises a VH region comprising a VH comprising the amino acid sequence of SEQ ID NO: 29. CDR1 , VH comprising the amino acid sequence of SEQ ID NO: 34 CDR2 and a VH comprising the amino acid sequence of SEQ ID NO: 41 CDR3 ; and a VL region comprising a VL comprising the amino acid sequence of SEQ ID NO: 42 CDR1 , VL containing the amino acid sequence of SEQ ID NO: 43 CDR2 and a VL comprising the amino acid sequence of SEQ ID NO: 47 CDR3 .

[0310] In some embodiments, the anti-CD3 antibody CD3-A10 comprises a VH region comprising the amino acid sequence set forth in SEQ ID NO:20 and a VL region comprising the amino acid sequence set forth in SEQ ID NO:26.

[0311] In some embodiments, the anti-CD3 antibody CD3-A11 comprises a VH region comprising a VH comprising the amino acid sequence of SEQ ID NO: 31. CDR1 , VH comprising the amino acid sequence of SEQ ID NO: 34 CDR2and a VH comprising the amino acid sequence of SEQ ID NO: 37 CDR3 ; and a VL region comprising a VL comprising the amino acid sequence of SEQ ID NO: 42 CDR1 , VL containing the amino acid sequence of SEQ ID NO: 43 CDR2 and a VL comprising the amino acid sequence of SEQ ID NO: 45 CDR3 .

[0312] In some embodiments, the anti-CD3 antibody CD3-A11 comprises a VH region comprising the amino acid sequence set forth in SEQ ID NO:21 and a VL region comprising the amino acid sequence set forth in SEQ ID NO:22.

[0313] In some embodiments, the anti-CD3 antibody CD3-A12 comprises a VH region comprising a VH comprising the amino acid sequence of SEQ ID NO: 29. CDR1 , VH comprising the amino acid sequence of SEQ ID NO: 34 CDR2 and a VH comprising the amino acid sequence of SEQ ID NO: 37 CDR3 ; and a VL region comprising a VL comprising the amino acid sequence of SEQ ID NO: 42 CDR1 , VL containing the amino acid sequence of SEQ ID NO: 43 CDR2 and a VL comprising the amino acid sequence of SEQ ID NO: 45 CDR3 .

[0314] In some embodiments, the anti-CD3 antibody CD3-A12 comprises a VH region comprising the amino acid sequence set forth in SEQ ID NO:13 and a VL region comprising the amino acid sequence set forth in SEQ ID NO:24.

[0315] In some embodiments, the anti-CD3 antibody CD3-A13 comprises a VH region comprising a VH comprising the amino acid sequence of SEQ ID NO: 29. CDR1 , VH comprising the amino acid sequence of SEQ ID NO: 34 CDR2 and a VH comprising the amino acid sequence of SEQ ID NO: 39 CDR3 ; and a VL region comprising a VL comprising the amino acid sequence of SEQ ID NO: 42 CDR1 , VL containing the amino acid sequence of SEQ ID NO: 43 CDR2 and a VL comprising the amino acid sequence of SEQ ID NO: 48 CDR3 .

[0316] In some embodiments, the anti-CD3 antibody CD3-A13 comprises a VH region comprising the amino acid sequence set forth in SEQ ID NO:16 and a VL region comprising the amino acid sequence set forth in SEQ ID NO:28.

[0317] Bispecific anti-CD3ε antibody

[0318] Provided herein are bispecific antibodies comprising a first antigen-binding domain that binds a first antigen (eg, CD3ε) and a second antigen-binding domain that binds a second antigen (eg, a disease-associated antigen).

[0319] In some embodiments, the bispecific antibody has the following structure: a) a first heavy chain polypeptide (H1) comprising a variable region (VH1) and a constant region (CH1) having a constant region 1 domain (CH1) H1 ), hinge region (H1H), constant region 2 domain (CH1 H2 ) and constant region 3 domain (CH1 H3 ); and a first light chain polypeptide (L1) comprising a variable region (VL1) and a constant region (CL1), and a second heavy chain polypeptide (H2) comprising a variable region (VH2) and a constant region (CH2), wherein the constant region (CH2) has a constant region 1 domain (CH2 H1 ), hinge region (H2H), constant region 2 domain (CH2 H2 ) and constant region 3 domain (CH2 H3 ); and a second light chain polypeptide (L2) comprising a variable region (VL2) and a constant region (CL2).

[0320] In some embodiments, the bispecific antibodies of the present disclosure comprise a first antigen binding domain (e.g., binds to CD3) comprising any one of the VH1 and VL1 sequences listed in Table 7. In Table 7, the underlined sequences are CDR sequences according to Kabat and the bolded sequences are CDR sequences according to Chothia.

[0321] In some embodiments, the bispecific antibodies of the present disclosure comprise a first antigen binding domain (e.g., binds to CD3ε) comprising: a) a heavy chain variable region (VH1) comprising a VH complementarity determining region 1 (VH1 CDR1 ), VH complementarity determining region 2 (VH1 CDR2 ) and VH complementarity determining region 3 (VH1 CDR3 ); and b) a light chain variable region (VL) comprising a VL complementarity determining region 1 (VL1 CDR1 ), VL complementarity determining region 2 (VL1 CDR2) and VL complementarity determining region 3 (VL1 CDR3 Tables 8 and 9 provide examples of CDR sequences of the anti-CD3 antibodies provided herein.

[0322] In some embodiments, the bispecific antibody comprises any one of the anti-CD3 antibodies disclosed herein. Exemplary anti-CD3 antibodies of the invention include CD3-A1, CD3-A2, CD3-A3, CD3-A4, CD3-A5, CD3-A6, CD3-A7, CD3-A8, CD3-A9, CD3-A10, CD3-A11, CD3-A12, and CD3-A13.

[0323] In some embodiments, the disclosure provides isolated antibodies (eg, monospecific antibodies or bispecific antibodies) that specifically bind to CD3ε and compete with any of the aforementioned antibodies.

[0324] In some embodiments, the invention provides antibodies (e.g., monospecific antibodies or bispecific antibodies) that bind to CD3ε and compete with the antibodies described herein, including CD3-A1, CD3-A2, CD3-A3, CD3-A4, CD3-A5, CD3-A6, CD3-A7, CD3-A8, CD3-A9, CD3-A10, CD3-A11, CD3-A12, and CD3-A13.

[0325] In some embodiments, the present invention also provides CDR portions of antibodies targeting CD3ε based on CDR contact regions. CDR contact regions are regions of an antibody that confer specificity to an antigen. Generally, CDR contact regions include residue positions within the CDRs and the Vernier region, which are constrained to maintain appropriate loop structure, allowing the antibody to bind to a specific antigen. See, for example, Makabe et al., J. Biol. Chem., 283:1156-1166, 2007. Determination of CDR contact regions is well within the skill of the art.

[0326] The binding affinity (K) of an anti-CD3ε antibody (eg, a monospecific antibody or a bispecific antibody) of the present invention to human CD3ε (eg, human CD3ε (eg, (SEQ ID NO: 419)) D ) can be from about 0.001 to about 5000 nM.

[0327] In some embodiments, the binding affinity is about any of 5000nM, 4500nM, 4000nM, 3500nM, 3000nM, 2500nM, 2000nM, 1789nM, 1583nM, 1540nM, 1500nM, 1490nM, 1064nM, 1000nM, 933nM, 894nM, 750nM, 705nM, 678nM, 532nM, 500nM, 494nM, 400nM, 349nM, 340nM, 353nM, 300nM, 250nM, 244nM, 231nM, 225nM, 207nM, 200nM, 186nM, M, 172nM, 136nM, 113nM, 104nM, 101nM, 100nM, 90nM, 83nM, 79nM, 74nM, 54 nM, 50nM, 45nM, 42nM, 40nM, 35nM, 32nM, 30nM, 25nM, 24nM, 22nM, 20nM, 19 nM, 18nM, 17nM, 16nM, 15nM, 12nM, 10nM, 9nM, 8nM, 7.5nM, 7nM, 6.5nM, 6nM , 5.5nM, 5nM, 4nM, 3nM, 2nM, 1nM, 0.5nM, 0.3nM, 0.1nM, 0.01nM or 0.001nM.

[0328] In some embodiments, the binding affinity is less than about any of 5000 nM, 4000 nM, 3000 nM, 2000 nM, 1000 nM, 900 nM, 800 nM, 250 nM, 200 nM, 100 nM, 50 nM, 30 nM, 20 nM, 10 nM, 7.5 nM, 7 nM, 6.5 nM, 6 nM, 5 nM, 4.5 nM, 4 nM, 3.5 nM, 3 nM, 2.5 nM, 2 nM, 1.5 nM, 1 nM, or 0.5 nM.

[0329] The binding affinity (K) of an anti-CD3ε antibody (e.g., a monospecific antibody or a bispecific antibody) of the present invention to cynomolgus monkey CD3ε (e.g., cynomolgus monkey CD3ε (e.g., (SEQ ID NO: 422)) D ) can be from about 0.001 to about 5000 nM.

[0330] In some embodiments, the binding affinity is about any of 5000nM, 4500nM, 4000nM, 3500nM, 3000nM, 2500nM, 2000nM, 1789nM, 1583nM, 1540nM, 1500nM, 1490nM, 1064nM, 1000nM, 933nM, 894nM, 750nM, 705nM, 678nM, 532nM, 500nM, 494nM, 400nM, 349nM, 340nM, 353nM, 300nM, 250nM, 244nM, 231nM, 225nM, 207nM, 200nM, 186nM, M, 172nM, 136nM, 113nM, 104nM, 101nM, 100nM, 90nM, 83nM, 79nM, 74nM, 54 nM, 50nM, 45nM, 42nM, 40nM, 35nM, 32nM, 30nM, 25nM, 24nM, 22nM, 20nM, 19 nM, 18nM, 17nM, 16nM, 15nM, 12nM, 10nM, 9nM, 8nM, 7.5nM, 7nM, 6.5nM, 6nM , 5.5nM, 5nM, 4nM, 3nM, 2nM, 1nM, 0.5nM, 0.3nM, 0.1nM, 0.01nM or 0.001nM.

[0331] In some embodiments, the binding affinity is less than about any of 5000 nM, 4000 nM, 3000 nM, 2000 nM, 1000 nM, 900 nM, 800 nM, 250 nM, 200 nM, 100 nM, 50 nM, 30 nM, 20 nM, 10 nM, 7.5 nM, 7 nM, 6.5 nM, 6 nM, 5 nM, 4.5 nM, 4 nM, 3.5 nM, 3 nM, 2.5 nM, 2 nM, 1.5 nM, 1 nM, or 0.5 nM.

[0332] In some embodiments, the present disclosure provides nucleic acids encoding any of the aforementioned isolated anti-CD3ε antibodies (e.g., monospecific antibodies or bispecific antibodies). In some embodiments, the present disclosure provides vectors comprising such nucleic acids. In some embodiments, the present disclosure provides host cells comprising such nucleic acids.

[0333] Disease-associated antigens

[0334] Provided herein are bispecific antibodies having a first antigen binding domain that binds a first antigen (eg, CD3ε) and a second antigen binding domain that binds a second antigen (eg, a cell surface antigen or DAA).

[0335] In some embodiments, the second biomolecule is a cell surface antigen. In some embodiments, the second biomolecule is a disease-associated antigen. Disease-associated antigens include but are not limited to ACVR1, ADAM21, AGL10, ALPPL2, APCDD1, ASPRV1, BCMA, BMPR1B, CD151, CD19, CD22, CD274, CD276, CD33, CD38, CD47, CD6, CD70, CD74, CD84, CD180, CDCP1, CDH17, CDH3, CDHR2, CDHR5, CEACAM5, CEACAM6, CEACAM7, CELSR1, CLCA2, CLDN1, CLDN18, CLDN6, CNGB1, CNGB3, COL11A1, C OL17A1, CRB1, CPSG4, CTAG2, CTAGE4, ​​CXADR, CXCR4, DCBLD2, DCST1, DLL3, DLL4, DPCR1, DSG3, DSG4, DUOX2, EBI3, EFNA4, EGFR, ENTPD1, ​​ENTPD2, E PCAM, EPHA10, EPHA6, EPHA8, EPHB3, EPS8L1, ERBB2, ERMP1, F11R, FAP, FAT1, FCER2, FCRL3, FER1L6, FGFR2, FLT3, FLVCR1, FN1, FXYD3, GABRA3, GG T2, GGT3P, GJB3, GLG1, GPC1, GPC2, GPNMB, GPC5A, GRIND2D, GUCY2C, HAVCR2, HEPHL1, HHLA1, IGSF3, IGSF9, IL2RB, IL3RA, ITGA2, ITGA6, ITGAV, I TGB4, ITGB6, LCN15, LILRB4, LNPEP, LRFN4, LRRC15, LY6D, LY75, MAL2, MET, MFI2, MICA, MICB, MMP13, MMP14, MPZL2, MS4A1, MSLN, MST1R, MTDH, MU C1, MUC13, MUC16, MUC17, NAALADL2, NCSTN, NIPAL4, NLGN1, NOTCH3, NOX1, OC90, OR10Q1, OR5l1, PAEP, PANX3, PCDH15, PDCHA9, PCDHB12, PCDHB2, PKD1L1, PODXL, POLR2J2, PROM1, PSMA, PTK7, PVR, PVRL1, PVRL4, RAET1E, RAET1G, RAET1L, ROR1, ROR2, SDC1, SDC4, SDK2, SHISA8, SIGLEC7, SIT1,SLAMF1, SLAMF6, SLAMF7, SLC11A2, SLC12A2, SLC15A1, SLC1A5, SLC22A25, SLC2A9, SLC34A2, SLC38A2, SLC39A4, SLC6A14, SLC7A11, SLC7A3, SLC7A5, SYT8, TAS2R5, TMEM132A, TMPRSS3, TMPRSS4, TMX1, TNFRSF17, TNFRSF21, TN FRSF9, TNFRSF11, TNFRSF15, TNFRSF4, TNFRSF9, TNMD, TP53l11, TPBG, TRPC5, TRPV2, TSPAN10, TSPAN8, UGT2A1, UGT3A2, ULBP1, ULBP2, ULBP3, UMODL1, UPK1B, VANGL1, VANGL2, VASN, VMP1, VSIG4, VTCN1, WNT16, YIF1B, and ZNRF4.

[0336] Exemplary disease-associated antigens include, but are not limited to, those shown in Table 22.

[0337] Table 22. Exemplary disease-associated antigens

[0338]

[0339]

[0340]

[0341]

[0342]

[0343] Multispecific antibodies are antibodies (e.g., monoclonal antibodies) that have binding specificity for at least two different sites. In some embodiments, the anti-CD3 CD3ε antibodies provided herein are multispecific antibodies (e.g., bispecific antibodies). In certain embodiments, bispecific antibodies can bind to two different epitopes of CD3 (e.g., CD3ε or CD3γ). In certain embodiments, one binding specificity is for CD3 (e.g., CD3ε or CD3γ) and the other binding specificity is for any other antigen (e.g., a second biomolecule, e.g., a cell surface antigen, e.g., a disease-associated antigen). Therefore, bispecific anti-CD3 antibodies can have binding specificity for CD3 and a second biomolecule, such as the second biomolecule listed in Table 22 and described in U.S. Publication No. 2010 / 0111856 and PCT Publication No. WO2016204966 A1 (e.g., a disease-associated antigen), all of which are incorporated herein by reference in their entirety.

[0344] In some cases, cell surface antigens (e.g., disease-associated antigens) can be expressed in low copy numbers on target cells (e.g., tumor cells). For example, in some cases, the cell surface antigen is expressed or present in less than 35,000 copies per target cell. In some embodiments, the low copy number cell surface antigen is present in between 100 and 35,000 copies per target cell; between 100 and 30,000 copies per target cell; between 100 and 25,000 copies per target cell; between 100 and 20,000 copies per target cell; between 100 and 15,000 copies per target cell; between 100 and 10,000 copies per target cell; between 100 and 5,000 copies per target cell; between 100 and 2,000 copies per target cell; between 100 and 1,000 copies per target cell; or between 100 and 500 copies per target cell. The copy number of the cell surface antigen can be determined, for example, using a standard scratch card plot.

[0345] UL16 binding protein

[0346] Exemplary UL16 binding proteins include, but are not limited to, ULBP1, ULBP2, ULBP3, RAET1E (ULBP4), RAET1G (ULBP5), and RAET1L (ULBP6). Defective regulation of ULBP1-6 is associated with diseases ranging from autoimmunity to cancer.

[0347] UL16 binding protein 2 (ULBP2) is a major histocompatibility complex (MHC) class I-associated molecule that binds to the NKG2D receptor on natural killer (NK) cells to trigger the release of multiple cytokines and chemokines, thereby contributing to NK cell recruitment and activation. The encoded protein undergoes further processing to produce the mature protein, which is either membrane-anchored via a glycosylphosphatidylinositol moiety or secreted. Many malignant cells secrete the encoded protein to evade NK cell immune surveillance. ULBP2 is widely and differentially expressed across multiple solid tumor indications. In particular, ULBP2 expression in melanoma and breast cancer is associated with poor prognosis and advanced disease.

[0348] Senescence is a stress-induced cellular state that prevents cell proliferation and promotes immune-mediated clearance of damaged cells by inducing the senescence-associated secretory phenotype (SASP), thereby limiting tumorigenesis (Rodier, F. et al. Persistent DNA damage signalling triggers senescence-associated inflammatory cytokinesecretion. Nat. Cell Biol. 11, 973-979 (2009)). Senescence is also associated with age-related tissue pathology, in which the accumulation of SASP-positive cells can induce tissue inflammation, leading to tissue dysfunction, which manifests as arthritis, autoimmunity, diabetes, fibrosis and delayed wound healing in elderly patients (Childs, BG et al. Senescent cells: an emerging target for diseases of ageing. Nat. Rev. Drug Discov. 16, 718-735 (2017)). SASP positive cells express a series of complex secretory proteins and cell surface proteins, including immune activation cytokines, tissue remodeling matrix metalloproteinases and cell surface proteins, including MHCI-like NKG2D ligands, which mediate the recognition and activation of NK and T cell effectors through NKG2D co-stimulatory receptors. Together, these proteins promote the elimination of senescent cells. However, age-related decline in immune cell activity and other factors such as cancer chemotherapy accelerate the induction of SASP positive cells in tissues and limit the clearance of senescent cells by the immune system (Jackola, DR, Ruger, JK & Miller, RA Age-associated changes in human T cell phenotype and function. Aging Clin. Exp. Res. 6, 25-34 (1994); Demaria, M. et al. Cellular Senescence Promotes Adverse Effects of Chemotherapy and Cancer Relapse. Cancer Discov. 7, 165-176 (2017)). Therapeutic strategies to eliminate SASP positive cells provide an opportunity to supplement aging immune surveillance and alleviate the potential causes of many age-related diseases and the persistent side effects of chemotherapy.Furthermore, clearing senescent cells not only reduces age-related diseases, but these anti-aging drugs also have the potential to extend lifespan (Baker, DJ et al. Naturally occurring p16Ink4a-positive cells shorten healthy lifespan. Nature 530, 184-189 (2016); Baker, DJ et al. Clearance of p16Ink4a-positive senescent cells delays ageing-associated disorders. Nature 479, 232-236 (2011)). ULBP2 is an MHCI-like ligand that has become a cell surface protein associated with stress-induced SASP-positive fibroblasts and cancer cells (Sagiv, A. et al. NKG2D ligands mediate immunosurveillance of senescent cells. Aging 8, 328-344 (2016); Ruscetti, M. et al. NK cell-mediated cytotoxicity contributes to tumor control by acytostatic drug combination. Science 362, 1416-1422 (2018);. DP et al. Targetable mechanisms driving immunoevasion of persistent senescent cells link chemotherapy-resistant cancer to aging. JCI Insight 5, e124716, 124716 (2019). In addition to eradicating cancer cells, ULBP2 or ULBP2 / 5 / 6-targeted drugs have the potential to eliminate SASP-positive cells from tissues and improve tissue function, thereby preventing age-related diseases and extending lifespan.

[0349] ULBP2 encompasses naturally occurring variants of ULBP2, including, for example, splice variants or allelic variants. ULBP2 includes, for example, the human ULBP2 protein (UniProt ID: Q9BZM5), which is 246 amino acids in length.

[0350] In one aspect, the present invention provides isolated antibodies that bind to ULBP2. In some cases, the anti-ULBP2 antibody binds to a human ULBP2 polypeptide or a portion thereof. In some embodiments, the human ULBP2 polypeptide comprises the amino acid sequence of SEQ ID NO: 421.

[0351] ULBP5 (RAET1G) encompasses naturally occurring variants of ULBP5, including, for example, splice variants or allelic variants. ULBP5 includes, for example, the human ULBP5 protein (UniProt ID: Q6H3X3), which is 334 amino acids in length.

[0352] In one aspect, the present invention provides isolated antibodies that bind to ULBP5 (RAET1G). In some cases, the anti-ULBP5 antibody binds to a human ULBP5 polypeptide or a portion thereof. In some embodiments, the human ULBP5 polypeptide comprises the amino acid sequence of SEQ ID NO: 422.

[0353] ULBP6 (RAET1L) encompasses naturally occurring variants of ULBP6, including, for example, splice variants or allelic variants. ULBP6 includes, for example, the human ULBP6 protein (UniProt ID: Q5VY80), which is 246 amino acids in length.

[0354] In one aspect, the present invention provides isolated antibodies that bind to ULBP6 (RAET1L). In some cases, the anti-ULBP6 antibody binds to a human ULBP6 polypeptide or a portion thereof. In some embodiments, the human ULBP6 polypeptide comprises the amino acid sequence of SEQ ID NO: 423.

[0355] Anti-ULBP2 / 5 / 6 antibodies

[0356] Antibodies that bind to anti-ULBP2 / 5 / 6 are provided herein. In some embodiments, the "E12" anti-ULBP2 / 5 / 6 antibody can be subjected to alanine scanning mutagenesis to generate affinity-modulated anti-ULBP2 / 5 / 6 antibodies. Antibodies that bind to anti-ULBP2 are also provided herein. In some embodiments, the "A06" anti-ULBP2 antibody can be subjected to alanine scanning mutagenesis to generate affinity-modulated anti-ULBP2 antibodies.

[0357] In some embodiments, the anti-ULBP2 / 5 / 6 antibodies of the present disclosure comprise any one of the VH and VL sequences listed in Table 10. In Table 10, the underlined sequences are CDR sequences according to Kabat and the bolded sequences are CDR sequences according to Chothia.

[0358] In some embodiments, the anti-ULBP2 antibodies of the present disclosure comprise: a) a heavy chain variable region (VH) comprising a VH complementarity determining region 1 (VH CDR1 ), VH complementarity determining region 2 (VH CDR2 ) and VH complementarity determining region 3 (VH CDR3 ); and b) a light chain variable region (VL), which comprises a VL complementarity determining region 1 (VL CDR1 ), VL complementarity determining region 2 (VL CDR2 ) and VL complementarity determining region 3 (VL CDR3 Tables 11 and 12 provide exemplary CDR sequences of the anti-ULBP2 antibodies provided herein.

[0359] Table 10. Anti-ULBP2 / 5 / 6 variable heavy chain and variable light chain domains

[0360]

[0361]

[0362] Table 11. Anti-ULBP2 / 5 / 6 Heavy Chain CDRs

[0363]

[0364] Table 12. Anti-ULBP2 / 5 / 6 Light Chain CDRs

[0365]

[0366] In some embodiments, the present disclosure provides antibodies (e.g., including antibody fragments, such as single-chain variable fragments (scFv) that specifically bind to ULBP2, wherein the antibody comprises a) a heavy chain variable region (VH) comprising i) a VH complementarity determining region 1 (VH) comprising the amino acid sequence of SEQ ID NO: 5 or 6; CDR1 ), ii) a VH complementarity determining region 2 (VH) comprising the amino acid sequence of SEQ ID NO: 7 or 8 CDR2 ), iii) a VH complementarity determining region 3 (VH) comprising the amino acid sequence of SEQ ID NO: 9 CDR3 ); and b) a light chain variable region (VL) comprising i) a VL complementarity determining region 1 (VL) comprising the amino acid sequence of SEQ ID NO: 10 CDR1 ), ii) a VL complementary determining region 2 (VL CDR2 ), iii) a VL complementary determining region 3 (VL CDR3 ).

[0367] Exemplary anti-ULBP2 antibodies of the present invention include ULBP2-01, ULBP2-02, E12, A06.

[0368] In some embodiments, the anti-ULBP2 antibody ULBP2-01 has a VH region comprising a VH comprising the amino acid sequence of SEQ ID NO: 5. CDR1 , VH comprising the amino acid sequence of SEQ ID NO: 7 CDR2 and a VH comprising the amino acid sequence of SEQ ID NO: 9 CDR3 ; and a VL region comprising a VL comprising the amino acid sequence of SEQ ID NO: 10 CDR1 , a VL comprising the amino acid sequence of SEQ ID NO: 11 CDR2 and a VL comprising the amino acid sequence of SEQ ID NO: 12 CDR3 .

[0369] In some embodiments, the anti-ULBP2 antibody ULBP2-01 has a VH region comprising the amino acid sequence set forth in SEQ ID NO:2 and a VL region comprising the amino acid sequence set forth in SEQ ID NO:1.

[0370] In some embodiments, the anti-ULBP2 antibody ULBP2-02 has a VH region comprising a VH comprising the amino acid sequence of SEQ ID NO: 5. CDR1 , VH comprising the amino acid sequence of SEQ ID NO: 7 CDR2 and a VH comprising the amino acid sequence of SEQ ID NO: 9 CDR3 ; and a VL region comprising a VL comprising the amino acid sequence of SEQ ID NO: 10 CDR1 , a VL comprising the amino acid sequence of SEQ ID NO: 11 CDR2 and a VL comprising the amino acid sequence of SEQ ID NO: 12 CDR3 .

[0371] In some embodiments, the anti-ULBP2 antibody ULBP2-02 has a VH region comprising the amino acid sequence set forth in SEQ ID NO:4 and a VL region comprising the amino acid sequence set forth in SEQ ID NO:3.

[0372] In some embodiments, the anti-ULBP2 antibody E12 has a VH region comprising a VH comprising the amino acid sequence of SEQ ID NO: 5. CDR1 , VH comprising the amino acid sequence of SEQ ID NO: 7 CDR2 and a VH comprising the amino acid sequence of SEQ ID NO: 9 CDR3; and a VL region comprising a VL comprising the amino acid sequence of SEQ ID NO: 10 CDR1 , a VL comprising the amino acid sequence of SEQ ID NO: 11 CDR2 and a VL comprising the amino acid sequence of SEQ ID NO: 12 CDR3 .

[0373] In some embodiments, the anti-ULBP2 antibody E12 has a VH region comprising the amino acid sequence of SEQ ID NO:425 and a VL region comprising the amino acid sequence of SEQ ID NO:424.

[0374] In some embodiments, the anti-ULBP2 antibody A06 has a VH region comprising a VH comprising the amino acid sequence of SEQ ID NO: 428. CDR1 , VH comprising the amino acid sequence of SEQ ID NO: 430 CDR2 and a VH comprising the amino acid sequence of SEQ ID NO: 432 CDR3 and a VL region comprising a VL comprising the amino acid sequence of SEQ ID NO: 433 CDR1 , a VL comprising the amino acid sequence of SEQ ID NO: 434 CDR2 and a VL comprising the amino acid sequence of SEQ ID NO: 435 CDR3 .

[0375] In some embodiments, the anti-ULBP2 antibody A06 has a VH region comprising the amino acid sequence of SEQ ID NO:427 and a VL region comprising the amino acid sequence of SEQ ID NO:426.

[0376] Bispecific anti-ULBP2 / 5 / 6 antibody

[0377] Provided herein are bispecific antibodies comprising a first antigen binding domain that binds a first antigen (eg, a cell surface antigen, CD3ε) and a second antigen binding domain that binds a second antigen (eg, ULBP2 / 5 / 6).

[0378] In some embodiments, the bispecific antibody has the following structure: a) a first heavy chain polypeptide (H1) comprising a variable region (VH1) and a constant region (CH1) having a constant region 1 domain (CH1) H1 ), hinge region (H1H), constant region 2 domain (CH1 H2 ) and constant region 3 domain (CH1 H3); and a first light chain polypeptide (L1) comprising a variable region (VL1) and a constant region (CL1), and a second heavy chain polypeptide (H2) comprising a variable region (VH2) and a constant region (CH2), wherein the constant region (CH2) has a constant region 1 domain (CH2 H1 ), hinge region (H2H), constant region 2 domain (CH2 H2 ) and constant region 3 domain (CH2 H3 ); and a second light chain polypeptide (L2) comprising a variable region (VL2) and a constant region (CL2).

[0379] In some embodiments, the bispecific antibodies of the present disclosure comprise a second antigen binding domain (e.g., binds to ULBP2 / 5 / 6) comprising any one of the VH2 and VL2 sequences listed in Table 10. In Table 10, the underlined sequences are CDR sequences according to Kabat and the bolded sequences are CDR sequences according to Chothia.

[0380] In some embodiments, the bispecific antibodies of the present disclosure comprise a second antigen-binding domain (e.g., binds to ULBP2), comprising: a) a heavy chain variable region (VH2) comprising a VH complementarity determining region 1 (VH2 CDR1 ), VH complementarity determining region 2 (VH2 CDR2 ) and VH2 complementarity determining region 3 (VH2 CDR3 ); and b) a light chain variable region (VL2) comprising a VL complementarity determining region 1 (VL2 CDR1 ), VL complementarity determining region 2 (VL2 CDR2 ) and VL complementarity determining region 3 (VL2 CDR3 Tables 11 and 12 provide exemplary CDR sequences of the anti-ULBP2 antibodies provided herein.

[0381] In some embodiments, the bispecific antibody comprises any one of the ULBP2 / 5 / 6 antibodies disclosed herein. Exemplary anti-ULBP2 / 5 / 6 antibodies of the present invention include ULBP2-01, ULBP2-02, and E12. In some embodiments, the bispecific antibody comprises any one of the anti-ULBP2 antibodies disclosed herein. Exemplary anti-ULBP2 antibodies of the present invention include A06.

[0382] In some embodiments, the present disclosure provides isolated antibodies (eg, monospecific antibodies or bispecific antibodies) that specifically bind to ULPB2 / 5 / 6 and compete with any of the aforementioned antibodies.

[0383] In some embodiments, the present invention provides antibodies (eg, monospecific antibodies or bispecific antibodies) that bind to ULBP2 / 5 / 6 and compete with the antibodies described herein, including ULBP2-01, ULBP2-02, A06, and E12.

[0384] In some embodiments, the present invention also provides CDR portions of antibodies targeting ULBP2 / 5 / 6 based on CDR contact regions. CDR contact regions are regions of an antibody that confer specificity to an antigen. Generally, CDR contact regions include residue positions within the CDRs and the Vernier region, which are constrained to maintain appropriate loop structure, allowing the antibody to bind to a specific antigen. See, for example, Makabe et al., J. Biol. Chem., 283:1156-1166, 2007. Determination of CDR contact regions is well within the skill of the art.

[0385] The binding affinity (K) of the ULBP2 / 5 / 6 antibodies (e.g., monospecific antibodies or bispecific antibodies) described herein to ULBP2 / 5 / 6 (e.g., human ULBP2 (e.g., (SEQ ID NO: 421), ULBP5 (SEQ ID NO: 422), ULBP6 (SEQ ID NO: 423)) D ) can be from about 0.001 to about 5000 nM.

[0386] In some embodiments, the binding affinity is about any of 5000nM, 4500nM, 4000nM, 3500nM, 3000nM, 2500nM, 2000nM, 1789nM, 1583nM, 1540nM, 1500nM, 1490nM, 1064nM, 1000nM, 933nM, 894nM, 750nM, 705nM, 678nM, 532nM, 500nM, 494nM, 400nM, 349nM, 340nM, 353nM, 300nM, 250nM, 244nM, 231nM, 225nM, 207nM, 200nM, 186nM, M, 172nM, 136nM, 113nM, 104nM, 101nM, 100nM, 90nM, 83nM, 79nM, 74nM, 54 nM, 50nM, 45nM, 42nM, 40nM, 35nM, 32nM, 30nM, 25nM, 24nM, 22nM, 20nM, 19 nM, 18nM, 17nM, 16nM, 15nM, 12nM, 10nM, 9nM, 8nM, 7.5nM, 7nM, 6.5nM, 6nM , 5.5nM, 5nM, 4nM, 3nM, 2nM, 1nM, 0.5nM, 0.3nM, 0.1nM, 0.01nM or 0.001nM.

[0387] In some embodiments, the binding affinity is less than about any of 5000 nM, 4000 nM, 3000 nM, 2000 nM, 1000 nM, 900 nM, 800 nM, 250 nM, 200 nM, 100 nM, 50 nM, 30 nM, 20 nM, 10 nM, 7.5 nM, 7 nM, 6.5 nM, 6 nM, 5 nM, 4.5 nM, 4 nM, 3.5 nM, 3 nM, 2.5 nM, 2 nM, 1.5 nM, 1 nM, or 0.5 nM.

[0388] In some embodiments, the present disclosure provides nucleic acids encoding any of the aforementioned isolated anti-ULBP2 / 5 / 6 antibodies (e.g., monospecific antibodies or bispecific antibodies). In some embodiments, the present disclosure provides vectors comprising such nucleic acids. In some embodiments, the present disclosure provides host cells comprising such nucleic acids.

[0389] With CD3 Ε Exemplary bispecific antibodies that bind to ULBP2 / 5 / 6

[0390] Provided herein are bispecific antibodies comprising a first antigen binding domain that binds a first antigen (eg, CD3ε) and a second antigen binding domain that binds a second antigen (eg, ULBP2 / 5 / 6).

[0391] In some embodiments, the bispecific antibody has the following structure: a) a first heavy chain polypeptide (H1) comprising a variable region (VH1) and a constant region (CH1) having a constant region 1 domain (CH1) H1 ), hinge region (H1H), constant region 2 domain (CH1 H2 ) and constant region 3 domain (CH1 H3 ); and a first light chain polypeptide (L1) comprising a variable region (VL1) and a constant region (CL1), and a second heavy chain polypeptide (H2) comprising a variable region (VH2) and a constant region (CH2), wherein the constant region (CH2) has a constant region 1 domain (CH2 H1 ), hinge region (H2H), constant region 2 domain (CH2 H2 ) and constant region 3 domain (CH2 H3) and a second light chain polypeptide (L2) comprising a variable region (VL2) and a constant region (CL2).

[0392] In some embodiments, the bispecific antibodies of the present disclosure comprise a first antigen binding domain (e.g., binds to CD3ε) comprising: a) a heavy chain variable region (VH1) comprising a VH complementarity determining region 1 (VH1 CDR1), VH complementarity determining region 2 (VH1 CDR2 ) and VH complementarity determining region 3 (VH1 CDR3 ); and b) a light chain variable region (VL) comprising a VL complementarity determining region 1 (VL1 CDR1 ), VL complementarity determining region 2 (VL1 CDR2 ) and VL complementarity determining region 3 (VL1 CDR3 ); and a second antigen binding domain (e.g., binds to ULBP2 / 5 / 6) comprising: a) a heavy chain variable region (VH2) comprising a VH complementarity determining region 1 (VH2 CDR1 ), VH complementarity determining region 2 (VH2 CDR2 ) and VH2 complementarity determining region 3 (VH2 CDR3 ); and b) a light chain variable region (VL2) comprising a VL complementarity determining region 1 (VL2 CDR1 ), VL complementarity determining region 2 (VL2 CDR2 ) and VL complementarity determining region 3 (VL2 CDR3 Tables 8 and 9 provide examples of CDR sequences for anti-CD3ε antibodies provided herein. Tables 11 and 12 provide examples of CDR sequences for anti-ULBP2 / 5 / 6 antibodies provided herein.

[0393] In some embodiments, the bispecific antibodies of the present disclosure comprise a first antigen binding domain (e.g., binds to CD3ε) comprising any of the VH1 and VL1 sequences listed in Table 7, and a second antigen binding domain (e.g., binds to ULBP2 / 5 / 6) comprising any of the VH2 and VL2 sequences listed in Table 10.

[0394] In some embodiments, the bispecific antibodies of the present disclosure comprise a first heavy chain polypeptide (H1) and a first light chain polypeptide (L1); and a second heavy chain polypeptide (H2) and a second light chain polypeptide (L2) comprising any one of the sequences listed in Tables 13 and 14. The italicized sequences are the heavy chain variable region and the light chain variable region. The underlined sequences are CDRs according to Kabat, while the bold sequences are CDRs according to Chothia.

[0395] In some embodiments, the bispecific antibodies of the present disclosure provided herein comprise an H1 that is 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%, at least 99%, or at least 100% identical to the amino acid sequence of the sequences listed in Tables 13 and 14.

[0396] In some embodiments, the bispecific antibodies of the present disclosure provided herein comprise an L1 that is 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%, at least 99%, or at least 100% identical to the amino acid sequence of the sequences listed in Tables 13 and 14.

[0397] In some embodiments, the bispecific antibodies of the present disclosure provided herein comprise an H2 that is 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%, at least 99%, or at least 100% identical to the amino acid sequence of the sequences listed in Tables 13 and 14.

[0398] In some embodiments, the bispecific antibodies of the present disclosure provided herein comprise an L2 that is 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%, at least 99%, or at least 100% identical to the amino acid sequence of the sequences listed in Tables 13 and 14.

[0399] In some embodiments, the H1 amino acid sequence is numbered according to SEQ ID NO: 439. In some embodiments, the L1 amino acid sequence is numbered according to SEQ ID NO: 438. In some embodiments, the H2 amino acid sequence is numbered according to SEQ ID NO: 437. In some embodiments, the L2 amino acid sequence is numbered according to SEQ ID NO: 436.

[0400] Table 13. Exemplary bispecific antibodies that bind CD3ε and ULBP2 / 5 / 6

[0401]

[0402]

[0403]

[0404]

[0405]

[0406]

[0407]

[0408]

[0409]

[0410]

[0411]

[0412]

[0413]

[0414]

[0415]

[0416]

[0417]

[0418]

[0419]

[0420]

[0421]

[0422]

[0423]

[0424]

[0425]

[0426] Exemplary CD3εxULBP2 / 5 / 6 bispecific antibodies of the invention include EIP0174, EIP0175, EIP0187, EIP0205, EIP0206, EIP0207, EIP0208, EIP0294, EIP0295, EIP0306, EIP0307, ​​EIP0318, EIP0340, EIP0342, EIP0354, EIP0356, EIP0367, EIP0377, EIP0404, EIP0406, EIP0473, and EIP0598.

[0427] The bispecific antibodies EIP0174, EIP0175, EIP0187, EIP0205, EIP0206, EIP0207, EIP0208, EIP0294, EIP0295, EIP0306, EIP0307, ​​EIP0318, EIP0340, EIP0342, EIP0354, EIP0356, EIP0367, EIP0377, EIP0404, EIP0406, EIP0473, and EIP0598 comprise a first antigen binding domain that binds CD3ε, the first antigen binding domain comprising a VH1 comprising a VH1 having the amino acid sequence of SEQ ID NO: 29 CDR1 ; VH1 having the amino acid sequence of SEQ ID NO: 34 CDR2 and VH1 having the amino acid sequence of SEQ ID NO: 37 CDR3 ; and VL1 comprising VL1 having the amino acid sequence of SEQ ID NO: 42 CDR1 ; VL1 having the amino acid sequence of SEQ ID NO: 43 CDR2 ; and VL1 having the amino acid sequence of SEQ ID NO: 45 CDR3 .

[0428] The bispecific antibodies EIP0174, EIP0175, EIP0187, EIP0205, EIP0206, EIP0207, EIP0208, EIP0294, EIP0295, EIP0306, EIP0307, ​​EIP0318, EIP0340, EIP0342, EIP0354, EIP0356, EIP0367, EIP0377, EIP0404, EIP0406, EIP0473, and EIP0598 comprise a second antigen binding domain that binds ULBP2 / 5 / 6, the second antigen binding domain comprising a VH2 comprising a VH2 having the amino acid sequence of SEQ ID NO: 5 CDR1 ; VH2 having the amino acid sequence of SEQ ID NO: 7 CDR2 and VH2 having the amino acid sequence of SEQ ID NO: 9 CDR3 ; and VL2, said VL2 comprising a VL2 having an amino acid sequence of SEQ ID NO: 10 CDR1 ; VL2 having the amino acid sequence of SEQ ID NO: 11 CDR2 ; and VL2 having the amino acid sequence of SEQ ID NO: 12 CDR3 .

[0429] In addition to the specific complementarity determining regions described above, the bispecific antibody EIP0174 comprises the following amino acid substitutions in H1 and L1: amino acid position 39 (Kabat numbering) of VH1 is K, and amino acid position 38 (Kabat numbering) of VL1 is D; H1 The amino acid at position 185 (EU numbering) of CL1 is K, and the amino acid at position 137 (EU numbering) of CL1 is D; H1 The amino acid at position 128 (EU numbering) of H1H is C, and the amino acid at position 118 (EU numbering) of CL1 is C; the amino acid at position 220 (EU numbering) of H1H is S, and the amino acid at position 214 (EU numbering) of CL1 is S; the amino acids at positions 234, 235, and 237 (EU numbering) of H1H are A; H3 The amino acid at position 349 (EU numbering) is C; CH1 H3 The amino acid at position 366 (EU numbering) is S; CH1 H3 The amino acid at position 368 (EU numbering) is A; CH1 H3 The amino acid at position 407 (EU numbering) is V; and when numbered according to the H1 amino acid sequence of SEQ NO: 439 and the L1 amino acid sequence of SEQ NO: 438, CH1 H3 The amino acid at position 447 (EU numbering) is deleted.

[0430] In addition to the specific complementarity determining regions described above, the bispecific antibody EIP0174 comprises the following amino acid substitutions in H2 and L2: amino acid position 39 (Kabat numbering) of VH2 is D, and amino acid position 38 (Kabat numbering) of VL2 is K; CH2 H1 The amino acid at position 147 (EU numbering) of CL2 is D, and the amino acid at position 180 (EU numbering) of CL2 is R; the amino acids at positions 234, 235, and 237 (EU numbering) of H2H are A; CH2 H3 The amino acid at position 354 (EU numbering) is C; CH2 H3 The amino acid at position 366 (EU numbering) is W; and when numbered according to the H2 amino acid sequence of SEQ NO: 437 and the L2 amino acid sequence of SEQ NO: 436, CH2 H3 The amino acid at position 447 (EU numbering) is deleted.

[0431] In some embodiments, the bispecific antibody EIP0174 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 13, a VL1 comprising the amino acid sequence of SEQ ID NO: 22, a VH2 comprising the amino acid sequence of SEQ ID NO: 2, and a VL2 comprising the amino acid sequence of SEQ ID NO: 1.

[0432] In some embodiments, the bispecific antibody EIP0174 comprises H1 comprising the amino acid sequence of SEQ ID NO:58, L1 comprising the amino acid sequence of SEQ ID NO:57, H2 comprising the amino acid sequence of SEQ ID NO:56, and L2 comprising the amino acid sequence of SEQ ID NO:55.

[0433] In addition to the specific complementarity determining regions described above, the bispecific antibody EIP0175 comprises the following amino acid substitutions in H1 and L1: amino acid position 39 (Kabat numbering) of VH1 is K, and amino acid position 38 (Kabat numbering) of VL1 is D; H1 The amino acid at position 185 (EU numbering) of CL1 is K, and the amino acid at position 137 (EU numbering) of CL1 is D; the amino acids at positions 234, 235, and 237 (EU numbering) of H1H are A; H3 The amino acid at position 349 (EU numbering) is C; CH1 H3 The amino acid at position 366 (EU numbering) is S; CH1 H3 The amino acid at position 368 (EU numbering) is A; CH1 H3 The amino acid at position 407 (EU numbering) is V; and when numbered according to the H1 amino acid sequence of SEQ NO: 439 and the L1 amino acid sequence of SEQ NO: 438, CH1 H3 The amino acid at position 447 (EU numbering) is deleted.

[0434] In addition to the specific complementary determining regions described above, the bispecific antibody EIP0175 comprises the following amino acid substitutions in H2 and L2: amino acid at position 39 (Kabat numbering) of VH2 is D, and amino acid at position 38 (Kabat numbering) of VL2 is K; CH2 H1 The amino acid at position 147 (EU numbering) of CL2 is D, and the amino acid at position 180 (EU numbering) of CL2 is R; CH2 H1The amino acid at position 134 (EU numbering) of H2H is C, and the amino acid at position 116 (EU numbering) of CL2 is C; the amino acid at position 220 (EU numbering) of H2H is S, and the amino acid at position 214 (EU numbering) of CL2 is S; the amino acids at positions 234, 235, and 237 (EU numbering) of H2H are A; CH2 H3 The amino acid at position 354 (EU numbering) is C; CH2 H3 The amino acid at position 366 (EU numbering) is W; and when numbered according to the H2 amino acid sequence of SEQ NO: 437 and the L2 amino acid sequence of SEQ NO: 436, CH2 H3 The amino acid at position 447 (EU numbering) is deleted.

[0435] In some embodiments, the bispecific antibody EIP0175 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 13, a VH2 comprising the amino acid sequence of SEQ ID NO: 2, a VL1 comprising the amino acid sequence of SEQ ID NO: 22, and a VL2 comprising the amino acid sequence of SEQ ID NO: 1.

[0436] In some embodiments, the bispecific antibody EIP0175 comprises H1 comprising the amino acid sequence of SEQ ID NO:62, H2 comprising the amino acid sequence of SEQ ID NO:60, L1 comprising the amino acid sequence of SEQ ID NO:61, and L2 comprising the amino acid sequence of SEQ ID NO:59.

[0437] In addition to the specific complementary determining regions described above, the bispecific antibody EIP0187 comprises the following amino acid substitutions in H1 and L1: amino acid position 39 (Kabat numbering) of VH1 is K, and amino acid position 38 (Kabat numbering) of VL1 is D; H1 The amino acid at position 185 (EU numbering) of CL1 is K, and the amino acid at position 137 (EU numbering) of CL1 is D; the amino acids at positions 234, 235, and 237 (EU numbering) of H1H are A; H3 The amino acid at position 349 (EU numbering) is C; CH1 H3 The amino acid at position 366 (EU numbering) is S; CH1 H3 The amino acid at position 368 (EU numbering) is A; CH1 H3 The amino acid at position 407 (EU numbering) is V; and when numbered according to the H1 amino acid sequence of SEQ NO: 439 and the L1 amino acid sequence of SEQ NO: 438, CH1 H3 The amino acid at position 447 (EU numbering) is deleted.

[0438] In addition to the specific complementary determining regions described above, the bispecific antibody EIP0187 comprises the following amino acid substitutions in H2 and L2: amino acid at position 39 (Kabat numbering) of VH2 is D, and amino acid at position 38 (Kabat numbering) of VL2 is K; CH2 H1 The amino acid at position 147 (EU numbering) of CL2 is D, and the amino acid at position 180 (EU numbering) of CL2 is R; CH2 H1 The amino acid at position 136 (EU numbering) of H2H is C, and the amino acid at position 114 (EU numbering) of CL2 is C; the amino acid at position 220 (EU numbering) of H2H is S, and the amino acid at position 214 (EU numbering) of CL2 is S; the amino acids at positions 234, 235, and 237 (EU numbering) of H2H are A; CH2 H3 The amino acid at position 354 (EU numbering) is C; CH2 H3 The amino acid at position 366 (EU numbering) is W; and when numbered according to the H2 amino acid sequence of SEQ NO: 437 and the L2 amino acid sequence of SEQ NO: 436, CH2 H3 The amino acid at position 447 (EU numbering) is deleted.

[0439] In some embodiments, the bispecific antibody EIP0187 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 13, a VH2 comprising the amino acid sequence of SEQ ID NO: 2, a VL1 comprising the amino acid sequence of SEQ ID NO: 22, and a VL2 comprising the amino acid sequence of SEQ ID NO: 1.

[0440] In some embodiments, the bispecific antibody EIP0187 comprises H1 comprising the amino acid sequence of SEQ ID NO:66, H2 comprising the amino acid sequence of SEQ ID NO:64, L1 comprising the amino acid sequence of SEQ ID NO:65, and L2 comprising the amino acid sequence of SEQ ID NO:63.

[0441] In addition to the specific complementarity determining regions described above, the bispecific antibody EIP0205 comprises the following amino acid substitutions in H1 and L1: amino acid position 39 (Kabat numbering) of VH1 is K, and amino acid position 38 (Kabat numbering) of VL1 is D; H1 The amino acid at position 147 (EU numbering) of CL1 is K, and the amino acid at position 131 (EU numbering) of CL1 is D; H1The amino acid at position 173 (EU numbering) of H1H is C, and the amino acid at position 162 (EU numbering) of CL1 is C; the amino acid at position 220 (EU numbering) of H1H is S, and the amino acid at position 214 (EU numbering) of CL1 is S; the amino acids at positions 234, 235, and 237 (EU numbering) of H1H are A; H3 The amino acid at position 349 (EU numbering) is C;

[0442] CH1 H3 The amino acid at position 366 (EU numbering) is S; CH1 H3 The amino acid at position 368 (EU numbering) is A; CH1 H3 The amino acid at position 407 (EU numbering) is V; and when numbered according to the H1 amino acid sequence of SEQ NO: 439 and the L1 amino acid sequence of SEQ NO: 438, CH1 H3 The amino acid at position 447 (EU numbering) is deleted.

[0443] In addition to the specific CDRs described above, the bispecific antibody EIP0205 comprises the following amino acid substitutions in H2 and L2: amino acid position 39 (Kabat numbering) of VH2 is D, and amino acid position 38 (Kabat numbering) of VL2 is K; CH2 H1 The amino acid at position 147 (EU numbering) of CL2 is D, and the amino acid at position 180 (EU numbering) of CL2 is R; the amino acids at positions 234, 235, and 237 (EU numbering) of H2H are A; CH2 H3 The amino acid at position 354 (EU numbering) is C; CH2 H3 The amino acid at position 366 (EU numbering) is W; and when numbered according to the H2 amino acid sequence of SEQ NO: 437 and the L2 amino acid sequence of SEQ NO: 436, CH2 H3 The amino acid at position 447 (EU numbering) is deleted.

[0444] In some embodiments, the bispecific antibody EIP0205 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 13, a VH2 comprising the amino acid sequence of SEQ ID NO: 2, a VL1 comprising the amino acid sequence of SEQ ID NO: 22, and a VL2 comprising the amino acid sequence of SEQ ID NO: 1.

[0445] In some embodiments, the bispecific antibody EIP0205 comprises H1 comprising the amino acid sequence of SEQ ID NO:70, H2 comprising the amino acid sequence of SEQ ID NO:68, L1 comprising the amino acid sequence of SEQ ID NO:69, and L2 comprising the amino acid sequence of SEQ ID NO:67.

[0446] In addition to the specific complementary determining regions described above, the bispecific antibody EIP0206 contains the following amino acid substitutions in H1 and L1: amino acid at position 39 (Kabat numbering) of VH1 is K, amino acid at position 38 (Kabat numbering) of VL1 is D; H1 The amino acid at position 185 (EU numbering) of CL1 is K, and the amino acid at position 137 (EU numbering) of CL1 is D; H1 The amino acid at position 173 (EU numbering) in H1H is C, and the amino acid at position 162 (EU numbering) in CL1 is C; the amino acid at position 220 (EU numbering) in H1H is S, and the amino acid at position 214 in CL1 is S;

[0447] The amino acids at positions 234, 235, and 237 (EU numbering) in H1H are A; CH1 H3 The amino acid at position 349 (EU numbering) is C; CH1 H3 The amino acid at position 366 (EU numbering) is S; CH1 H3 The amino acid at position 368 (EU numbering) is A; CH1 H3 The amino acid at position 407 (EU numbering) is V; and

[0448] When numbering according to the H1 amino acid sequence of SEQ NO: 439 and the L1 amino acid sequence of SEQ NO: 438, CH1 H3 The amino acid at position 447 (EU numbering) is deleted.

[0449] In addition to the specific complementary determining regions described above, the bispecific antibody EIP0206 comprises the following amino acid substitutions in H2 and L2: amino acid position 39 (Kabat numbering) of VH2 is D, and amino acid position 38 (Kabat numbering) of VL2 is K; CH2 H1 The amino acid at position 147 (EU numbering) of CL2 is D, and the amino acid at position 180 (EU numbering) of CL2 is R; CH2 H1 The amino acid at position 134 (EU numbering) of CL2 is C, and the amino acid at position 116 (EU numbering) of CL2 is C; the amino acids at positions 234, 235, and 237 (EU numbering) of H2H are A; CH2 H3 The amino acid at position 354 (EU numbering) is C; CH2H3 The amino acid at position 366 (EU numbering) is W; and when numbered according to the H2 amino acid sequence of SEQ NO: 437 and the L2 amino acid sequence of SEQ NO: 436, CH2 H3 The amino acid at position 447 (EU numbering) is deleted.

[0450] In some embodiments, the bispecific antibody EIP0206 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 13, a VH2 comprising the amino acid sequence of SEQ ID NO: 2, a VL1 comprising the amino acid sequence of SEQ ID NO: 22, and a VL2 comprising the amino acid sequence of SEQ ID NO: 1.

[0451] In some embodiments, the bispecific antibody EIP0206 comprises H1 comprising the amino acid sequence of SEQ ID NO:74, H2 comprising the amino acid sequence of SEQ ID NO:72, L1 comprising the amino acid sequence of SEQ ID NO:73, and L2 comprising the amino acid sequence of SEQ ID NO:71.

[0452] In addition to the specific complementary determining regions described above, the bispecific antibody EIP0207 comprises the following amino acid substitutions in H1 and L1: amino acid position 39 (Kabat numbering) of VH1 is K, and amino acid position 38 (Kabat numbering) of VL1 is D; H1 The amino acid at position 185 (EU numbering) of CL1 is K, and the amino acid at position 137 (EU numbering) of CL1 is D; the amino acids at positions 234, 235, and 237 (EU numbering) of H1H are A; H3 The amino acid at position 349 (EU numbering) is C; CH1 H3 The amino acid at position 366 (EU numbering) is S; CH1 H3 The amino acid at position 368 (EU numbering) is A; CH1 H3 The amino acid at position 407 (EU numbering) is V; and when numbered according to the H1 amino acid sequence of SEQ NO: 439 and the L1 amino acid sequence of SEQ NO: 438, CH1 H3 The amino acid at position 447 (EU numbering) is deleted.

[0453] In addition to the specific complementary determining regions described above, the bispecific antibody EIP0207 comprises the following amino acid substitutions in H2 and L2: amino acid at position 39 (Kabat numbering) of VH2 is D, and amino acid at position 38 (Kabat numbering) of VL2 is K; CH2 H1The amino acid at position 147 (EU numbering) of CL2 is D, and the amino acid at position 180 (EU numbering) of CL2 is R; CH2 H1 The amino acid at position 131 (EU numbering) of H2H is C, and the amino acid at position 114 (EU numbering) of CL2 is C; the amino acid at position 220 (EU numbering) of H2H is S, and the amino acid at position 214 (EU numbering) of CL2 is S; the amino acids at positions 234, 235, and 237 (EU numbering) of H2H are A; CH2 H3 The amino acid at position 354 (EU numbering) is C; CH2 H3 The amino acid at position 366 (EU numbering) is W; and when numbered according to the H2 amino acid sequence of SEQ NO: 437 and the L2 amino acid sequence of SEQ NO: 436, CH2 H3 The amino acid at position 447 (EU numbering) is deleted.

[0454] In some embodiments, the bispecific antibody EIP0207 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 13, a VH2 comprising the amino acid sequence of SEQ ID NO: 2, a VL1 comprising the amino acid sequence of SEQ ID NO: 22, and a VL2 comprising the amino acid sequence of SEQ ID NO: 1.

[0455] In some embodiments, the bispecific antibody EIP0207 comprises H1 comprising the amino acid sequence of SEQ ID NO:78, H2 comprising the amino acid sequence of SEQ ID NO:76, L1 comprising the amino acid sequence of SEQ ID NO:77, and L2 comprising the amino acid sequence of SEQ ID NO:75.

[0456] In addition to the specific complementary determining regions described above, the bispecific antibody EIP0208 comprises the following amino acid substitutions in H1 and L1: amino acid position 39 (Kabat numbering) of VH1 is K, and amino acid position 38 (Kabat numbering) of VL1 is D; H1 The amino acid at position 185 (EU numbering) of CL1 is K, and the amino acid at position 137 (EU numbering) of CL1 is D; the amino acids at positions 234, 235, and 237 (EU numbering) of H1H are A; H3 The amino acid at position 349 (EU numbering) is C; CH1 H3 The amino acid at position 366 (EU numbering) is S; CH1 H3 The amino acid at position 368 (EU numbering) is A; CH1 H3The amino acid at position 407 (EU numbering) is V; and when numbered according to the H1 amino acid sequence of SEQ NO: 439 and the L1 amino acid sequence of SEQ NO: 438, CH1 H3 The amino acid at position 447 (EU numbering) is deleted.

[0457] In addition to the specific complementarity determining regions described above, the bispecific antibody EIP0208 comprises the following amino acid substitutions in H2 and L2: amino acid position 39 (Kabat numbering) of VH2 is D, and amino acid position 38 (Kabat numbering) of VL2 is K; CH2 H1 The amino acid at position 187 (EU numbering) of CL2 is D, and the amino acid at position 138 (EU numbering) of CL2 is K; CH2 H1 The amino acid at position 170 (EU numbering) of H2H is C, and the amino acid at position 162 (EU numbering) of CL2 is C; the amino acid at position 220 (EU numbering) of H2H is S, and the amino acid at position 214 (EU numbering) of CL2 is S; the amino acids at positions 234, 235, and 237 (EU numbering) of H2H are A; CH2 H3 The amino acid at position 354 (EU numbering) is C; CH2 H3 The amino acid at position 366 (EU numbering) is W; and when numbered according to the H2 amino acid sequence of SEQ NO: 437 and the L2 amino acid sequence of SEQ NO: 436, CH2 H3 The amino acid at position 447 (EU numbering) is deleted.

[0458] In some embodiments, the bispecific antibody EIP0208 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 13, a VH2 comprising the amino acid sequence of SEQ ID NO: 2, a VL1 comprising the amino acid sequence of SEQ ID NO: 22, and a VL2 comprising the amino acid sequence of SEQ ID NO: 1.

[0459] In some embodiments, the bispecific antibody EIP0208 comprises H1 comprising the amino acid sequence of SEQ ID NO:82, H2 comprising the amino acid sequence of SEQ ID NO:80, L1 comprising the amino acid sequence of SEQ ID NO:81, and L2 comprising the amino acid sequence of SEQ ID NO:79.

[0460] In addition to the specific complementary determining regions described above, the bispecific antibody EIP0294 comprises the following amino acid substitutions in H1 and L1: amino acid position 39 (Kabat numbering) of VH1 is K, and amino acid position 38 (Kabat numbering) of VL1 is D; H1The amino acid at position 185 (EU numbering) of CL1 is E, and the amino acid at position 137 (EU numbering) of CL1 is K; and the amino acid at position 179 (EU numbering) of CL1 is E; the amino acids at positions 234, 235, and 237 (EU numbering) of H1H are A; CH1 H3 The amino acid at position 349 (EU numbering) is C; CH1 H3 The amino acid at position 366 (EU numbering) is S; CH1 H3 The amino acid at position 368 (EU numbering) is A; CH1 H3 The amino acid at position 407 (EU numbering) is V; and

[0461] When numbering according to the H1 amino acid sequence of SEQ NO: 439 and the L1 amino acid sequence of SEQ NO: 438, CH1 H3 The amino acid at position 447 (EU numbering) is deleted.

[0462] In addition to the specific CDRs described above, the bispecific antibody EIP0294 comprises the following amino acid substitutions in H2 and L2: amino acid position 39 (Kabat numbering) of VH2 is D, and amino acid position 38 (Kabat numbering) of VL2 is K; CH2 H1 The amino acid at position 187 (EU numbering) of CL2 is D, and the amino acid at position 138 (EU numbering) of CL2 is K; CH2 H1 The amino acid at position 171 (EU numbering) of H2H is C, and the amino acid at position 162 (EU numbering) of CL2 is C; the amino acid at position 220 (EU numbering) of H2H is S, and the amino acid at position 214 (EU numbering) of CL2 is S; the amino acids at positions 234, 235, and 237 (EU numbering) of H2H are A; CH2 H3 The amino acid at position 354 (EU numbering) is C; CH2 H3 The amino acid at position 366 (EU numbering) is W; and when numbered according to the H2 amino acid sequence of SEQ NO: 437 and the L2 amino acid sequence of SEQ NO: 436, CH2 H3 The amino acid at position 447 (EU numbering) is deleted.

[0463] In some embodiments, the bispecific antibody EIP0294 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 13, a VH2 comprising the amino acid sequence of SEQ ID NO: 2, a VL1 comprising the amino acid sequence of SEQ ID NO: 22, and a VL2 comprising the amino acid sequence of SEQ ID NO: 1.

[0464] In some embodiments, the bispecific antibody EIP0294 comprises H1 comprising the amino acid sequence of SEQ ID NO:86, H2 comprising the amino acid sequence of SEQ ID NO:84, L1 comprising the amino acid sequence of SEQ ID NO:85, and L2 comprising the amino acid sequence of SEQ ID NO:83.

[0465] In addition to the specific complementarity determining regions described above, the bispecific antibody EIP0295 comprises the following amino acid substitutions in H1 and L1: amino acid position 39 (Kabat numbering) of VH1 is K, and amino acid position 38 (Kabat numbering) of VL1 is D; H1 The amino acid at position 185 (EU numbering) of CL1 is K, and the amino acid at position 137 (EU numbering) of CL1 is D; the amino acids at positions 234, 235, and 237 (EU numbering) of H1H are A; H3 The amino acid at position 349 (EU numbering) is C; CH1 H3 The amino acid at position 366 (EU numbering) is S; CH1 H3 The amino acid at position 368 (EU numbering) is A; CH1 H3 The amino acid at position 407 (EU numbering) is V; and when numbered according to the H1 amino acid sequence of SEQ NO: 439 and the L1 amino acid sequence of SEQ NO: 438, CH1 H3 The amino acid at position 447 (EU numbering) is deleted.

[0466] In addition to the specific complementarity determining regions described above, the bispecific antibody EIP0295 comprises the following amino acid substitutions in H2 and L2: amino acid position 39 (Kabat numbering) of VH2 is D, and amino acid position 38 (Kabat numbering) of VL2 is K; CH2 H1 The amino acid at position 187 (EU numbering) of CL2 is D, and the amino acid at position 138 (EU numbering) of CL2 is K; CH2 H1 The amino acid at position 171 (EU numbering) of H2H is C, and the amino acid at position 162 (EU numbering) of CL2 is C; the amino acid at position 220 (EU numbering) of H2H is S, and the amino acid at position 214 (EU numbering) of CL2 is S; the amino acids at positions 234, 235, and 237 (EU numbering) of H2H are A; CH2 H3 The amino acid at position 354 (EU numbering) is C; CH2 H3 The amino acid at position 366 (EU numbering) is W; and when numbered according to the H2 amino acid sequence of SEQ NO: 437 and the L2 amino acid sequence of SEQ NO: 436, CH2 H3The amino acid at position 447 (EU numbering) is deleted.

[0467] In some embodiments, the bispecific antibody EIP0295 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 13, a VH2 comprising the amino acid sequence of SEQ ID NO: 2, a VL1 comprising the amino acid sequence of SEQ ID NO: 22, and a VL2 comprising the amino acid sequence of SEQ ID NO: 1.

[0468] In some embodiments, the bispecific antibody EIP0295 comprises H1 comprising the amino acid sequence of SEQ ID NO:90, H2 comprising the amino acid sequence of SEQ ID NO:88, L1 comprising the amino acid sequence of SEQ ID NO:89, and L2 comprising the amino acid sequence of SEQ ID NO:87.

[0469] In addition to the specific complementarity determining regions described above, the bispecific antibody EIP0306 comprises the following amino acid substitutions in H1 and L1: amino acid at position 39 (Kabat numbering) of VH1 is K, and amino acid at position 38 (Kabat numbering) of VL1 is D; H1 The amino acid at position 185 (EU numbering) of CL1 is E, the amino acid at position 137 (EU numbering) of CL1 is K, and the amino acid at position 179 (EU numbering) of CL1 is E; CH1 H1 The amino acid at position 185 (EU numbering) of CL1 is E, and the amino acid at position 179 (EU numbering) of CL1 is E; the amino acids at positions 234, 235, and 237 (EU numbering) of H1H are A; H3 The amino acid at position 349 (EU numbering) is C; CH1 H3 The amino acid at position 366 (EU numbering) is S;

[0470] CH1 H3 The amino acid at position 368 (EU numbering) is A; CH1 H3 The amino acid at position 407 (EU numbering) is V; and when numbered according to the H1 amino acid sequence of SEQ NO: 439 and the L1 amino acid sequence of SEQ NO: 438, CH1 H3 The amino acid at position 447 (EU numbering) is deleted.

[0471] In addition to the specific complementary determining regions described above, the bispecific antibody EIP0306 comprises the following amino acid substitutions in H2 and L2: amino acid position 39 (Kabat numbering) of VH2 is D, and amino acid position 38 (Kabat numbering) of VL2 is K; CH2 H1The amino acid at position 147 (EU numbering) of CL2 is D, and the amino acid at position 180 (EU numbering) of CL2 is R; CH2 H1 The amino acid at position 171 (EU numbering) of H2H is C, and the amino acid at position 162 (EU numbering) of CL2 is C; the amino acid at position 220 (EU numbering) of H2H is S, and the amino acid at position 214 (EU numbering) of CL2 is S; the amino acids at positions 234, 235, and 237 (EU numbering) of H2H are A; CH2 H3 The amino acid at position 354 (EU numbering) is C; CH2 H3 The amino acid at position 366 (EU numbering) is W; and when numbered according to the H2 amino acid sequence of SEQ NO: 437 and the L2 amino acid sequence of SEQ NO: 436, CH2 H3 The amino acid at position 447 (EU numbering) is deleted.

[0472] In some embodiments, the bispecific antibody EIP0306 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 13, a VH2 comprising the amino acid sequence of SEQ ID NO: 2, a VL1 comprising the amino acid sequence of SEQ ID NO: 22, and a VL2 comprising the amino acid sequence of SEQ ID NO: 1.

[0473] In some embodiments, the bispecific antibody EIP0306 comprises H1 comprising the amino acid sequence of SEQ ID NO:94, H2 comprising the amino acid sequence of SEQ ID NO:92, L1 comprising the amino acid sequence of SEQ ID NO:93, and L2 comprising the amino acid sequence of SEQ ID NO:91.

[0474] In addition to the specific complementary determining regions described above, the bispecific antibody EIP0307 comprises the following amino acid substitutions in H1 and L1: amino acid position 39 (Kabat numbering) of VH1 is K, and amino acid position 38 (Kabat numbering) of VL1 is D; H1 The amino acid at position 185 (EU numbering) of CL1 is K, and the amino acid at position 137 (EU numbering) of CL1 is D; the amino acids at positions 234, 235, and 237 (EU numbering) of H1H are A; H3 The amino acid at position 349 (EU numbering) is C; CH1 H3 The amino acid at position 366 (EU numbering) is S; CH1 H3 The amino acid at position 368 (EU numbering) is A; CH1 H3The amino acid at position 407 (EU numbering) is V; and when numbered according to the H1 amino acid sequence of SEQ NO: 439 and the L1 amino acid sequence of SEQ NO: 438, CH1 H3 The amino acid at position 447 (EU numbering) is deleted.

[0475] In addition to the specific complementary determining regions described above, the bispecific antibody EIP0307 contains the following amino acid substitutions in H2 and L2: amino acid at position 39 (Kabat numbering) of VH2 is D, amino acid at position 38 (Kabat numbering) of VL2 is K; CH2 H1 The amino acid at position 147 (EU numbering) of CL2 is D, and the amino acid at position 180 (EU numbering) of CL2 is R; CH2 H1 The amino acid at position 171 (EU numbering) of H2H is C, and the amino acid at position 162 (EU numbering) of CL2 is C; the amino acid at position 220 (EU numbering) of H2H is S, and the amino acid at position 214 (EU numbering) of CL2 is S; the amino acids at positions 234, 235, and 237 (EU numbering) of H2H are A; CH2 H3 The amino acid at position 354 (EU numbering) is C; CH2 H3 The amino acid at position 366 (EU numbering) is W; and when numbered according to the H2 amino acid sequence of SEQ NO: 437 and the L2 amino acid sequence of SEQ NO: 436, CH2 H3 The amino acid at position 447 (EU numbering) is deleted.

[0476] In some embodiments, the bispecific antibody EIP0307 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 13, a VH2 comprising the amino acid sequence of SEQ ID NO: 2, a VL1 comprising the amino acid sequence of SEQ ID NO: 22, and a VL2 comprising the amino acid sequence of SEQ ID NO: 1.

[0477] In some embodiments, the bispecific antibody EIP0307 comprises H1 comprising the amino acid sequence of SEQ ID NO:98, H2 comprising the amino acid sequence of SEQ ID NO:96, L1 comprising the amino acid sequence of SEQ ID NO:97, and L2 comprising the amino acid sequence of SEQ ID NO:95.

[0478] In addition to the specific complementary determining regions described above, the bispecific antibody EIP0318 comprises the following amino acid substitutions in H1 and L1: amino acid position 39 (Kabat numbering) of VH1 is K, and amino acid position 38 (Kabat numbering) of VL1 is D; H1The amino acid at position 147 (EU numbering) of CL1 is K, and the amino acid at position 131 (EU numbering) of CL1 is D; H1 The amino acid at position 185 (EU numbering) of CL1 is K, and the amino acid at position 137 (EU numbering) of CL1 is D; the amino acids at positions 234, 235, and 237 (EU numbering) of H1H are A; CH1 H3 The amino acid at position 349 (EU numbering) is C; CH1 H3 The amino acid at position 366 (EU numbering) is S; CH1 H3 The amino acid at position 368 (EU numbering) is A.

[0479] CH1 H3 The amino acid at position 407 (EU numbering) is V; and when numbered according to the H1 amino acid sequence of SEQ NO: 439 and the L1 amino acid sequence of SEQ NO: 438, CH1 H3 The amino acid at position 447 (EU numbering) is deleted.

[0480] In addition to the specific complementary determining regions described above, the bispecific antibody EIP0318 contains the following amino acid substitutions in H2 and L2: amino acid D at position 39 (Kabat numbering) of VH2, amino acid K at position 38 (Kabat numbering) of VL2; H1 The amino acid at position 147 (EU numbering) of CL2 is D, and the amino acid at position 180 (EU numbering) of CL2 is R; CH2 H1 The amino acid at position 171 (EU numbering) of H2H is C, and the amino acid at position 162 (EU numbering) of CL2 is C; the amino acid at position 220 (EU numbering) of H2H is S, and the amino acid at position 214 (EU numbering) of CL2 is S; the amino acids at positions 234, 235, and 237 (EU numbering) of H2H are A; CH2 H3 The amino acid at position 354 (EU numbering) is C; CH2 H3 The amino acid at position 366 (EU numbering) is W; and when numbered according to the H2 amino acid sequence of SEQ NO: 437 and the L2 amino acid sequence of SEQ NO: 436, CH2 H3 The amino acid at position 447 (EU numbering) is deleted.

[0481] In some embodiments, the bispecific antibody EIP0318 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 13, a VH2 comprising the amino acid sequence of SEQ ID NO: 2, a VL1 comprising the amino acid sequence of SEQ ID NO: 22, and a VL2 comprising the amino acid sequence of SEQ ID NO: 1.

[0482] In some embodiments, the bispecific antibody EIP0318 comprises H1 comprising the amino acid sequence of SEQ ID NO: 102, H2 comprising the amino acid sequence of SEQ ID NO: 100, L1 comprising the amino acid sequence of SEQ ID NO: 101, and L2 comprising the amino acid sequence of SEQ ID NO: 99.

[0483] In addition to the specific complementary determining regions described above, the bispecific antibody EIP0340 comprises the following amino acid substitutions in H1 and L1: amino acid 39 (Kabat numbering) of VH1 is D, and amino acid 38 (Kabat numbering) of VL1 is K; CH1 H1 The amino acid at position 185 (EU numbering) of CL1 is E, and the amino acid at position 137 (EU numbering) of CL1 is K; the amino acids at positions 234, 235, and 237 (EU numbering) of H1H are A; H3 The amino acid at position 349 (EU numbering) is C; CH1 H3 The amino acid at position 366 (EU numbering) is S; CH1 H3 The amino acid at position 368 (EU numbering) is A; CH1 H3 The amino acid at position 407 (EU numbering) is V; and when numbered according to the H1 amino acid sequence of SEQ NO: 439 and the L1 amino acid sequence of SEQ NO: 438, CH1 H3 The amino acid at position 447 (EU numbering) is deleted.

[0484] In addition to the specific complementary determining regions described above, the bispecific antibody EIP0340 comprises the following amino acid substitutions in H2 and L2: amino acid position 39 (Kabat numbering) of VH2 is K, and amino acid position 38 (Kabat numbering) of VL2 is D; CH2 H1 The amino acid at position 187 (EU numbering) of CL2 is D, and the amino acid at position 138 (EU numbering) of CL2 is K; CH2 H1 The amino acid at position 136 (EU numbering) of H2H is C, and the amino acid at position 114 (EU numbering) of CL2 is C; the amino acid at position 220 (EU numbering) of H2H is S, and the amino acid at position 214 (EU numbering) of CL2 is S; the amino acids at positions 234, 235, and 237 (EU numbering) of H2H are A; CH2 H3 The amino acid at position 354 (EU numbering) is C; CH2 H3 The amino acid at position 366 (EU numbering) is W; and when numbered according to the H2 amino acid sequence of SEQ NO: 437 and the L2 amino acid sequence of SEQ NO: 436, CH2 H3The amino acid at position 447 (EU numbering) is deleted.

[0485] In some embodiments, the bispecific antibody EIP0340 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 14, a VH2 comprising the amino acid sequence of SEQ ID NO: 4, a VL1 comprising the amino acid sequence of SEQ ID NO: 23, and a VL2 comprising the amino acid sequence of SEQ ID NO: 3.

[0486] In some embodiments, the bispecific antibody EIP0340 comprises H1 comprising the amino acid sequence of SEQ ID NO: 106, H2 comprising the amino acid sequence of SEQ ID NO: 104, L1 comprising the amino acid sequence of SEQ ID NO: 105, and L2 comprising the amino acid sequence of SEQ ID NO: 103.

[0487] In addition to the specific complementarity determining regions described above, the bispecific antibody EIP0342 comprises the following amino acid substitutions in H1 and L1: amino acid position 39 (Kabat numbering) of VH1 is D, and amino acid position 38 (Kabat numbering) of VL1 is K; H1 The amino acid at position 185 (EU numbering) of CL1 is E, and the amino acid at position 137 (EU numbering) of CL1 is K; the amino acids at positions 234, 235, and 237 (EU numbering) of H1H are A; H3 The amino acid at position 349 (EU numbering) is C; CH1 H3 The amino acid at position 366 (EU numbering) is S; CH1 H3 The amino acid at position 368 (EU numbering) is A; CH1 H3 The amino acid at position 407 (EU numbering) is V; and when numbered according to the H1 amino acid sequence of SEQ NO: 439 and the L1 amino acid sequence of SEQ NO: 438, CH1 H3 The amino acid at position 447 (EU numbering) is deleted.

[0488] In addition to the specific complementary determining regions described above, the bispecific antibody EIP0342 comprises the following amino acid substitutions in H2 and L2: amino acid position 39 (Kabat numbering) of VH2 is K, and amino acid position 38 (Kabat numbering) of VL2 is D; CH2 H1 The amino acid at position 187 (EU numbering) of CL2 is D, and the amino acid at position 138 (EU numbering) of CL2 is K; CH2 H1The amino acid at position 171 (EU numbering) of H2H is C, and the amino acid at position 162 (EU numbering) of CL2 is C; the amino acid at position 220 (EU numbering) of H2H is S, and the amino acid at position 214 (EU numbering) of CL2 is S; the amino acids at positions 234, 235, and 237 (EU numbering) of H2H are A; CH2 H3 The amino acid at position 354 (EU numbering) is C; CH2 H3 The amino acid at position 366 (EU numbering) is W; and when numbered according to the H2 amino acid sequence of SEQ NO: 437 and the L2 amino acid sequence of SEQ NO: 436, CH2 H3 The amino acid at position 447 (EU numbering) is deleted.

[0489] In some embodiments, the bispecific antibody EIP0342 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 14, a VH2 comprising the amino acid sequence of SEQ ID NO: 4, a VL1 comprising the amino acid sequence of SEQ ID NO: 23, and a VL2 comprising the amino acid sequence of SEQ ID NO: 3.

[0490] In some embodiments, the bispecific antibody EIP0342 comprises H1 comprising the amino acid sequence of SEQ ID NO: 110, H2 comprising the amino acid sequence of SEQ ID NO: 108, L1 comprising the amino acid sequence of SEQ ID NO: 109, and L2 comprising the amino acid sequence of SEQ ID NO: 107.

[0491] In addition to the specific complementary determining regions described above, the bispecific antibody EIP0354 comprises the following amino acid substitutions in H1 and L1: amino acid position 39 (Kabat numbering) of VH1 is D, and amino acid position 38 (Kabat numbering) of VL1 is K; H1 The amino acid at position 185 (EU numbering) of CL1 is K, and the amino acid at position 137 (EU numbering) of CL1 is D; the amino acids at positions 234, 235, and 237 (EU numbering) of H1H are A; H3 The amino acid at position 349 (EU numbering) is C; CH1 H3 The amino acid at position 366 (EU numbering) is S; CH1 H3 The amino acid at position 368 (EU numbering) is A; CH1 H3 The amino acid at position 407 (EU numbering) is V; and when numbered according to the H1 amino acid sequence of SEQ NO: 439 and the L1 amino acid sequence of SEQ NO: 438, CH1 H3 The amino acid at position 447 (EU numbering) is deleted.

[0492] In addition to the specific complementary determining regions described above, the bispecific antibody EIP0354 comprises the following amino acid substitutions in H2 and L2: amino acid position 39 (Kabat numbering) of VH2 is K, and amino acid position 38 (Kabat numbering) of VL2 is D; CH2 H1 The amino acid at position 187 (EU numbering) of CL2 is D, and the amino acid at position 138 (EU numbering) of CL2 is K; CH2 H1 The amino acid at position 171 (EU numbering) of H2H is C, and the amino acid at position 162 (EU numbering) of CL2 is C; the amino acid at position 220 (EU numbering) of H2H is S, and the amino acid at position 214 (EU numbering) of CL2 is S; the amino acids at positions 234, 235, and 237 (EU numbering) of H2H are A; CH2 H3 The amino acid at position 354 (EU numbering) is C; CH2 H3 The amino acid at position 366 (EU numbering) is W; and when numbered according to the H2 amino acid sequence of SEQ NO: 437 and the L2 amino acid sequence of SEQ NO: 436, CH2 H3 The amino acid at position 447 (EU numbering) is deleted.

[0493] In some embodiments, the bispecific antibody EIP0354 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 14, a VH2 comprising the amino acid sequence of SEQ ID NO: 4, a VL1 comprising the amino acid sequence of SEQ ID NO: 23, and a VL2 comprising the amino acid sequence of SEQ ID NO: 3.

[0494] In some embodiments, the bispecific antibody EIP0354 comprises H1 comprising the amino acid sequence of SEQ ID NO: 114, H2 comprising the amino acid sequence of SEQ ID NO: 112, L1 comprising the amino acid sequence of SEQ ID NO: 113, and L2 comprising the amino acid sequence of SEQ ID NO: 111.

[0495] In addition to the specific complementarity determining regions described above, the bispecific antibody EIP0356 comprises the following amino acid substitutions in H1 and L1: amino acid position 39 (Kabat numbering) of VH1 is D, and amino acid position 38 (Kabat numbering) of VL1 is K; H1 The amino acid at position 147 (EU numbering) of CL1 is K, and the amino acid at position 131 (EU numbering) of CL1 is D; H1 The amino acid at position 185 (EU numbering) of CL1 is E, and the amino acid at position 137 (EU numbering) of CL1 is K;H1 The amino acid at position 145 (EU numbering) of CL1 is S, and the amino acid at position 180 (EU numbering) of CL1 is E; the amino acids at positions 234, 235, and 237 (EU numbering) of H1H are A; CH1 H3 The amino acid at position 349 (EU numbering) is C; CH1 H3 The amino acid at position 366 (EU numbering) is S; CH1 H3 The amino acid at position 368 (EU numbering) is A; CH1 H3 The amino acid at position 407 (EU numbering) is V; and when numbered according to the H1 amino acid sequence of SEQ NO: 439 and the L1 amino acid sequence of SEQ NO: 438, CH1 H3 The amino acid at position 447 (EU numbering) is deleted.

[0496] In addition to the specific complementary determining regions described above, the bispecific antibody EIP0356 comprises the following amino acid substitutions in H2 and L2: amino acid position 39 (Kabat numbering) of VH2 is K, and amino acid position 38 (Kabat numbering) of VL2 is D; CH2 H1 The amino acid at position 187 (EU numbering) of CL2 is D, and the amino acid at position 138 (EU numbering) of CL2 is K; CH2 H1 The amino acid at position 170 (EU numbering) of H2H is C, and the amino acid at position 162 (EU numbering) of CL2 is C; the amino acid at position 220 (EU numbering) of H2H is S, and the amino acid at position 214 (EU numbering) of CL2 is S; the amino acids at positions 234, 235, and 237 (EU numbering) of H2H are A; CH2 H3 The amino acid at position 354 (EU numbering) is C; CH2 H3 The amino acid at position 366 (EU numbering) is W; and when numbered according to the H2 amino acid sequence of SEQ NO: 437 and the L2 amino acid sequence of SEQ NO: 436, CH2 H3 The amino acid at position 447 (EU numbering) is deleted.

[0497] In some embodiments, the bispecific antibody EIP0356 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 14, a VH2 comprising the amino acid sequence of SEQ ID NO: 4, a VL1 comprising the amino acid sequence of SEQ ID NO: 23, and a VL2 comprising the amino acid sequence of SEQ ID NO: 3.

[0498] In some embodiments, the bispecific antibody EIP0356 comprises H1 comprising the amino acid sequence of SEQ ID NO: 118, H2 comprising the amino acid sequence of SEQ ID NO: 116, L1 comprising the amino acid sequence of SEQ ID NO: 117, and L2 comprising the amino acid sequence of SEQ ID NO: 115.

[0499] In addition to the specific complementary determining regions described above, the bispecific antibody EIP0367 comprises the following amino acid substitutions in H1 and L1: amino acid at position 39 (Kabat numbering) of VH1 is K, and amino acid at position 38 (Kabat numbering) of VL1 is D; H1 The amino acid at position 147 (EU numbering) of CL1 is D, and the amino acid at position 131 (EU numbering) of CL1 is K; H1 The amino acid at position 185 (EU numbering) of CL1 is E, and the amino acid at position 137 (EU numbering) of CL1 is D; the amino acids at positions 234, 235, and 237 (EU numbering) of H1H are A; CH1 H3 The amino acid at position 349 (EU numbering) is C; CH1 H3 The amino acid at position 366 (EU numbering) is S; CH1 H3 The amino acid at position 368 (EU numbering) is A; CH1 H3 The amino acid at position 407 (EU numbering) is V; and when numbered according to the H1 amino acid sequence NO: 439 of SEQ and the L1 amino acid sequence NO: 438 of SEQ, CH1 H3 The amino acid at position 447 (EU numbering) is deleted.

[0500] In addition to the specific complementary determining regions described above, the bispecific antibody EIP0367 comprises the following amino acid substitutions in H2 and L2: amino acid position 39 (Kabat numbering) of VH2 is D, and amino acid position 38 (Kabat numbering) of VL2 is K; CH2 H1 The amino acid at position 187 (EU numbering) of CL2 is D, the amino acid at position 137 (EU numbering) of CL2 is K, and the amino acid at position 138 (EU numbering) of CL2 is R; CH2 H1 The amino acid at position 170 (EU numbering) of H2H is C, and the amino acid at position 162 (EU numbering) of CL2 is C; the amino acid at position 220 (EU numbering) of H2H is S, and the amino acid at position 214 (EU numbering) of CL2 is S; the amino acids at positions 234, 235, and 237 (EU numbering) of H2H are A; CH2 H3 The amino acid at position 354 (EU numbering) is C; CH2 H3The amino acid at position 366 (EU numbering) is W; and when numbered according to the H2 amino acid sequence of SEQ NO: 437 and the L2 amino acid sequence of SEQ NO: 436, CH2 H3 The amino acid at position 447 (EU numbering) is deleted.

[0501] In some embodiments, the bispecific antibody EIP0367 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 13, a VH2 comprising the amino acid sequence of SEQ ID NO: 2, a VL1 comprising the amino acid sequence of SEQ ID NO: 22, and a VL2 comprising the amino acid sequence of SEQ ID NO: 1.

[0502] In some embodiments, the bispecific antibody EIP0367 comprises H1 comprising the amino acid sequence of SEQ ID NO: 122, H2 comprising the amino acid sequence of SEQ ID NO: 120, L1 comprising the amino acid sequence of SEQ ID NO: 121, and L2 comprising the amino acid sequence of SEQ ID NO: 119.

[0503] In addition to the specific complementary determining regions described above, the bispecific antibody EIP0377 comprises the following amino acid substitutions in H1 and L1: amino acid position 39 (Kabat numbering) of VH1 is K, and amino acid position 38 (Kabat numbering) of VL1 is D; H1 The amino acid at position 147 (EU numbering) is D, and CH1 H1 The amino acid at position 145 (EU numbering) of CL1 is S; the amino acid at position 131 (EU numbering) of CL1 is K; the amino acids at positions 234, 235, and 237 (EU numbering) of H1H are A; H3 The amino acid at position 349 (EU numbering) is C; CH1 H3 The amino acid at position 366 (EU numbering) is S; CH1 H3 The amino acid at position 368 (EU numbering) is A; CH1 H3 The amino acid at position 407 (EU numbering) is V; and

[0504] When numbering according to the H1 amino acid sequence of SEQ NO: 439 and the L1 amino acid sequence of SEQ NO: 438, CH1 H3 The amino acid at position 447 (EU numbering) is deleted.

[0505] In addition to the specific complementarity determining regions described above, the bispecific antibody EIP0377 comprises the following amino acid substitutions in H2 and L2: amino acid position 39 (Kabat numbering) of VH2 is D, and amino acid position 38 (Kabat numbering) of VL2 is K; CH2 H1 The amino acid at position 187 (EU numbering) of CL2 is D, and the amino acid at position 138 (EU numbering) of CL2 is K; CH2 H1 The amino acid at position 170 (EU numbering) of H2H is C, and the amino acid at position 162 (EU numbering) of CL2 is C; the amino acid at position 220 (EU numbering) of H2H is S, and the amino acid at position 214 (EU numbering) of CL2 is S; the amino acids at positions 234, 235, and 237 (EU numbering) of H2H are A; CH2 H3 The amino acid at position 354 (EU numbering) is C; CH2 H3 The amino acid at position 366 (EU numbering) is W; and when numbered according to the H2 amino acid sequence of SEQ NO: 437 and the L2 amino acid sequence of SEQ NO: 436, CH2 H3 The amino acid at position 447 (EU numbering) is deleted.

[0506] In some embodiments, the bispecific antibody EIP0377 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 13, a VH2 comprising the amino acid sequence of SEQ ID NO: 2, a VL1 comprising the amino acid sequence of SEQ ID NO: 22, and a VL2 comprising the amino acid sequence of SEQ ID NO: 1.

[0507] In some embodiments, the bispecific antibody EIP0377 comprises H1 comprising the amino acid sequence of SEQ ID NO: 126, H2 comprising the amino acid sequence of SEQ ID NO: 124, L1 comprising the amino acid sequence of SEQ ID NO: 125, and L2 comprising the amino acid sequence of SEQ ID NO: 123.

[0508] In addition to the specific complementarity determining regions described above, the bispecific antibody EIP0404 comprises the following amino acid substitutions in H1 and L1: amino acid position 39 (Kabat numbering) of VH1 is D, and amino acid position 38 (Kabat numbering) of VL1 is K; H1 The amino acid at position 147 (EU numbering) of CL1 is K, and the amino acid at position 131 (EU numbering) of CL1 is D; H1 The amino acid at position 185 (EU numbering) of CL1 is E, and the amino acid at position 137 (EU numbering) of CL1 is K; the amino acids at positions 234, 235, and 237 (EU numbering) of H1H are A; CH1H3 The amino acid at position 349 (EU numbering) is C; CH1 H3 The amino acid at position 366 (EU numbering) is S; CH1 H3 The amino acid at position 368 (EU numbering) is A; CH1 H3 The amino acid at position 407 (EU numbering) is V; and when numbered according to the H1 amino acid sequence of SEQ NO: 439 and the L1 amino acid sequence of SEQ NO: 438, CH1 H3 The amino acid at position 447 (EU numbering) is deleted.

[0509] In addition to the specific complementary determining regions described above, the bispecific antibody EIP0404 comprises the following amino acid substitutions in H2 and L2: amino acid position 39 (Kabat numbering) of VH2 is K, and amino acid position 38 (Kabat numbering) of VL2 is D; CH2 H1 The amino acid at position 147 (EU numbering) of CL2 is D, and the amino acid at position 180 (EU numbering) of CL2 is R; CH2 H1 The amino acid at position 136 (EU numbering) of H2H is C, and the amino acid at position 114 (EU numbering) of CL2 is C; the amino acid at position 220 (EU numbering) of H2H is S, and the amino acid at position 214 (EU numbering) of CL2 is S; the amino acids at positions 234, 235, and 237 (EU numbering) of H2H are A; CH2 H3 The amino acid at position 354 (EU numbering) is C; CH2 H3 The amino acid at position 366 (EU numbering) is W; and when numbered according to the H2 amino acid sequence of SEQ NO: 437 and the L2 amino acid sequence of SEQ NO: 436, CH2 H3 The amino acid at position 447 (EU numbering) is deleted.

[0510] In some embodiments, the bispecific antibody EIP0404 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 13, a VH2 comprising the amino acid sequence of SEQ ID NO: 2, a VL1 comprising the amino acid sequence of SEQ ID NO: 24, and a VL2 comprising the amino acid sequence of SEQ ID NO: 1.

[0511] In some embodiments, the bispecific antibody EIP0404 comprises H1 comprising the amino acid sequence of SEQ ID NO: 130, H2 comprising the amino acid sequence of SEQ ID NO: 128, L1 comprising the amino acid sequence of SEQ ID NO: 129, and L2 comprising the amino acid sequence of SEQ ID NO: 127.

[0512] In addition to the specific complementarity determining regions described above, the bispecific antibody EIP0406 comprises the following amino acid substitutions in H1 and L1: amino acid K at position 39 (Kabat numbering) of VH1 and amino acid E at position 38 (Kabat numbering) of VL1; H1 The amino acid at position 147 (EU numbering) of CL1 is K, and the amino acid at position 131 (EU numbering) of CL1 is D; H1 The amino acid at position 185 (EU numbering) of CL1 is E, and the amino acid at position 137 (EU numbering) of CL1 is K; the amino acids at positions 234, 235, and 237 (EU numbering) of H1H are A; CH1 H3 The amino acid at position 349 (EU numbering) is C; CH1 H3 The amino acid at position 366 (EU numbering) is S; CH1 H3 The amino acid at position 368 (EU numbering) is A; CH1 H3 The amino acid at position 407 (EU numbering) is V; and when numbered according to the H1 amino acid sequence NO: 439 of SEQ and the L1 amino acid sequence NO: 438 of SEQ, CH1 H3 The amino acid at position 447 (EU numbering) is deleted.

[0513] In addition to the specific complementary determining regions described above, the bispecific antibody EIP0406 comprises the following amino acid substitutions in H2 and L2: amino acid position 39 (Kabat numbering) of VH2 is D, and amino acid position 38 (Kabat numbering) of VL2 is K; CH2 H1 The amino acid at position 187 (EU numbering) of CL2 is D, and the amino acid at position 138 (EU numbering) of CL2 is K; CH2 H1 The amino acid at position 136 (EU numbering) of H2H is C, and the amino acid at position 114 (EU numbering) of CL2 is C; the amino acid at position 220 (EU numbering) of H2H is S, and the amino acid at position 214 (EU numbering) of CL2 is S; the amino acids at positions 234, 235, and 237 (EU numbering) of H2H are A; CH2 H3 The amino acid at position 354 (EU numbering) is C; CH2 H3 The amino acid at position 366 (EU numbering) is W; and when numbered according to the H2 amino acid sequence of SEQ NO: 437 and the L2 amino acid sequence of SEQ NO: 436, CH2 H3 The amino acid at position 447 (EU numbering) is deleted.

[0514] In some embodiments, the bispecific antibody EIP0406 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 13, a VH2 comprising the amino acid sequence of SEQ ID NO: 2, a VL1 comprising the amino acid sequence of SEQ ID NO: 24, and a VL2 comprising the amino acid sequence of SEQ ID NO: 1.

[0515] In some embodiments, the bispecific antibody EIP0406 comprises H1 comprising the amino acid sequence of SEQ ID NO: 134, H2 comprising the amino acid sequence of SEQ ID NO: 132, L1 comprising the amino acid sequence of SEQ ID NO: 133, and L2 comprising the amino acid sequence of SEQ ID NO: 131.

[0516] In addition to the specific complementarity determining regions described above, the bispecific antibody EIP0473 comprises the following amino acid substitutions in H1 and L1: amino acid position 39 (Kabat numbering) of VH1 is K, and amino acid position 38 (Kabat numbering) of VL1 is D; H1 The amino acid at position 147 (EU numbering) of CL1 is D, and the amino acid at position 131 (EU numbering) of CL1 is K; H1 The amino acid at position 185 (EU numbering) of CL1 is D, and the amino acid at position 137 (EU numbering) of CL1 is K; the amino acids at positions 234, 235, and 237 (EU numbering) of H1H are A; CH1 H3 The amino acid at position 349 (EU numbering) is C; CH1 H3 The amino acid at position 366 (EU numbering) is S; CH1 H3 The amino acid at position 368 (EU numbering) is A; CH1 H3 The amino acid at position 407 (EU numbering) is V; and when numbered according to the H1 amino acid sequence of SEQ NO: 439 and the L1 amino acid sequence of SEQ NO: 438, CH1 H3 The amino acid at position 447 (EU numbering) is deleted.

[0517] In addition to the specific CDRs described above, the bispecific antibody EIP0473 comprises the following amino acid substitutions in H2 and L2: amino acid position 39 (Kabat numbering) of VH2 is D, and amino acid position 38 (Kabat numbering) of VL2 is K; CH2 H1 The amino acid at position 187 (EU numbering) of CL2 is D, and the amino acid at position 138 (EU numbering) of CL2 is K; CH2 H1The amino acid at position 171 (EU numbering) of H2H is C, and the amino acid at position 162 (EU numbering) of CL2 is C; the amino acid at position 220 (EU numbering) of H2H is S, and the amino acid at position 214 (EU numbering) of CL2 is S; the amino acids at positions 234, 235, and 237 (EU numbering) of H2H are A; CH2 H3 The amino acid at position 354 (EU numbering) is C; CH2 H3 The amino acid at position 366 (EU numbering) is W; and when numbered according to the H2 amino acid sequence of SEQ NO: 437 and the L2 amino acid sequence of SEQ NO: 436, CH2 H3 The amino acid at position 447 (EU numbering) is deleted.

[0518] In some embodiments, the bispecific antibody EIP0473 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 13, a VH2 comprising the amino acid sequence of SEQ ID NO: 4, a VL1 comprising the amino acid sequence of SEQ ID NO: 22, and a VL2 comprising the amino acid sequence of SEQ ID NO: 3.

[0519] In some embodiments, the bispecific antibody EIP0473 comprises H1 comprising the amino acid sequence of SEQ ID NO: 138, H2 comprising the amino acid sequence of SEQ ID NO: 136, L1 comprising the amino acid sequence of SEQ ID NO: 137, and L2 comprising the amino acid sequence of SEQ ID NO: 135.

[0520] In addition to the specific complementary determining regions described above, the bispecific antibody EIP0598 comprises the following amino acid substitutions in H1 and L1: amino acid position 39 (Kabat numbering) of VH1 is K, and amino acid position 38 (Kabat numbering) of VL1 is D; H1 The amino acid at position 170 (EU numbering) of CL1 is S, and the amino acid at position 131 (EU numbering) of CL1 is D; H1 The amino acid at position 173 (EU numbering) of H1H is C, and the amino acid at position 162 (EU numbering) of CL1 is C; the amino acid at position 220 (EU numbering) of H1H is S, and the amino acid at position 214 (EU numbering) of CL1 is S; the amino acids at positions 234, 235, and 237 (EU numbering) of H1H are A; H3 The amino acid at position 349 (EU numbering) is C; CH1 H3 The amino acid at position 366 (EU numbering) is S; CH1 H3 The amino acid at position 368 (EU numbering) is A; CH1 H3The amino acid at position 407 (EU numbering) is V; and when numbered according to the H1 amino acid sequence of SEQ NO: 439 and the L1 amino acid sequence of SEQ NO: 438, CH1 H3 The amino acid at position 447 (EU numbering) is deleted.

[0521] In addition to the specific CDRs described above, the bispecific antibody EIP0598 comprises the following amino acid substitutions in H2 and L2: amino acid position 39 (Kabat numbering) of VH2 is D, and amino acid position 38 (Kabat numbering) of VL2 is K; CH2 H1 The amino acid at position 147 (EU numbering) of CL2 is D, and the amino acid at position 180 (EU numbering) of CL2 is R; the amino acids at positions 234, 235, and 237 (EU numbering) of H2H are A; CH2 H3 The amino acid at position 354 (EU numbering) is C; CH2 H3 The amino acid at position 366 (EU numbering) is W; and when numbered according to the H2 amino acid sequence of SEQ NO: 437 and the L2 amino acid sequence of SEQ NO: 436, CH2 H3 The amino acid at position 447 (EU numbering) is deleted.

[0522] In some embodiments, the bispecific antibody EIP0598 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 13, a VH2 comprising the amino acid sequence of SEQ ID NO: 2, a VL1 comprising the amino acid sequence of SEQ ID NO: 22, and a VL2 comprising the amino acid sequence of SEQ ID NO: 1.

[0523] In some embodiments, the bispecific antibody EIP0598 comprises H1 comprising the amino acid sequence of SEQ ID NO: 142, H2 comprising the amino acid sequence of SEQ ID NO: 140, L1 comprising the amino acid sequence of SEQ ID NO: 141, and L2 comprising the amino acid sequence of SEQ ID NO: 139.

[0524] Any of the bispecific antibodies shown in Table 13 above can be further modified by replacing any of the anti-CD3ε antigen-binding regions with any of the anti-CD3ε binding regions shown in Tables 7-9. For example, the anti-CD3ε antigen-binding region of the bispecific antibody "EIP0205" can be replaced with any of the anti-CD3ε binding regions shown in Tables 7-9 to generate the bispecific antibodies of the present invention. Exemplary antibodies are shown in Table 14. The underlined sequences are CDR sequences according to Kabat, and the bold sequences are CDR sequences according to Chothia.

[0525] Table 14. Exemplary bispecific antibodies that bind to CD3ε and ULBP2 / 5 / 6

[0526]

[0527]

[0528]

[0529]

[0530]

[0531]

[0532]

[0533]

[0534]

[0535]

[0536]

[0537]

[0538]

[0539]

[0540]

[0541]

[0542]

[0543]

[0544]

[0545]

[0546]

[0547] In some embodiments, exemplary CD3εxULBP2 / 5 / 6 bispecific antibodies of the invention include EIP0527, EIP0624, EIP0486, EIP0626, EIP0483, EIP0623, EIP0621, EIP0625, EIP0622, and EIP0525.

[0548] Bispecific antibodies EIP0527, EIP0624, EIP0486, EIP0626, EIP0483, EIP0623, EIP0621, EIP0625, EIP0622, and EIP0525 comprise the following amino acid substitutions in H1 and L1: amino acid at position 39 (Kabat numbering) of VH1 is K, amino acid at position 38 (Kabat numbering) of VL1 is D;

[0549] CH1 H1 The 147th amino acid (EU numbering) of CL1 is K, and the 131st amino acid (EU numbering) of CL2 is D; H1 The amino acid at position 173 (EU numbering) of H1H is C, and the amino acid at position 162 (EU numbering) of CL1 is C; the amino acid at position 220 (EU numbering) of H1H is S, and the amino acid at position 214 (EU numbering) of CL1 is S; the amino acids at positions 234, 235, and 237 (EU numbering) of H1H are A; H3 The amino acid at position 349 (EU numbering) is C; CH1 H3 The amino acid at position 366 (EU numbering) is S; CH1 H3 The amino acid at position 368 (EU numbering) is A; CH1 H3 The amino acid at position 407 (EU numbering) is V; and when numbered according to the H1 amino acid sequence NO: 439 of SEQ and the L1 amino acid sequence NO: 438 of SEQ, CH1 H3 The amino acid at position 447 (EU numbering) is deleted.

[0550] The bispecific antibodies EIP0527, EIP0624, EIP0486, EIP0626, EIP0483, EIP0623, EIP0621, EIP0625, EIP0622, and EIP0525 have a second antigen-binding domain that binds to ULBP2 / 5 / 6, comprising a VH2 comprising a VH2 having the amino acid sequence of SEQ ID NO: 5 CDR1 , VH2 having the amino acid sequence of SEQ ID NO: 7 CDR2 and VH2 having the amino acid sequence of SEQ ID NO: 9 CDR3 ; and VL2 comprising a VL2 having an amino acid sequence of SEQ ID NO: 10 CDR1 , VL2 having the amino acid sequence of SEQ ID NO: 11 CDR2 and VL2 having the amino acid sequence of SEQ ID NO: 12CDR3 .

[0551] The bispecific antibodies EIP0527, EIP0624, EIP0486, EIP0626, EIP0483, EIP0623, EIP0621, EIP0625, EIP0622, and EIP0525 comprise a VH2 comprising the amino acid sequence of SEQ ID NO: 2; and a VL2 comprising the amino acid sequence of SEQ ID NO: 1.

[0552] The bispecific antibodies EIP0527, EIP0624, EIP0486, EIP0626, EIP0483, EIP0623, EIP0621, EIP0625, EIP0622, and EIP0525 have H2 comprising the amino acid sequence of SEQ ID NO: 68; and L2 comprising the amino acid sequence of SEQ ID NO: 67.

[0553] In addition to the VH2, VL2, H2, L2 sequences and amino acid substitutions shown above, the bispecific antibody EIP0527 further comprises a first antigen-binding domain that binds to CD3ε, comprising VH1, wherein VH1 comprises a VH1 having an amino acid sequence of SEQ ID NO: 29. CDR1 ; VH1 having the amino acid sequence of SEQ ID NO: 34 CDR2 and VH1 having the amino acid sequence of SEQ ID NO: 37 CDR3 ; and VL1 comprising a VL1 having an amino acid sequence of SEQ ID NO: 42 CDR1 ; VL1 having the amino acid sequence of SEQ ID NO: 43 CDR2 and VL1 having the amino acid sequence of SEQ ID NO: 46 CDR3 .

[0554] In some embodiments, the bispecific antibody EIP0527 comprises a VH1 having the amino acid sequence of SEQ ID NO: 13 and a VL1 having the amino acid sequence of SEQ ID NO: 25. In some embodiments, the bispecific antibody EIP0527 comprises an H1 having the amino acid sequence of SEQ ID NO: 146 and an L1 having the amino acid sequence of SEQ ID NO: 145.

[0555] In addition to the VH2, VL2, H2, L2 sequences and amino acid substitutions shown above, the bispecific antibody EIP0624 further comprises a first antigen-binding domain that binds to CD3ε, comprising VH1, wherein the VH1 comprises a VH1 having an amino acid sequence of SEQ ID NO: 29. CDR1 ; VH1 having the amino acid sequence of SEQ ID NO: 34 CDR2 and VH1 having the amino acid sequence of SEQ ID NO: 38 CDR3 ; and VL1 comprising a VL1 having an amino acid sequence of SEQ ID NO: 42 CDR1 ; VL1 having the amino acid sequence of SEQ ID NO: 43 CDR2 and VL1 having the amino acid sequence of SEQ ID NO: 47 CDR3 .

[0556] In some embodiments, the bispecific antibody EIP0624 comprises a VH1 having the amino acid sequence of SEQ ID NO: 15 and a VL1 having the amino acid sequence of SEQ ID NO: 26. In some embodiments, the bispecific antibody EIP0624 comprises an H1 having the amino acid sequence of SEQ ID NO: 150 and an L1 having the amino acid sequence of SEQ ID NO: 149.

[0557] In addition to the VH2, VL2, H2, L2 sequences and amino acid substitutions shown above, the bispecific antibody EIP0486 further comprises a first antigen-binding domain that binds to CD3ε, the first antigen-binding domain comprising VH1, the VH1 comprising a VH1 having an amino acid sequence of SEQ ID NO: 29 CDR1 ; VH1 having the amino acid sequence of SEQ ID NO: 34 CDR2 and VH1 having the amino acid sequence of SEQ ID NO: 37 CDR3 ; and VL1 comprising a VL1 having an amino acid sequence of SEQ ID NO: 42 CDR1 ; VL1 having the amino acid sequence of SEQ ID NO: 44 CDR2 and VL1 having the amino acid sequence of SEQ ID NO: 45 CDR3 .

[0558] In some embodiments, the bispecific antibody EIP0486 comprises a VH1 having the amino acid sequence of SEQ ID NO: 13 and a VL1 having the amino acid sequence of SEQ ID NO: 27. In some embodiments, the bispecific antibody EIP0486 comprises an H1 having the amino acid sequence of SEQ ID NO: 154 and an L1 having the amino acid sequence of SEQ ID NO: 153.

[0559] In addition to the VH2, VL2, H2, L2 sequences and amino acid substitutions shown above, the bispecific antibody EIP0626 further comprises a first antigen-binding domain that binds to CD3ε, comprising VH1, wherein the VH1 comprises a VH1 having an amino acid sequence of SEQ ID NO: 29. CDR1 ; VH1 having the amino acid sequence of SEQ ID NO: 34 CDR2 and VH1 having the amino acid sequence of SEQ ID NO: 37 CDR3 ; and VL1 comprising a VL1 having an amino acid sequence of SEQ ID NO: 42 CDR1 ; VL1 having the amino acid sequence of SEQ ID NO: 43 CDR2 and VL1 having the amino acid sequence of SEQ ID NO: 47 CDR3 .

[0560] In some embodiments, the bispecific antibody EIP0626 comprises a VH1 having the amino acid sequence of SEQ ID NO: 16 and a VL1 having the amino acid sequence of SEQ ID NO: 26. In some embodiments, the bispecific antibody EIP0626 comprises an H1 having the amino acid sequence of SEQ ID NO: 158 and an L1 having the amino acid sequence of SEQ ID NO: 157.

[0561] In addition to the VH2, VL2, H2, L2 sequences and amino acid substitutions shown above, the bispecific antibody EIP0483 further comprises a first antigen-binding domain that binds to CD3ε, comprising VH1, wherein the VH1 comprises a VH1 having an amino acid sequence of SEQ ID NO: 30. CDR1 ; VH1 having the amino acid sequence of SEQ ID NO: 34 CDR2 and VH1 having the amino acid sequence of SEQ ID NO: 37 CDR3 ; and VL1 comprising a VL1 having an amino acid sequence of SEQ ID NO: 42 CDR1 ; VL1 having the amino acid sequence of SEQ ID NO: 43 CDR2and VL1 having the amino acid sequence of SEQ ID NO: 45 CDR3 .

[0562] In some embodiments, the bispecific antibody EIP0483 comprises a VH1 having the amino acid sequence of SEQ ID NO: 17 and a VL1 having the amino acid sequence of SEQ ID NO: 22. In some embodiments, the bispecific antibody EIP0483 comprises an H1 having the amino acid sequence of SEQ ID NO: 162 and an L1 having the amino acid sequence of SEQ ID NO: 161.

[0563] In addition to the VH2, VL2, H2, L2 sequences and amino acid substitutions shown above, the bispecific antibody EIP0623 further comprises a first antigen-binding domain that binds to CD3ε, comprising VH1, wherein the VH1 comprises a VH1 having an amino acid sequence of SEQ ID NO: 29. CDR1 ; VH1 having the amino acid sequence of SEQ ID NO: 35 CDR2 and VH1 having the amino acid sequence of SEQ ID NO: 38 CDR3 ; and VL1 comprising a VL1 having an amino acid sequence of SEQ ID NO: 42 CDR1 ; VL1 having the amino acid sequence of SEQ ID NO: 43 CDR2 and VL1 having the amino acid sequence of SEQ ID NO: 47 CDR3 .

[0564] In some embodiments, the bispecific antibody EIP0623 comprises a VH1 having the amino acid sequence of SEQ ID NO: 18 and a VL1 having the amino acid sequence of SEQ ID NO: 26. In some embodiments, the bispecific antibody EIP0623 comprises an H1 having the amino acid sequence of SEQ ID NO: 166 and an L1 having the amino acid sequence of SEQ ID NO: 165.

[0565] In addition to the VH2, VL2, H2, L2 sequences and amino acid substitutions shown above, the bispecific antibody EIP0621 further comprises a first antigen-binding domain that binds to CD3ε, the first antigen-binding domain comprising VH1, the VH1 comprising a VH1 having an amino acid sequence of SEQ ID NO: 29 CDR1 ; VH1 having the amino acid sequence of SEQ ID NO: 34 CDR2 and VH1 having the amino acid sequence of SEQ ID NO: 40 CDR3; and VL1 comprising a VL1 having an amino acid sequence of SEQ ID NO: 42 CDR1 ; VL1 having the amino acid sequence of SEQ ID NO: 43 CDR2 and VL1 having the amino acid sequence of SEQ ID NO: 47 CDR3 .

[0566] In some embodiments, the bispecific antibody EIP0621 comprises a VH1 having the amino acid sequence of SEQ ID NO: 19 and a VL1 having the amino acid sequence of SEQ ID NO: 26. In some embodiments, the bispecific antibody EIP0621 comprises an H1 having the amino acid sequence of SEQ ID NO: 170 and an L1 having the amino acid sequence of SEQ ID NO: 169.

[0567] In addition to the VH2, VL2, H2, L2 sequences and amino acid substitutions shown above, the bispecific antibody EIP0625 further comprises a first antigen-binding domain that binds to CD3ε, comprising VH1, wherein the VH1 comprises a VH1 having an amino acid sequence of SEQ ID NO: 29. CDR1 ; VH1 having the amino acid sequence of SEQ ID NO: 35 CDR2 and VH1 having the amino acid sequence of SEQ ID NO: 38 CDR3 ; and VL1 comprising a VL1 having an amino acid sequence of SEQ ID NO: 42 CDR1 ; VL1 having the amino acid sequence of SEQ ID NO: 43 CDR2 and VL1 having the amino acid sequence of SEQ ID NO: 45 CDR3 .

[0568] In some embodiments, the bispecific antibody EIP0625 comprises a VH1 having the amino acid sequence of SEQ ID NO: 18 and a VL1 having the amino acid sequence of SEQ ID NO: 22. In some embodiments, the bispecific antibody EIP0625 comprises an H1 having the amino acid sequence of SEQ ID NO: 174 and an L1 having the amino acid sequence of SEQ ID NO: 173.

[0569] In addition to the VH2, VL2, H2, L2 sequences and amino acid substitutions shown above, the bispecific antibody EIP0622 further comprises a first antigen-binding domain that binds to CD3ε, comprising VH1, wherein the VH1 comprises a VH1 having an amino acid sequence of SEQ ID NO: 29. CDR1; VH1 having the amino acid sequence of SEQ ID NO: 34 CDR2 and VH1 having the amino acid sequence of SEQ ID NO: 41 CDR3 ; and VL1 comprising a VL1 having an amino acid sequence of SEQ ID NO: 42 CDR1 ; VL1 having the amino acid sequence of SEQ ID NO: 43 CDR2 and VL1 having the amino acid sequence of SEQ ID NO: 47 CDR3 .

[0570] In some embodiments, the bispecific antibody EIP0622 comprises a VH1 having the amino acid sequence of SEQ ID NO: 20 and a VL1 having the amino acid sequence of SEQ ID NO: 26. In some embodiments, the bispecific antibody EIP0622 comprises an H1 having the amino acid sequence of SEQ ID NO: 178 and an L1 having the amino acid sequence of SEQ ID NO: 177.

[0571] In addition to the VH2, VL2, H2, L2 sequences and amino acid substitutions shown above, the bispecific antibody EIP0525 further comprises a first antigen-binding domain that binds to CD3ε, the first antigen-binding domain comprising VH1, the VH1 comprising a VH1 having an amino acid sequence of SEQ ID NO: 31 CDR1 ; VH1 having the amino acid sequence of SEQ ID NO: 34 CDR2 and VH1 having the amino acid sequence of SEQ ID NO: 37 CDR3 ; and VL1 comprising a VL1 having an amino acid sequence of SEQ ID NO: 42 CDR1 ; VL1 having the amino acid sequence of SEQ ID NO: 43 CDR2 and VL1 having the amino acid sequence of SEQ ID NO: 45 CDR3 .

[0572] In some embodiments, the bispecific antibody EIP0525 comprises a VH1 having the amino acid sequence of SEQ ID NO: 21 and a VL1 having the amino acid sequence of SEQ ID NO: 22. In some embodiments, the bispecific antibody EIP0525 comprises an H1 having the amino acid sequence of SEQ ID NO: 182 and an L1 having the amino acid sequence of SEQ ID NO: 181.

[0573] In some embodiments, exemplary CD3εxULBP2 / 5 / 6 bispecific antibodies of the invention include EIP0630, EIP0540, EIP0628, EIP0542, EIP0627, EIP0515, EIP0477, EIP0541, EIP0513, and EIP0629.

[0574] The bispecific antibodies EIP0630, EIP0540, EIP0628, EIP0542, EIP0627, EIP0515, EIP0477, EIP0541, EIP0513, and EIP0629 have the following amino acid substitutions in H1 and L1: amino acid at position 39 (Kabat numbering) of VH1 is K, and amino acid at position 38 (Kabat numbering) of VL1 is D; H1 The amino acid at position 170 (EU numbering) of CL1 is S, and the amino acid at position 131 (EU numbering) of CL1 is D; H1 The amino acid at position 173 (EU numbering) of H1H is C, and the amino acid at position 162 (EU numbering) of CL1 is C; the amino acid at position 220 (EU numbering) of H1H is S, and the amino acid at position 214 (EU numbering) of CL1 is S; the amino acids at positions 234, 235, and 237 (EU numbering) of H1H are A; H3 The amino acid at position 349 (EU numbering) is C; CH1 H3 The amino acid at position 366 (EU numbering) is S; CH1 H3 The amino acid at position 368 (EU numbering) is A; CH1 H3 The amino acid at position 407 (EU numbering) is V; and when numbered according to the H1 amino acid sequence of SEQ NO: 439 and the L1 amino acid sequence of SEQ NO: 438, CH1 H3 The amino acid at position 447 (EU numbering) is deleted.

[0575] The bispecific antibodies EIP0630, EIP0540, EIP0628, EIP0542, EIP0627, EIP0515, EIP0477, EIP0541, EIP0513, and EIP0629 have a second antigen-binding domain that binds to ULBP2 / 5 / 6, comprising a VH2 having a VH2 having the amino acid sequence of SEQ ID NO: 5 CDR1 ; VH2 comprising the amino acid sequence of SEQ ID NO: 7 CDR2 and VH2 having the amino acid sequence of SEQ ID NO: 9 CDR3; and VL2 having a VL2 having an amino acid sequence of SEQ ID NO: 10 CDR1 ; VL2 having the amino acid sequence of SEQ ID NO: 11 CDR2 and VL2 having the amino acid sequence of SEQ ID NO: 12 CDR3 .

[0576] The bispecific antibodies EIP0630, EIP0540, EIP0628, EIP0542, EIP0627, EIP0515, EIP0477, EIP0541, EIP0513, and EIP0629 have: a VH2 having the amino acid sequence of SEQ ID NO: 2; and a VL2 having the amino acid sequence of SEQ ID NO: 1.

[0577] The bispecific antibodies EIP0630, EIP0540, EIP0628, EIP0542, EIP0627, EIP0515, EIP0477, EIP0541, EIP0513, and EIP0629 have: H2 having the amino acid sequence of SEQ ID NO: 140; and L2 having the amino acid sequence of SEQ ID NO: 139.

[0578] In addition to the VH2, VL2, H2, L2 sequences and amino acid substitutions shown above, the bispecific antibody EIP0630 further comprises a first antigen-binding domain that binds to CD3ε, comprising VH1, wherein the VH1 comprises a VH1 having an amino acid sequence of SEQ ID NO: 29. CDR1 ; VH1 having the amino acid sequence of SEQ ID NO: 34 CDR2 and VH1 having the amino acid sequence of SEQ ID NO: 37 CDR3 ; and VL1 comprising a VL1 having an amino acid sequence of SEQ ID NO: 42 CDR1 ; VL1 having the amino acid sequence of SEQ ID NO: 43 CDR2 and VL1 having the amino acid sequence of SEQ ID NO: 46 CDR3 .

[0579] In some embodiments, the bispecific antibody EIP0630 comprises a VH1 having the amino acid sequence of SEQ ID NO: 13 and a VL1 having the amino acid sequence of SEQ ID NO: 25. In some embodiments, the bispecific antibody EIP0630 comprises an H1 having the amino acid sequence of SEQ ID NO: 186 and an L1 having the amino acid sequence of SEQ ID NO: 185.

[0580] In addition to the VH2, VL2, H2, L2 sequences and amino acid substitutions shown above, the bispecific antibody EIP0540 further comprises a first antigen-binding domain that binds to CD3ε, comprising VH1, wherein the VH1 comprises a VH1 having an amino acid sequence of SEQ ID NO: 29. CDR1 ; VH1 having the amino acid sequence of SEQ ID NO: 34 CDR2 and VH1 having the amino acid sequence of SEQ ID NO: 38 CDR3 ; and VL1 comprising a VL1 having an amino acid sequence of SEQ ID NO: 42 CDR1 ; VL1 having the amino acid sequence of SEQ ID NO: 43 CDR2 and VL1 having the amino acid sequence of SEQ ID NO: 47 CDR3 .

[0581] In some embodiments, the bispecific antibody EIP0540 comprises a VH1 having the amino acid sequence of SEQ ID NO: 15 and a VL1 having the amino acid sequence of SEQ ID NO: 26. In some embodiments, the bispecific antibody EIP0540 comprises an H1 having the amino acid sequence of SEQ ID NO: 190 and an L1 having the amino acid sequence of SEQ ID NO: 189.

[0582] In addition to the VH2, VL2, H2, L2 sequences and amino acid substitutions shown above, the bispecific antibody EIP0628 further comprises a first antigen-binding domain that binds to CD3ε, comprising VH1, wherein the VH1 comprises a VH1 having an amino acid sequence of SEQ ID NO: 29. CDR1 ; VH1 having the amino acid sequence of SEQ ID NO: 34 CDR2 and VH1 having the amino acid sequence of SEQ ID NO: 37 CDR3 ; and VL1 comprising a VL1 having an amino acid sequence of SEQ ID NO: 42 CDR1 ; VL1 having the amino acid sequence of SEQ ID NO: 44 CDR2 and VL1 having the amino acid sequence of SEQ ID NO: 45 CDR3 .

[0583] In some embodiments, the bispecific antibody EIP0628 comprises a VH1 having the amino acid sequence of SEQ ID NO: 13 and a VL1 having the amino acid sequence of SEQ ID NO: 27. In some embodiments, the bispecific antibody EIP0628 comprises an H1 having the amino acid sequence of SEQ ID NO: 194 and an L1 having the amino acid sequence of SEQ ID NO: 193.

[0584] In addition to the VH2, VL2, H2, L2 sequences and amino acid substitutions shown above, the bispecific antibody EIP0542 further comprises a first antigen-binding domain that binds to CD3ε, comprising VH1, wherein the VH1 comprises a VH1 having an amino acid sequence of SEQ ID NO: 29. CDR1 ; VH1 having the amino acid sequence of SEQ ID NO: 34 CDR2 and VH1 having the amino acid sequence of SEQ ID NO: 39 CDR3 ; and VL1 comprising a VL1 having an amino acid sequence of SEQ ID NO: 42 CDR1 ; VL1 having the amino acid sequence of SEQ ID NO: 43 CDR2 and VL1 having the amino acid sequence of SEQ ID NO: 47 CDR3 .

[0585] In some embodiments, the bispecific antibody EIP0542 comprises a VH1 having the amino acid sequence of SEQ ID NO: 16 and a VL1 having the amino acid sequence of SEQ ID NO: 26. In some embodiments, the bispecific antibody EIP0542 comprises an H1 having the amino acid sequence of SEQ ID NO: 198 and an L1 having the amino acid sequence of SEQ ID NO: 197.

[0586] In addition to the VH2, VL2, H2, L2 sequences and amino acid substitutions shown above, the bispecific antibody EIP0627 further comprises a first antigen-binding domain that binds to CD3ε, the first antigen-binding domain comprising VH1, the VH1 comprising a VH1 having an amino acid sequence of SEQ ID NO: 30 CDR1 ; VH1 having the amino acid sequence of SEQ ID NO: 34 CDR2 and VH1 having the amino acid sequence of SEQ ID NO: 37 CDR3 ; and VL1 comprising a VL1 having an amino acid sequence of SEQ ID NO: 42 CDR1 ; VL1 having the amino acid sequence of SEQ ID NO: 43 CDR2and VL1 having the amino acid sequence of SEQ ID NO: 45 CDR3 .

[0587] In some embodiments, the bispecific antibody EIP0627 comprises a VH1 having the amino acid sequence of SEQ ID NO: 17 and a VL1 having the amino acid sequence of SEQ ID NO: 22. In some embodiments, the bispecific antibody EIP0627 comprises an H1 having the amino acid sequence of SEQ ID NO: 202 and an L1 having the amino acid sequence of SEQ ID NO: 201.

[0588] In addition to the VH2, VL2, H2, L2 sequences and amino acid substitutions shown above, the bispecific antibody EIP0515 further comprises a first antigen-binding domain that binds to CD3ε, comprising VH1, wherein the VH1 comprises a VH1 having an amino acid sequence of SEQ ID NO: 29. CDR1 ; VH1 having the amino acid sequence of SEQ ID NO: 35 CDR2 and VH1 having the amino acid sequence of SEQ ID NO: 38 CDR3 ; and VL1 comprising a VL1 having an amino acid sequence of SEQ ID NO: 42 CDR1 ; VL1 having the amino acid sequence of SEQ ID NO: 43 CDR2 and VL1 having the amino acid sequence of SEQ ID NO: 47 CDR3 .

[0589] In some embodiments, the bispecific antibody EIP0515 comprises a VH1 having the amino acid sequence of SEQ ID NO: 18 and a VL1 having the amino acid sequence of SEQ ID NO: 26. In some embodiments, the bispecific antibody EIP0515 comprises an H1 having the amino acid sequence of SEQ ID NO: 206 and an L1 having the amino acid sequence of SEQ ID NO: 205.

[0590] In addition to the VH2, VL2, H2, L2 sequences and amino acid substitutions shown above, the bispecific antibody EIP0477 further comprises a first antigen-binding domain that binds to CD3ε, comprising VH1, wherein VH1 comprises a VH1 having an amino acid sequence of SEQ ID NO: 29. CDR1 ; VH1 having the amino acid sequence of SEQ ID NO: 34 CDR2 and VH1 having the amino acid sequence of SEQ ID NO: 40 CDR3; and VL1 comprising a VL1 having an amino acid sequence of SEQ ID NO: 42 CDR1 ; VL1 having the amino acid sequence of SEQ ID NO: 43 CDR2 and VL1 having the amino acid sequence of SEQ ID NO: 47 CDR3 .

[0591] In some embodiments, the bispecific antibody EIP0477 comprises a VH1 having the amino acid sequence of SEQ ID NO: 19 and a VL1 having the amino acid sequence of SEQ ID NO: 26. In some embodiments, the bispecific antibody EIP0477 comprises an H1 having the amino acid sequence of SEQ ID NO: 210 and an L1 having the amino acid sequence of SEQ ID NO: 209.

[0592] In addition to the VH2, VL2, H2, L2 sequences and amino acid substitutions shown above, the bispecific antibody EIP0541 further comprises a first antigen-binding domain that binds to CD3ε, comprising VH1, wherein the VH1 comprises a VH1 having an amino acid sequence of SEQ ID NO: 29. CDR1 ; VH1 having the amino acid sequence of SEQ ID NO: 35 CDR2 and VH1 having the amino acid sequence of SEQ ID NO: 38 CDR3 ; and VL1 comprising a VL1 having an amino acid sequence of SEQ ID NO: 42 CDR1 ; VL1 having the amino acid sequence of SEQ ID NO: 43 CDR2 and VL1 having the amino acid sequence of SEQ ID NO: 45 CDR3 .

[0593] In some embodiments, the bispecific antibody EIP0541 comprises a VH1 having the amino acid sequence of SEQ ID NO: 18 and a VL1 having the amino acid sequence of SEQ ID NO: 22. In some embodiments, the bispecific antibody EIP0541 comprises an H1 having the amino acid sequence of SEQ ID NO: 214 and an L1 having the amino acid sequence of SEQ ID NO: 213.

[0594] In addition to the VH2, VL2, H2, L2 sequences and amino acid substitutions shown above, the bispecific antibody EIP0513 further comprises a first antigen-binding domain that binds to CD3ε, comprising VH1, wherein the VH1 comprises a VH1 having an amino acid sequence of SEQ ID NO: 29. CDR1; VH1 having the amino acid sequence of SEQ ID NO: 34 CDR2 and VH1 having the amino acid sequence of SEQ ID NO: 41 CDR3 ; and VL1 comprising a VL1 having an amino acid sequence of SEQ ID NO: 42 CDR1 ; VL1 having the amino acid sequence of SEQ ID NO: 43 CDR2 and VL1 having the amino acid sequence of SEQ ID NO: 47 CDR3 .

[0595] In some embodiments, the bispecific antibody EIP0513 comprises a VH1 having the amino acid sequence of SEQ ID NO: 20 and a VL1 having the amino acid sequence of SEQ ID NO: 26. In some embodiments, the bispecific antibody EIP0513 comprises an H1 having the amino acid sequence of SEQ ID NO: 218 and an L1 having the amino acid sequence of SEQ ID NO: 217.

[0596] In addition to the VH2, VL2, H2, L2 sequences and amino acid substitutions shown above, the bispecific antibody EIP0629 further comprises a first antigen-binding domain that binds to CD3ε, comprising VH1, wherein the VH1 comprises a VH1 having an amino acid sequence of SEQ ID NO: 31. CDR1 ; VH1 having the amino acid sequence of SEQ ID NO: 34 CDR2 and VH1 having the amino acid sequence of SEQ ID NO: 37 CDR3 ; and VL1 comprising a VL1 having an amino acid sequence of SEQ ID NO: 42 CDR1 ; VL1 having the amino acid sequence of SEQ ID NO: 43 CDR2 and VL1 having the amino acid sequence of SEQ ID NO: 45 CDR3 .

[0597] In some embodiments, the bispecific antibody EIP0629 comprises a VH1 having the amino acid sequence of SEQ ID NO: 21 and a VL1 having the amino acid sequence of SEQ ID NO: 22. In some embodiments, the bispecific antibody EIP0629 comprises an H1 having the amino acid sequence of SEQ ID NO: 222 and an L1 having the amino acid sequence of SEQ ID NO: 221.

[0598] In some embodiments, exemplary CD3εxULBP2 / 5 / 6 bispecific antibodies of the invention include EIP0820.

[0599] In some embodiments, the bispecific antibody EIP0820 comprises the following amino acid substitutions in H1 and L1: the amino acid at position 39 (Kabat numbering) of VH1 is K, and the amino acid at position 38 (Kabat numbering) of VL1 is D;

[0600] CH1 H1 The 147th amino acid (EU numbering) of CL1 is K, and the 131st amino acid (EU numbering) of CL2 is D; H1 The amino acid at position 173 (EU numbering) of H1H is C, and the amino acid at position 162 (EU numbering) of CL1 is C; the amino acid at position 220 (EU numbering) of H1H is S, and the amino acid at position 214 (EU numbering) of CL1 is S; the amino acids at positions 234, 235, and 237 (EU numbering) of H1H are A; H3 The amino acid at position 349 (EU numbering) is C; CH1 H3 The amino acid at position 366 (EU numbering) is S; CH1 H3 The amino acid at position 368 (EU numbering) is A; CH1 H3 The amino acid at position 407 (EU numbering) is V; and when numbered according to the H1 amino acid sequence NO: 439 of SEQ and the L1 amino acid sequence NO: 438 of SEQ, CH1 H3 The amino acid at position 447 (EU numbering) is deleted.

[0601] In some embodiments, the bispecific antibody EIP0820 has a second antigen binding domain that binds to ULBP2 / 5 / 6, wherein the second antigen binding domain comprises VH2, wherein the VH2 comprises a VH2 having an amino acid sequence of SEQ ID NO: 5. CDR1 ; VH2 having the amino acid sequence of SEQ ID NO: 7 CDR2 and VH2 having the amino acid sequence of SEQ ID NO: 9 CDR3 ; and VL2 comprising a VL2 having an amino acid sequence of SEQ ID NO: 10 CDR1 ; VL2 having the amino acid sequence of SEQ ID NO: 11 CDR2 and VL2 having the amino acid sequence of SEQ ID NO: 12 CDR3 .

[0602] In some embodiments, the bispecific antibody EIP0820 comprises a VH2 comprising the amino acid sequence of SEQ ID NO: 2; and a VL2 comprising the amino acid sequence of SEQ ID NO: 1.

[0603] In some embodiments, the bispecific antibody EIP0820 comprises a VH2 comprising the amino acid sequence of SEQ ID NO: 629; and a VL2 comprising the amino acid sequence of SEQ ID NO: 1.

[0604] In some embodiments, the bispecific antibody EIP0820 comprises H2 comprising the amino acid sequence of SEQ ID NO: 621; and L2 comprising the amino acid sequence of SEQ ID NO: 67.

[0605] In addition to the VH2, VL2, H2, L2 sequences and amino acid substitutions shown above, the bispecific antibody EIP0820 comprises a first antigen-binding domain that binds to CD3ε, comprising VH1, comprising a VH1 having an amino acid sequence of SEQ ID NO: 30. CDR1 ; VH1 having the amino acid sequence of SEQ ID NO: 34 CDR2 and VH1 having the amino acid sequence of SEQ ID NO: 37 CDR3 ; and VL1 comprising a VL1 having an amino acid sequence of SEQ ID NO: 42 CDR1 ; VL1 having the amino acid sequence of SEQ ID NO: 43 CDR2 and VL1 having the amino acid sequence of SEQ ID NO: 45 CDR3 .

[0606] In some embodiments, the bispecific antibody EIP0820 comprises a VH1 having the amino acid sequence of SEQ ID NO: 17 and a VL1 having the amino acid sequence of SEQ ID NO: 22. In some embodiments, the bispecific antibody EIP0820 comprises an H1 having the amino acid sequence of SEQ ID NO: 622 and an L1 having the amino acid sequence of SEQ ID NO: 69.

[0607] In some embodiments, exemplary CD3εxULBP2 / 5 / 6 bispecific antibodies of the present disclosure comprise CH1 H3 and / or CH2 H3 In some embodiments, wherein the exemplary bispecific antibody comprises CH1 H3 and / or CH2 H3 446 (EU numbering) of amino acid sequence SEQ ID NO: ... H3 and / or CH2 H3The amino acid at position 446 (EU numbering) of the bispecific antibody further comprises CH1 H3 and / or amino acid 447 (EU numbering) of CH2H3. In some embodiments, wherein the exemplary bispecific antibody comprises CH1 H3 and / or CH2 H3 The amino acid at position 447 (EU numbering) is K.

[0608] In some embodiments, exemplary CD3εxULBP2 / 5 / 6 bispecific antibodies of the present disclosure comprise CH1 H3 and / or CH2 H3 In some embodiments, the bispecific antibody comprises CH1 H3 and CH2 H3 In some embodiments, the bispecific antibody comprises CH1 H3 In some embodiments, the bispecific antibody comprises CH2 H3 The amino acid at position 446 (EU numbering) of .

[0609] In some embodiments, wherein the bispecific antibody comprises CH1 H3 and / or CH2 H3 446 (EU numbering) of the amino acid sequence of the bispecific antibody, wherein the amino acid sequence of the bispecific antibody is G. In some embodiments, wherein the bispecific antibody comprises CH1 H3 In some embodiments, the bispecific antibody comprises a CH2 H3 446 (EU numbering) of the amino acid sequence of the bispecific antibody, wherein the amino acid sequence of the bispecific antibody is G. In some embodiments, wherein the bispecific antibody comprises CH1 H3 and CH2 H3 The 446th amino acid (EU numbering) of H3 and CH2 H3 The amino acid at position 446 (EU numbering) is G.

[0610] In some embodiments, exemplary CD3εxULBP2 / 5 / 6 bispecific antibodies of the present disclosure comprise CH1 H3 and / or CH2 H3 In some embodiments, the bispecific antibody comprises CH1 H3 or CH2 H3In some embodiments, the bispecific antibody comprises CH1 H3 and CH2 H3 In some embodiments, the bispecific antibody comprises CH1 H3 In some embodiments, the bispecific antibody comprises CH2 H3 The amino acid at position 447 (EU numbering) of .

[0611] In some embodiments, wherein the CD3εxULBP2 / 5 / 6 bispecific antibody comprises CH1 H3 and / or CH2 H3 The amino acid at position 447 (EU numbering) of is K. In some embodiments, wherein the bispecific antibody comprises CH1 H3 The amino acid at position 447 (EU numbering) of the bispecific antibody is K. In some embodiments, wherein the bispecific antibody comprises CH2 H3 The amino acid at position 447 (EU numbering) is K.

[0612] Fusion peptide

[0613] Provided herein are antibodies (eg, monospecific antibodies or bispecific antibodies) having a fusion peptide fused to the N-terminus or C-terminus of a first heavy chain polypeptide or a second heavy chain polypeptide.

[0614] Crucial to the initial T cell response is the ability of T cells to detect foreign and mutant proteins through their T cell receptors. This response, often referred to as T cell activation signal 1, occurs when the T cell receptor engages a cell displaying a foreign or mutant protein fragment or antigen in a specific protein complex called major histocompatibility complex I (MHCI). Activation of the T cell receptor itself has both activating and self-regulatory effects on T cells. Strong binding of the TCR to the MHCI complex leads to chronic activation of the TCR. This form of signaling is associated with T cells that respond to self-antigens. When T cells undergo this form of activation, they are programmed to be inactivated. T cells with a weaker but sufficient TCR for activation experience acute signaling and are likely to remain active and differentiate into memory T cells. This is becoming an important consideration in the design of T cell therapies.

[0615] T cell cytokine activation, commonly referred to as signal 3, is important in the transition of T cells from a non-dividing to a rapidly dividing state or from one phenotypic state to another. T cell cytokine receptors bind to cytokines produced by both immune and non-immune cells and, depending on the cytokine and the state of the T cell at the time of cytokine signaling, can induce cell proliferation, maintain viability, or induce T cells to differentiate into specialized cell states suitable for sustained activation or inactivation following infection.

[0616] One example is the transformation that naive cells undergo through cytokines, which can induce naive T cell proliferation and promote T cell differentiation into memory T cells. Exemplary cytokines include but are not limited to IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, and IL-21.

[0617] Costimulatory receptor activation, known as signal 2, provides a specific context for cell-to-cell reinforcement of T cell activation. The most recognized form of costimulation occurs when T cells interact with activated antigen-presenting cells through the T cell costimulatory receptor CD28 found on APCs and its ligands CD80 and CD86. These interactions can "prime" specific T cells with T cell receptors that respond to pathogens or cancer proteins.

[0618] Less understood is the costimulation induced at sites of infection and malignancy. This includes costimulation acting through CD2 and NKG2D receptors, which respond to ligands such as CD58 and UL16-binding proteins (e.g., ULBP2 / 5 / 6) induced in immune and epithelial cells following viral infection. These signals not only potentiate T cell activation but also confirm that lethal effector activities of T cells can be targeted with single-cell precision. Although many costimulatory receptors have been discovered, the importance of the specific context of each receptor and the impact of simultaneous signaling by multiple costimulatory receptors remain largely unknown, an area that could greatly advance our understanding of T cell biology and create possibilities for the development of novel tumor-targeted T cell therapies.

[0619] Co-stimulatory ligands include, but are not limited to, CD48, CD58, CD86, TNFSF9, OX40L, 4-1BBL, GITL, CD70, CD80, MR1, TNFSF4, ICOSL, or ICOSLG.

[0620] CD58 is preferred over other costimulatory ligands because it is the major costimulatory pathway available at the tumor site, as tumor-infiltrating T lymphocytes often lose expression of other costimulatory receptors (such as CD28) or are unable to adequately activate T cells due to low tumor cell immunogenicity, thus limiting the potential for inducible costimulatory receptors (such as 41BB).

[0621] As previously described, the anti-CD3ε antibodies of the present disclosure induce different levels of T cell receptor activation, which leads to changes in T cell viability and cytokine production. Therefore, the fusion of the costimulatory ligand CD58 with the anti-CD3ε bispecific antibody provides integrated costimulatory T cell activation to achieve optimal T cell activation.

[0622] In some embodiments, the bispecific antibody has a peptide fused to the N-terminus of the first heavy chain polypeptide (H1). In some embodiments, the bispecific antibody has a peptide fused to the C-terminus of the first heavy chain polypeptide (H1). In some embodiments, the bispecific antibody has a polypeptide fused to the N-terminus of the second heavy chain polypeptide (H2). In some embodiments, the bispecific antibody has a polypeptide fused to the C-terminus of the second heavy chain polypeptide (H2). Exemplary peptides include but are not limited to IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21 or a portion thereof. Exemplary peptides include but are not limited to CD48, CD58, CD86, TNFSF9, OX40L, 4-1BBL, GITL, CD70, CD80, MR1, TNFSF4, ICOSL, ICOSLG or a portion thereof. Exemplary peptide sequences fused to bispecific antibodies include but are not limited to the peptide sequences listed in Table 15.1.

[0623] Table 15.1. Exemplary Fusion Peptide Sequences

[0624]

[0625] In some embodiments, the CD3 x ULBP2 / 5 / 6 bispecific antibodies of the invention have a CD58 fusion peptide comprising any one of the CD58 sequences in Table 15.2. In some embodiments, the CD3 x ULBP2 / 5 / 6 bispecific antibodies of the invention have a CD58 fusion peptide comprising any one of SEQ ID NOs: 624-628.

[0626]

[0627] In some embodiments, the polypeptide is fused directly to the bispecific antibody. In some embodiments, the polypeptide is fused indirectly via a linker. In some embodiments, the bispecific antibody fused to the peptide comprises a linker sequence. Exemplary linker sequences include, but are not limited to, those listed in Table 16.1.

[0628] Table 16.1 Exemplary Linker Sequences

[0629] domain Amino acid sequence SEQ ID NO: Connector 1 GGGGS SEQ ID NO:52 Connector 2 GGGGSGGGGS SEQ ID NO:53 Connector 3 GGGGSGGGGSGGGGS SEQ ID NO:54

[0630] In some embodiments, the CD3 x ULBP2 / 5 / 6 bispecific antibodies of the present invention have a CD58 fusion peptide (SEQ ID NO: 49) indirectly fused to the C-terminus of the first heavy chain polypeptide (H1) using Linker-1 (SEQ ID NO: 52). In some embodiments, the CD3 x ULBP2 / 5 / 6 bispecific antibodies of the present invention have a CD58v* fusion peptide (SEQ ID NO: 50) indirectly fused to the C-terminus of the first heavy chain polypeptide (H1) using Linker-1 (SEQ ID NO: 52). In some embodiments, the CD3 x ULBP2 / 5 / 6 bispecific antibodies of the present invention have an IL-7 fusion peptide (SEQ ID NO: 51) indirectly fused to the C-terminus of the first heavy chain polypeptide (H1) using Linker-1 (SEQ ID NO: 52).

[0631] In some embodiments, the CD3 x ULBP2 / 5 / 6 bispecific antibodies of the present invention have a CD58 fusion peptide (SEQ ID NO: 49) indirectly fused to the C-terminus of the first heavy chain polypeptide (H1) using Linker-2 (SEQ ID NO: 53). In some embodiments, the CD3 x ULBP2 / 5 / 6 bispecific antibodies of the present invention have a CD58v* fusion peptide (SEQ ID NO: 50) indirectly fused to the C-terminus of the first heavy chain polypeptide (H1) using Linker-2 (SEQ ID NO: 53). In some embodiments, the CD3 x ULBP2 / 5 / 6 bispecific antibodies of the present invention have an IL-7 fusion peptide (SEQ ID NO: 51) indirectly fused to the C-terminus of the first heavy chain polypeptide (H1) using Linker-2 (SEQ ID NO: 53).

[0632] In some embodiments, the CD3 x ULBP2 / 5 / 6 bispecific antibodies of the present invention have a CD58 fusion peptide (SEQ ID NO: 49) indirectly fused to the C-terminus of the first heavy chain polypeptide (H1) using Linker-3 (SEQ ID NO: 54). In some embodiments, the CD3 x ULBP2 / 5 / 6 bispecific antibodies of the present invention have a CD58v* fusion peptide (SEQ ID NO: 50) indirectly fused to the C-terminus of the first heavy chain polypeptide (H1) using Linker-3 (SEQ ID NO: 54). In some embodiments, the CD3 x ULBP2 / 5 / 6 bispecific antibodies of the present invention have an IL-7 fusion peptide (SEQ ID NO: 51) indirectly fused to the C-terminus of the first heavy chain polypeptide (H1) using Linker-3 (SEQ ID NO: 54).

[0633] In some embodiments, the CD3 x ULBP2 / 5 / 6 bispecific antibodies of the invention have a hinge sequence comprising any one of the linker sequences in Table 16.2.

[0634] In some embodiments, the CD3 x ULBP2 / 5 / 6 bispecific antibody of the invention has a linker sequence comprising any one of SEQ ID NOs: 530-552.

[0635]

[0636]

[0637] In some embodiments, the CD3 x ULBP2 / 5 / 6 bispecific antibodies of the invention have a hinge sequence comprising any one of the linker sequences in Table 16.3.

[0638] In some embodiments, the CD3 x ULBP2 / 5 / 6 bispecific antibodies of the invention have a linker sequence comprising any one of SEQ ID NOs: 553-606.

[0639]

[0640]

[0641] Exemplary CD3 x ULBP2 / 5 / 6 bispecific antibodies with CD58, CD58v*, and IL-7 fusions are shown in Tables 17 and 18.

[0642] Table 17. Exemplary bispecific fusion molecules

[0643]

[0644] Table 18. Exemplary bispecific antibodies that bind to CD3ε and ULBP2 / 5 / 6 with C-terminal fusion peptides

[0645]

[0646]

[0647]

[0648]

[0649]

[0650]

[0651]

[0652]

[0653]

[0654]

[0655]

[0656]

[0657]

[0658]

[0659]

[0660]

[0661]

[0662]

[0663]

[0664]

[0665]

[0666]

[0667]

[0668]

[0669]

[0670]

[0671]

[0672]

[0673]

[0674]

[0675]

[0676]

[0677]

[0678]

[0679]

[0680] Table 19. Exemplary bispecific antibodies that bind to CD3ε and a second antigen

[0681] (italics - variable regions, italics and underlined - Kabat CDR definitions, italics and bold - Chothia CDR definitions, underlined - CD58 fusions, underlined - cysteine ​​mutations)

[0682]

[0683]

[0684]

[0685]

[0686]

[0687]

[0688]

[0689]

[0690]

[0691]

[0692]

[0693]

[0694]

[0695]

[0696]

[0697]

[0698]

[0699]

[0700]

[0701]

[0702]

[0703]

[0704]

[0705]

[0706]

[0707] In some embodiments, the bispecific antibody EIP0373 comprises L2 comprising the amino acid sequence of SEQ ID NO: 450, H2 comprising the amino acid sequence of SEQ ID NO: 451, L1 comprising the amino acid sequence of SEQ ID NO: 452, and H1 comprising the amino acid sequence of SEQ ID NO: 453.

[0708] In some embodiments, the bispecific antibody EIP0535 comprises L2 comprising the amino acid sequence of SEQ ID NO: 454, H2 comprising the amino acid sequence of SEQ ID NO: 455, L1 comprising the amino acid sequence of SEQ ID NO: 456, and H1 comprising the amino acid sequence of SEQ ID NO: 457.

[0709] In some embodiments, the bispecific antibody EIP0506 comprises L2 comprising the amino acid sequence of SEQ ID NO: 458, H2 comprising the amino acid sequence of SEQ ID NO: 459, L1 comprising the amino acid sequence of SEQ ID NO: 460, and H1 comprising the amino acid sequence of SEQ ID NO: 461.

[0710] In some embodiments, the bispecific antibody EIP0534 comprises L2 comprising the amino acid sequence of SEQ ID NO: 462, H2 comprising the amino acid sequence of SEQ ID NO: 463, L1 comprising the amino acid sequence of SEQ ID NO: 464, and H1 comprising the amino acid sequence of SEQ ID NO: 465.

[0711] In some embodiments, the bispecific antibody EIP0702 comprises L2 comprising the amino acid sequence of SEQ ID NO: 466, H2 comprising the amino acid sequence of SEQ ID NO: 467, L1 comprising the amino acid sequence of SEQ ID NO: 468, and H1 comprising the amino acid sequence of SEQ ID NO: 469.

[0712] In some embodiments, the bispecific antibody EIP0703 comprises L2 comprising the amino acid sequence of SEQ ID NO: 470, H2 comprising the amino acid sequence of SEQ ID NO: 471, L1 comprising the amino acid sequence of SEQ ID NO: 472, and H1 comprising the amino acid sequence of SEQ ID NO: 473.

[0713] In some embodiments, the bispecific antibody EIP0765 comprises L2 comprising the amino acid sequence of SEQ ID NO: 474, H2 comprising the amino acid sequence of SEQ ID NO: 475, L1 comprising the amino acid sequence of SEQ ID NO: 476, and H1 comprising the amino acid sequence of SEQ ID NO: 477.

[0714] In some embodiments, the bispecific antibody EIP0766 comprises L2 comprising the amino acid sequence of SEQ ID NO: 478, H2 comprising the amino acid sequence of SEQ ID NO: 479, L1 comprising the amino acid sequence of SEQ ID NO: 480, and H1 comprising the amino acid sequence of SEQ ID NO: 481.

[0715] In some embodiments, the bispecific antibody EIP0990 comprises L2 comprising the amino acid sequence of SEQ ID NO: 482, H2 comprising the amino acid sequence of SEQ ID NO: 483, L1 comprising the amino acid sequence of SEQ ID NO: 484, and H1 comprising the amino acid sequence of SEQ ID NO: 485.

[0716] In some embodiments, the bispecific antibody EIP0991 comprises L2 comprising the amino acid sequence of SEQ ID NO: 486, H2 comprising the amino acid sequence of SEQ ID NO: 487, L1 comprising the amino acid sequence of SEQ ID NO: 488, and H1 comprising the amino acid sequence of SEQ ID NO: 489.

[0717] In some embodiments, the bispecific antibody EIP0991 comprises L2 comprising the amino acid sequence of SEQ ID NO: 486, H2 comprising the amino acid sequence of SEQ ID NO: 487, L1 comprising the amino acid sequence of SEQ ID NO: 488, and H1 comprising the amino acid sequence of SEQ ID NO: 489.

[0718] In some embodiments, the bispecific antibody EIP0992 comprises L2 comprising the amino acid sequence of SEQ ID NO: 490, H2 comprising the amino acid sequence of SEQ ID NO: 491, L1 comprising the amino acid sequence of SEQ ID NO: 492, and H1 comprising the amino acid sequence of SEQ ID NO: 493.

[0719] In some embodiments, the bispecific antibody EIP0993 comprises L2 comprising the amino acid sequence of SEQ ID NO: 494, H2 comprising the amino acid sequence of SEQ ID NO: 495, L1 comprising the amino acid sequence of SEQ ID NO: 496, and H1 comprising the amino acid sequence of SEQ ID NO: 497.

[0720] In some embodiments, the bispecific antibody EIP0869 comprises L2 comprising the amino acid sequence of SEQ ID NO: 498, H2 comprising the amino acid sequence of SEQ ID NO: 499, L1 comprising the amino acid sequence of SEQ ID NO: 500, and H1 comprising the amino acid sequence of SEQ ID NO: 501.

[0721] In some embodiments, the bispecific antibody EIP0870 comprises L2 comprising the amino acid sequence of SEQ ID NO: 502, H2 comprising the amino acid sequence of SEQ ID NO: 503, L1 comprising the amino acid sequence of SEQ ID NO: 504, and H1 comprising the amino acid sequence of SEQ ID NO: 505.

[0722] In some embodiments, the bispecific antibody EIP0871 comprises L2 comprising the amino acid sequence of SEQ ID NO: 506, H2 comprising the amino acid sequence of SEQ ID NO: 507, L1 comprising the amino acid sequence of SEQ ID NO: 508, and H1 comprising the amino acid sequence of SEQ ID NO: 509.

[0723] In some embodiments, the bispecific antibody EIP0872 comprises L2 comprising the amino acid sequence of SEQ ID NO: 510, H2 comprising the amino acid sequence of SEQ ID NO: 511, L1 comprising the amino acid sequence of SEQ ID NO: 512, and H1 comprising the amino acid sequence of SEQ ID NO: 513.

[0724] In some embodiments, the bispecific antibody EIP0546 comprises L2 comprising the amino acid sequence of SEQ ID NO: 514, H2 comprising the amino acid sequence of SEQ ID NO: 515, L1 comprising the amino acid sequence of SEQ ID NO: 516, and H1 comprising the amino acid sequence of SEQ ID NO: 517.

[0725] In some embodiments, the bispecific antibody EIP0607 comprises L2 comprising the amino acid sequence of SEQ ID NO: 518, H2 comprising the amino acid sequence of SEQ ID NO: 519, L1 comprising the amino acid sequence of SEQ ID NO: 520, and H1 comprising the amino acid sequence of SEQ ID NO: 521.

[0726] In some embodiments, the bispecific antibody EIP0614 comprises L2 comprising the amino acid sequence of SEQ ID NO: 522, H2 comprising the amino acid sequence of SEQ ID NO: 523, L1 comprising the amino acid sequence of SEQ ID NO: 524, and H1 comprising the amino acid sequence of SEQ ID NO: 525.

[0727] Table 20. Bispecific antibodies of the present disclosure

[0728]

[0729]

[0730]

[0731]

[0732]

[0733]

[0734]

[0735]

[0736] Table 21. Exemplary bispecific antibodies of the present disclosure

[0737]

[0738]

[0739] Production method

[0740] Various methods known in the art can be used to produce polyclonal or monoclonal antibodies against a given target, such as ULBP2 / 5 / 6, disease-associated antigens, or other targets, or against derivatives, fragments, analogs, homologs, or orthologs thereof (see, for example, Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, incorporated herein by reference).

[0741] Antibodies are purified by well-known techniques, such as affinity chromatography using protein A or protein G, which primarily provide the IgG fraction of immune serum. Subsequently or alternatively, the specific antigen or epitope thereof that is the target of the immunoglobulin being sought can be immobilized on a column to purify the immunospecific antibodies by immunoaffinity chromatography. For example, D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia PA, Vol. 14, No. 8 (April 17, 2000), pp. 25-28) discusses the purification of immunoglobulins.

[0742] In some embodiments, the antibodies of the present invention are monoclonal antibodies. For example, monoclonal antibodies are generated using the procedures described in the Examples provided herein. Antibodies can also be generated by, for example, immunizing BALB / c mice with cell transfectants that express a given target at high levels on their surfaces. Hybridomas produced by myeloma / B cell fusions are then screened for their reactivity to the selected target.

[0743] Monoclonal antibodies can be prepared, for example, using a hybridoma method such as that described in Kohler and Milstein, Nature, 256:495 (1975). In the hybridoma method, mice, hamsters, or other appropriate host animals are typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that specifically bind to the immunizing agent. Alternatively, lymphocytes can be immunized in vitro.

[0744] The immunizing agent will generally include a protein antigen, a fragment thereof, or a fusion protein thereof. Generally, if cells of human origin are desired, peripheral blood lymphocytes are used, and if cells of non-human mammalian origin are desired, spleen cells or lymph node cells are used. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent (such as polyethylene glycol) to form hybridoma cells (Goding et al., 2001). Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103). Immortalized cell lines are typically transformed mammalian cells, particularly myeloma cells of rodent, bovine, and human origin. Rat or mouse myeloma cell lines are commonly used. Hybridoma cells can be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridomas will typically include hypoxanthine, aminopterin, and thymidine ("HAT medium"), which can prevent the growth of HGPRT-deficient cells.

[0745] Preferred immortalized cell lines are those that can effectively fuse, support the selected antibody-producing cells to stably express antibodies at high levels, and are sensitive to culture media such as HAT culture media. A more preferred immortalized cell line is a mouse myeloma cell line, which can be obtained from, for example, the Salk Institute Cell Distribution Center in San Diego, California, and the American Type Culture Collection in Manassas, Virginia. Human myeloma and mouse-human heteromyeloma cell lines have also been described for producing monoclonal antibodies. (See Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp.51-63)).

[0746] The presence of monoclonal antibodies against antigens in the culture medium of the hybridoma cells can then be determined. Preferably, the binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Such techniques and assays are known in the art. For example, the binding affinity of the monoclonal antibody can be measured by Scatchard analysis of Munson and Pollard, Anal. Biochem., 107:220 (1980). In addition, in therapeutic applications of monoclonal antibodies, identification of antibodies with high specificity and high binding affinity to the target antigen is performed.

[0747] After the desired hybridoma cells are identified, the clones can be subcloned by limiting dilution procedures and grown by standard methods. (See Goding, Monoclonal Antibodies:Principles and Practice, Academic Press, (1986) pp. 59-103). Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, hybridoma cells can be grown in vivo as ascites in a mammal.

[0748] The monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0749] Monoclonal antibodies can also be prepared by recombinant DNA methods such as those described in U.S. Patent No. 4,816,567. DNA encoding the monoclonal antibodies of the present invention can be easily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of mouse antibodies). The hybridoma cells of the present invention are a preferred source of such DNA. Once isolated, the DNA can be placed in an expression vector and then transfected into host cells that do not otherwise produce immunoglobulins, such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells, to synthesize monoclonal antibodies in recombinant host cells. The DNA can also be modified, for example, by replacing the coding sequence of the human heavy and light chain constant domains with the homologous mouse sequence (see U.S. Patent No. 4,816,567; Morrison, Nature 368, 812-13 (1994)) or by covalently linking all or part of the coding sequence of a non-immunoglobulin polypeptide to the immunoglobulin coding sequence. Such non-immunoglobulin polypeptides can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-binding site of an antibody of the invention to create a chimeric bivalent antibody.

[0750] The monoclonal antibodies of the present invention include humanized antibodies or human antibodies. These antibodies are suitable for administration to humans and do not cause an immune response in humans to the administered immunoglobulins. The humanized form of an antibody is a chimeric immunoglobulin, an immunoglobulin chain, or a fragment thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of an antibody), which is primarily composed of sequences of human immunoglobulins and contains minimal sequences derived from non-human immunoglobulins. Humanization is, for example, performed according to the methods of Winter et al. (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)) by replacing the corresponding sequences of a human antibody with rodent CDRs or CDR sequences. (See also U.S. Patent No. 5,225,539). In some cases, the Fv framework residues of human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies also include residues that are not found in, for example, the CDR or framework sequences of receptor antibodies or inputs. In general, humanized antibodies include substantially all of at least one, usually two variable domains, wherein all or substantially all of the CDR regions correspond to those of non-human immunoglobulins, and all or substantially all of the framework regions are those of human immunoglobulin consensus sequences. Humanized antibodies optionally also will include at least a portion of an immunoglobulin constant region (Fc), typically at least a portion of an immunoglobulin constant region (Fc) of a human immunoglobulin (Jones et al., 1986; Riechmann et al., 1988; and Presta, Curr. Op. Struct. Biol., 2: 593-596 (1992)).

[0751] A fully human antibody is an antibody molecule in which the entire sequence of the light and heavy chains (including the CDRs) is derived from human genes. Such antibodies are referred to herein as "human antibodies" or "fully human antibodies." Monoclonal antibodies can be produced by using tri-hybridization techniques, human B cell hybridoma technology (see Kozbor et al., 1983 Immunol Today 4:72), and EBV hybridoma technology for producing monoclonal antibodies (see Cole et al., 1985, in M ONOCLONAL A NTIBODIES AND C ANCER T HERAPY, Alan R. Liss, Inc., pp. 77-96). Monoclonal antibodies can be used, or they can be produced using human hybridomas (see Cote et al., 1983. Proc Natl Acad Sci USA 80: 2026-2030) or by transforming human B cells with Epstein-Barr virus in vitro (see Cole et al., 1985, in: M ONOCLONAL A NTIBODIES AND C ANCER THERAPY, Lan R. Liss, Inc., pp. 77-96).

[0752] In addition, human antibodies can also be produced using other technologies, including phage display libraries. (See Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)). Similarly, human antibodies can be prepared by introducing human immunoglobulin loci into transgenic animals (e.g., mice in which endogenous immunoglobulin genes have been partially or completely inactivated). Upon challenge, the production of human antibodies is observed, which is very similar to that observed in humans in all aspects, including gene rearrangement, assembly, and antibody repertoires. This approach is described, for example, in U.S. Pat. Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, 5,661,016, and in Marks et al., Bio / Technology 10, 779-783 (1992); Lonberg et al., Nature 368, 856-859 (1994); Morrison, Nature 368, 812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51 (1996); Neuberger, Nature Biotechnology 14, 826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13, 65-93 (1995).

[0753] Transgenic non-human animals can also be used to produce human antibodies in addition, which have been modified to produce fully human antibodies rather than endogenous antibodies in response to antigenic challenge. (See PCT Publication WO94 / 02602). The endogenous genes encoding heavy and light chain immunoglobulins in the non-human host have been disabled, and the active loci encoding human heavy and light chain immunoglobulins are inserted into the genome of the host. For example, yeast artificial chromosomes containing the desired human DNA fragments are used to integrate the human genes. Then, by crossbreeding, intermediate transgenic animals containing less than the modified full complement are obtained to obtain animals that provide all the desired modifications as offspring. An example of such a non-human animal is a so-called Xenomouse as disclosed in PCT Publications WO 96 / 33735 and WO 96 / 34096. TM Such animals produce B cells that secrete fully human immunoglobulins. Antibodies can be obtained directly from the animal after immunization with the immunogen of interest, for example as a preparation of polyclonal antibodies, or from immortalized B cells derived from the animal, such as hybridomas that produce monoclonal antibodies. In addition, genes encoding immunoglobulins with human variable regions can be recovered and expressed to obtain antibodies directly, or can be further modified to obtain antibody analogs, such as single-chain Fv (scFv) molecules.

[0754] U.S. Patent No. 5,939,598 discloses an example of a method for producing a non-human host (e.g., mouse) lacking endogenous immunoglobulin heavy chain expression. It can be obtained by a method comprising deleting a J segment gene from at least one endogenous heavy chain locus in an embryonic stem cell to prevent rearrangement of the locus and the formation of transcripts of the rearranged immunoglobulin heavy chain locus, wherein the deletion is achieved by a targeting vector containing a gene encoding a selectable marker; and generating a transgenic mouse from the embryonic stem cell whose somatic and germ cells contain a gene encoding the selectable marker.

[0755] U.S. Patent No. 5,916,771 discloses a method for producing an antibody of interest (e.g., a human antibody). This method comprises introducing an expression vector containing a nucleotide sequence encoding a heavy chain into one mammalian host cell in culture, introducing an expression vector containing a nucleotide sequence encoding a light chain into another mammalian host cell, and fusing the two cells to form a hybrid cell. The hybrid cell expresses an antibody comprising both heavy and light chains.

[0756] In a further improvement on this approach, PCT Publication WO 99 / 53049 discloses methods for identifying clinically relevant epitopes on immunogens, and related methods for selecting antibodies that specifically bind to the relevant epitopes with high affinity.

[0757] The antibody can be expressed by a vector containing a DNA segment encoding the above-mentioned single-chain antibody.

[0758] These may include vectors, liposomes, naked DNA, adjuvant-assisted DNA, gene guns, catheters, etc. Vectors include chemical conjugates such as those described in WO 93 / 64701, which have a targeting moiety (e.g., a ligand for a cell surface receptor) and a nucleic acid binding moiety (e.g., polylysine), viral vectors (e.g., DNA or RNA viral vectors), fusion proteins such as those described in PCT / US95 / 02140 (WO95 / 22618), which are fusion proteins containing a targeting moiety (e.g., an antibody specific for a target cell) and a nucleic acid binding moiety (e.g., protamine), plasmids, phages, etc. Vectors may be chromosomal, non-chromosomal, or synthetic.

[0759] Preferred vectors include viral vectors, fusion proteins, and chemical conjugates. Retroviral vectors include Moloney murine leukemia virus. DNA viral vectors are preferred. These vectors include pox vectors such as orthopox or fowlpox vectors, herpes virus vectors such as herpes simplex virus type I (HSV) vectors (see Geller, AI et al., J. Neurochem, 64:487 (1995); Lim, F., et al., in DNA Cloning: Mammalian Systems, D. Glover, ed. (Oxford Univ. Press, Oxford England) (1995); Geller, AI et al., Proc Natl. Acad. Sci.: USA 90:7603 (1993); Geller, AI et al., Proc Natl. Acad. Sci USA 87:1149 (1990), adenovirus vectors (see LeGal LaSalle et al., Science, 259:988 (1993); Davidson et al., Nat. Genet. 3:219 (1993); Yang et al., J. Virol. 69:2004 (1995) and adeno-associated virus vectors (see Kaplitt, MG et al., Nat. Genet. 8:148 (1994).

[0760] Poxvirus vectors introduce genes into the cytoplasm of cells. Fowlpoxvirus vectors only result in short-term expression of nucleic acids. Adenovirus vectors, adeno-associated virus vectors and herpes simplex virus (HSV) vectors are preferred for introducing nucleic acids into neural cells. The expression time of adenovirus vectors (about 2 months) is shorter than that of adeno-associated virus (about 4 months), which in turn is shorter than that of HSV vectors. The specific vector selected will depend on the target cell and the disease to be treated. Introduction can be carried out by standard techniques such as infection, transfection, transduction or transformation. Examples of gene transfer modes include, for example, naked DNA, (Ca) 2 (PO 4 ) 3 precipitation, DEAE dextran, electroporation, protoplast fusion, lipofection, cell microinjection and viral vectors.

[0761] The vector can be used to target essentially any desired target cell. For example, a vector (e.g., adenovirus, HSV) can be guided to the desired location using stereotactic injection. In addition, particles can be delivered by intracerebroventricular (icv) infusion using a micropump infusion system (e.g., SynchroMed infusion system). Methods based on volume flow (called convection) have also been shown to be effective in delivering macromolecules to extended areas of the brain and can be used to deliver vectors to target cells. (See Bobo et al., Proc. Natl. Acad. Sci. USA 91: 2076-2080 (1994); Morrison et al., Am. J. Physiol. 266: 292-305 (1994)). Other methods that can be used include catheter, intravenous, parenteral, intraperitoneal and subcutaneous injections, and oral or other known routes of administration.

[0762] Bispecific antibodies are monoclonal antibodies that have binding specificities for at least two different antigens. In this case, one of the binding specificities is for the first target, such as CD3ε or any fragment thereof, and the second binding target is a disease-associated antigen, such as ULBP2 / 5 / 6 or any fragment thereof.

[0763] Methods for preparing bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain / light chain pairs, wherein the two heavy chains have different specificities (Milstein and Cuello, Nature, 305:537-539 (1983)). Due to the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadrama) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule is usually completed by an affinity chromatography step. Similar procedures are disclosed in WO 93 / 08829 and Traunecker et al., EMBO J., 10:3655-3659 (1991), published on May 13, 1993.

[0764] The bispecific and / or monovalent antibodies of the present invention can be prepared using any of a variety of techniques recognized in the art, including the technology disclosed in co-pending application WO 2012 / 023053, filed on August 16, 2011, which is incorporated herein by reference in its entirety. The method described in WO 2012 / 023053 produces bispecific antibodies with a structure identical to that of human immunoglobulins. This type of molecule consists of two copies of a unique heavy chain polypeptide, a first light chain variable region fused to a constant kappa domain, and a second light chain variable region fused to a constant lambda domain. Each binding site exhibits a different antigen specificity, to which both the heavy chain and the light chain contribute. The light chain variable region can belong to the lambda or kappa family and is preferably fused to the lambda and kappa constant domains, respectively. This is preferred to avoid the generation of non-natural polypeptide connections. However, the bispecific antibodies of the present invention can also be obtained by fusing a kappa light chain variable domain to a constant lambda domain to obtain a first specificity, and fusing a lambda light chain variable domain to a constant kappa domain to obtain a second specificity. The bispecific antibodies described in WO 2012 / 023053, termed IgGκλ antibodies or "κλ bodies," represent a novel, fully human bispecific IgG format. This κλ body format enables affinity purification of bispecific antibodies that are indistinguishable from standard IgG molecules and possess properties indistinguishable from standard monoclonal antibodies, offering advantages over previous formats.

[0765] An important step of this method is to identify two antibody Fv regions (each consisting of a variable light chain and a variable heavy chain domain) with different antigenic specificities but sharing the same heavy chain variable domain. A variety of methods for producing monoclonal antibodies and fragments thereof have been described. (See, for example, Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, incorporated herein by reference). Fully human antibodies are antibody molecules in which the sequences of light and heavy chains (including CDR 1 and 2) are all from human genes. The CDR3 region can be of human origin or can be designed synthetically. Such antibodies are referred to herein as "human antibodies" or "fully human antibodies." Human monoclonal antibodies can be produced by using tri-hybridization technology, human B cell hybridoma technology (see Kozbor et al., 1983 Immunol Today 4:72) and EBV hybridoma technology (see Cole et al., 1985, in M ​​et al.) for producing human monoclonal antibodies. ONOCLONAL A NTIBODIES ANDCANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). Human monoclonal antibodies are available and can be produced by using human hybridomas (see Cote et al., 1983. Proc Natl Acad Sci USA 80: 2026-2030) or by transforming human B cells with Epstein-Barr virus in vitro (see Cole et al., 1985: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96).

[0766] Monoclonal antibodies are produced, for example, by immunizing an animal with a target antigen or an immunogenic fragment, derivative, or variant thereof. Alternatively, an animal is immunized with cells transfected with a vector containing a nucleic acid molecule encoding the target antigen, such that the target antigen is expressed and associates with the surface of the transfected cells. Various techniques for producing xenogeneic non-human animals are well known in the art. For example, see U.S. Patent Nos. 6,075,181 and 6,150,584, which are incorporated herein by reference in their entirety.

[0767] Alternatively, antibodies are obtained by screening a library comprising antibody or antigen binding domain sequences that bind to the target antigen. For example, such a library is prepared in phage as a protein or peptide fusion to a phage coat protein that is expressed on the surface of the assembled phage particle and encodes a DNA sequence contained within the phage particle (i.e., a "phage display library"). Alternatively, a library can be prepared in yeast as a protein or peptide fused to a cell wall protein on the surface of the yeast cell and the encoding DNA sequence contained within the yeast cell (i.e., a "yeast display library").

[0768] The hybridomas produced by the myeloma / B cell fusion are then screened for reactivity to the target antigen. Monoclonal antibodies can be prepared, for example, using a hybridoma method such as that described in Kohler and Milstein, Nature, 256:495 (1975). In the hybridoma method, mice, hamsters, or other appropriate host animals are typically immunized with an immunizing agent to induce lymphocytes that produce or are capable of producing antibodies that specifically bind to the immunizing agent. Alternatively, lymphocytes can be immunized in vitro.

[0769] Although not completely impossible, it is extremely unlikely that different antibodies with the same heavy chain variable domain but targeting different antigens will be identified by chance. In fact, in most cases, the heavy chain contributes a large part to the antigen binding surface and its sequence is also the most variable. Specifically, CDR3 on the heavy chain is the most diverse CDR in terms of sequence, length and structure. Therefore, two antibodies targeting different antigens almost always carry different heavy chain variable domains.

[0770] The method disclosed in co-pending application WO 2012 / 023053 overcomes this limitation and greatly promotes the separation of antibodies with the same heavy chain variable domain by using antibody libraries, wherein the heavy chain variable domain is the same for all library members, so diversity is limited to the light chain variable domain. Such libraries are described in, for example, co-pending applications WO 2010 / 135558 and WO 2011 / 084255, each of which is incorporated herein by reference in its entirety. However, since the light chain variable domain is co-expressed with the heavy chain variable domain, both domains can promote antigen binding. To further promote this process, antibody libraries containing the same heavy chain variable domain and multiple lambda variable light chains or kappa variable light chains can be used in parallel to select antibodies against different antigens in vitro. This method can identify two antibodies with a common heavy chain but one carrying a lambda light chain variable domain and the other carrying a kappa light chain variable domain, which can be used as building blocks for generating bispecific antibodies in the form of complete immunoglobulins of the present invention. The bispecific antibodies of the present invention can be of different isotypes, and their Fc portions can be modified to alter binding properties to different Fc receptors, thereby modifying the effector functions and pharmacokinetic properties of the antibodies. Various methods for modifying the Fc portion have been described and are applicable to the antibodies of the present invention. (See, for example, Strohl, WR Curr Opin Biotechnol 2009(6):685-91; U.S. Patent No. 6,528,624; PCT / US2009 / 0191199 filed January 9, 2009). The methods of the present invention can also be used to produce bispecific antibodies and antibody mixtures in the form of F(ab')2 that lack the Fc portion.

[0771] A common heavy chain and two different light chains are co-expressed in a single cell to allow assembly of the bispecific antibodies of the present invention. If all polypeptides are expressed at the same level and assemble equally well to form immunoglobulin molecules, the ratio of monospecific (same light chain) to bispecific (two different light chains) should be 50%. However, it is possible that different light chains are expressed at different levels and / or do not assemble with the same efficiency. Therefore, methods for regulating the relative expression of different polypeptides are used to compensate for their inherent expression characteristics or different tendencies to ass...

Claims

1. An antibody comprising the following structure: a. A first heavy chain polypeptide (H1) comprising a variable region (VH1) and a constant region (CH1), wherein the constant region (CH1) has a constant region 1 domain (CH1 H1 ), hinge region (H1H), constant region 2 domain (CH1 H2 ) and constant region 3 domain (CH1 H3 ); and a first light chain polypeptide (L1) comprising a variable region (VL1) and a constant region (CL1), b. A second heavy chain polypeptide (H2) comprising a variable region (VH2) and a constant region (CH2), wherein the constant region (CH2) has a constant region 1 domain (CH2 H1 ), hinge region (H2H), constant region 2 domain (CH2 H2 ) and constant region 3 domain (CH2 H3 ); and a second light chain polypeptide (L2) comprising a variable region (VL2) and a constant region (CL2), in i. the amino acid at position 39 (Kabat numbering) of VH1 and VH2 is a charged or polar amino acid residue, and the amino acid at position 38 (Kabat numbering) of VL1 and VL2 is an oppositely charged or polar amino acid residue compared to the amino acid at position 39 of VH1 and VH2; or the amino acid at position 100 (Kabat numbering) of VH1 and VH2 is a charged or polar amino acid residue, and the amino acid at position 44 (Kabat numbering) of VL1 and VL2 is an oppositely charged or polar amino acid residue compared to the amino acid at position 100 (Kabat numbering) of VH1 and VH2; ii.CH1 H1 and CH1 H2 The amino acid at position 147 (EU numbering) of CL1 or CL2 is a charged or polar amino acid residue, and one of the amino acids at position 131, 179, or 180 (EU numbering) of CL1 or CL2 is incompatible with CH1 H1 and CH1 H2 An amino acid residue with an opposite charge or polarity compared to the amino acid at position 147 (EU numbering); iii.CH1 H1 and CH1 H2 The amino acid at position 185 (EU numbering) of CL1 and CL2 is a charged or polar amino acid residue, and the amino acid at position 137 (EU numbering) of CL1 and CL2 is aligned with CH1. H1 and CH1 H2 The amino acid residue at position 185 (EU numbering) of CH1 is an amino acid residue with an opposite charge or polarity compared to the amino acid at position 185 (EU numbering); or CH1 H1 and CH1 H2 The amino acid at position 187 (EU numbering) of CL1 is a charged or polar amino acid residue, and one of the amino acids at position 137 or 138 (EU numbering) of CL1 and CL2 is incompatible with CH1. H1 and CH1 H2 is an amino acid residue with an opposite charge or polarity compared to the amino acid at position 187 (EU numbering) of iv.CH1 H1 and CH1 H2 The amino acid at position 145 (EU numbering) of CL1 or CL2 is a charged or polar amino acid residue, and the amino acid at position 131 (EU numbering) of CL1 or CL2 is aligned with the amino acid at position 145 (EU numbering) of CH1 H1 and CH1 H2 The amino acid residue at position 145 (EU numbering) is an amino acid residue with an opposite charge or polarity compared to the amino acid at position 145 (EU numbering).

2. The antibody of claim 1, wherein the charged amino acid residue is a naturally occurring amino acid or a non-naturally occurring amino acid.

3. The antibody of claim 1, wherein the naturally occurring charged amino acid residue is arginine, lysine, histidine, glutamic acid or aspartic acid.

4. The antibody of claim 1 , wherein the H1 amino acid at position 39, 100, 147, 185, 187 or 145 is positively charged and the L1 amino acid at position 38, 44, 131, 179, 180, 137 or 138 is negatively charged; and the H2 amino acid at position 39, 100, 147, 185, 187 or 145 is negatively charged and the L2 amino acid at position 38, 44, 131, 179, 180, 137 or 138 is positively charged.

5. The antibody of claim 1 , wherein the H1 amino acid at positions 39, 100, 147, 185, 187 and 145 is negatively charged, and the L1 amino acid at positions 38, 44, 131, 179, 180, 137 or 138 is positively charged; and the H2 amino acid at positions 39, 100, 147, 185, 187 or 145 is positively charged, and the L2 amino acid at positions 38, 44, 131, 179, 180, 137 or 138 is negatively charged.

6. The antibody of any one of the preceding claims, wherein L1 and L2 are lambda or kappa light chains.

7. The antibody of claim 6, wherein L1 is a λ light chain and L2 is a κ light chain; or L1 is a κ light chain and L2 is a λ light chain.

8. The antibody of any one of the preceding claims, wherein the antibody is of IgGl, IgG2 or IgG4 isotype.

9. The antibody of any one of the preceding claims, wherein the antibody is a chimeric antibody, a human antibody or a humanized antibody.

10. The antibody of any one of the preceding claims, wherein the antibody is a bispecific antibody.

11. The antibody of claim 10, wherein the bispecific antibody comprises i) a first antigen binding domain that binds to a cell surface antigen, wherein the cell surface antigen is expressed on a T cell, a NK cell, a neutrophil, a B cell, or a dendritic cell engager; and ii) a second antigen binding domain that binds to a disease associated antigen (DAA).

12. The antibody of claim 11, wherein the cell surface antigen is expressed on T cells.

13. The antibody of claim 12, wherein the cell surface antigen expressed on T cells is CD3.

14. The antibody of claim 11, wherein the DAA is UL16 binding protein 2 (ULBP2) or is encoded by the gene RAET1G (UL16 binding protein 5; ULBP5) or is encoded by the gene RAET1L (UL16 binding protein 6; ULBP6).

15. The antibody of any one of the preceding claims, wherein the polypeptide is fused to the N-terminus or C-terminus of H1 and / or H2.

16. The antibody of claim 15, wherein the polypeptide is CD58, IL-17 or a fragment thereof.

17. The antibody of claim 15, wherein the polypeptide is fused via a linker peptide.

18. The antibody of claim 17, wherein the linker peptide is at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 amino acid residues in length.

19. The antibody of claim 17, wherein the linker peptide comprises the amino acid sequence of SEQ ID NOs: 52-54.

20. The antibody of any one of the preceding claims, wherein: i) the amino acid at position 87 (Kabat numbering) of VH1 and / or VH2 is G; and ii) the amino acid at position 45 (Kabat numbering) of VL1 and / or VL2 is W.

21. The antibody of any one of the preceding claims, wherein: i) CH1 H3 has C at position 349, S at position 366, A at position 368, and V at position 407 (EU numbering); and the CH2 H3 C at position 354 and W at position 366 (EU numbering); ii) the CH2 H3 has C at position 349, S at position 366, A at position 368, and V at position 407 (EU numbering); and said CH1 H3 C at position 354 and W at position 366 (EU numbering); iii) the CH1 H3 has C at position 354, S at position 366, A at position 368, and V at position 407 (EU numbering); and the CH2 H3 C at position 349 and W at position 366 (EU numbering); or iv) the CH2 H3 has C at position 354, S at position 366, A at position 368, and V at position 407 (EU numbering); and said CH1 H3 It has a C at position 349 and a W at position 366 (EU numbering).

22. The antibody of any one of the preceding claims, further comprising said CH1 H3 and / or the CH2 H3 the amino acid at position 446 (EU numbering) and / or the amino acid at position 447 (EU numbering); optionally wherein the amino acid at position 446 (EU numbering) is G; and optionally wherein the amino acid at position 447 (EU numbering) is K.

23. The antibody of any one of the preceding claims, wherein: i) H1H and / or H2H have A at positions 234 and 235 (EU numbering); i) said H1H and / or said H2H has A at positions 234, 235 and 237 (EU numbering); or iii) said H1H and / or said H2H has A at position 234 and 235, and G at position 329 (EU numbering).

24. An antibody as claimed in any one of the preceding claims, wherein i) CH1 H3 and / or the CH2 H3 has an A at position 297 (EU numbering); ii) CH1 H3 and / or the CH2 H3 with a G at position 297 (EU numbering); or iii) the CH1 H3 and / or the CH2 H3 There is S at position 297 (EU numbering).

25. The antibody of any one of the preceding claims, wherein the CH1 H3 and / or the CH2 H3 There is S at position 331 (EU numbering).

26. An antibody as claimed in any one of the preceding claims, wherein i) CH1 H2 and / or the CH2 H2 has a C at position 370 (Kabat numbering); and i) CH1 H2 and / or the CH2 H2 There is a C at position 375 (Kabat numbering).

27. The antibody of claim 1, wherein a) The H1 and L1 include the following: i. the amino acid at position 39 (Kabat numbering) of the VH1 is K and the amino acid at position 38 (Kabat numbering) of the VL1 is D; ii. the CH1 H1 The amino acid at position 185 (EU numbering) of is K and the amino acid at position 137 (EU numbering) of CL1 is D; iii. The CH1 H1 The amino acid at position 128 (EU numbering) of is C and the amino acid at position 118 (EU numbering) of CL1 is C; and iv. the amino acid at position 220 (EU numbering) in the H1H is S and the amino acid at position 214 (EU numbering) in the CL1 is S; and b) said H2 and said L2 include the following: i. the amino acid at position 39 (Kabat numbering) of the VH2 is D and the amino acid at position 38 (Kabat numbering) of the VL2 is K; and ii. the CH2 H1 The 147th amino acid (EU numbering) of is D and the 180th amino acid (EU numbering) of CL2 is R.

28. The antibody of claim 1, wherein a) The H1 and L1 include the following: i. the amino acid at position 39 (Kabat numbering) of the VH1 is K and the amino acid at position 38 (Kabat numbering) of the VL1 is D; and ii. the CH1 H1 The amino acid at position 185 (EU numbering) of CL1 is K and the amino acid at position 137 (EU numbering) of CL1 is D; and b) said H2 and said L2 include the following: i. the amino acid at position 39 (Kabat numbering) of the VH2 is D and the amino acid at position 38 (Kabat numbering) of the VL2 is K; ii. the CH2 H1 The amino acid at position 147 (EU numbering) of CL2 is D and the amino acid at position 180 (EU numbering) of CL2 is R; iii. the CH2 H1 The amino acid at position 134 (EU numbering) of CL is C and the amino acid at position 116 (EU numbering) of CL2 is C; and iv. the amino acid at position 220 (EU numbering) in the H2H is S and the amino acid at position 214 (EU numbering) in the CL2 is S.

29. The antibody of claim 1, wherein a) The H1 and L1 include the following: i. the amino acid at position 39 (Kabat numbering) of the VH1 is K and the amino acid at position 38 (Kabat numbering) of the VL1 is D; and ii. the CH1 H1 The amino acid at position 185 (EU numbering) of CL1 is K and the amino acid at position 137 (EU numbering) of CL1 is D; and b) said H2 and said L2 include the following: i. the amino acid at position 39 (Kabat numbering) of the VH2 is D and the amino acid at position 38 (Kabat numbering) of the VL2 is K; ii. the CH2 H1 The amino acid at position 147 (EU numbering) of CL2 is D and the amino acid at position 180 (EU numbering) of CL2 is R; iii. the CH2 H1 The amino acid at position 136 (EU numbering) of CL is C and the amino acid at position 114 (EU numbering) of CL2 is C; and iv. the amino acid at position 220 (EU numbering) in the H2H is S and the amino acid at position 214 (EU numbering) in the CL2 is S.

30. The antibody of claim 1, wherein a) The H1 and L1 include the following: i. the amino acid at position 39 (Kabat numbering) of the VH1 is K and the amino acid at position 38 (Kabat numbering) of the VL1 is D; ii. the CH1 H1 The amino acid at position 147 (EU numbering) of is K and the amino acid at position 131 (EU numbering) of CL1 is D; iii. The CH1 H1 The 173rd amino acid (EU numbering) of is C and the 162nd amino acid (EU numbering) of CL1 is C; iv. the amino acid at position 220 (EU numbering) in the H1H is S and the amino acid at position 214 (EU numbering) in the CL1 is S; and b) said H2 and said L2 include the following: i. the amino acid at position 39 (Kabat numbering) of the VH2 is D and the amino acid at position 38 (Kabat numbering) of the VL2 is K; and ii. the CH2 H1 The 147th amino acid (EU numbering) of is D and the 180th amino acid (EU numbering) of CL2 is R.

31. The antibody of claim 1, wherein a) The H1 and L1 include the following: i. the amino acid at position 39 (Kabat numbering) of the VH1 is K and the amino acid at position 38 (Kabat numbering) of the VL1 is D; ii. the CH1 H1 The amino acid at position 185 (EU numbering) of is K and the amino acid at position 137 (EU numbering) of CL1 is D; iii. The CH1 H1 The amino acid at position 173 (EU numbering) of is C and the amino acid at position 162 (EU numbering) of CL1 is C; and iv. the amino acid at position 220 (EU numbering) in the H1H is S and the amino acid at position 214 (EU numbering) in the CL1 is S; and b) said H2 and said L2 include the following: i. the amino acid at position 39 (Kabat numbering) of the VH2 is D and the amino acid at position 38 (Kabat numbering) of the VL2 is K; and ii. the CH2 H1 The 147th amino acid (EU numbering) of is D and the 180th amino acid (EU numbering) of CL2 is R.

32. The antibody of claim 1, wherein a) The H1 and L1 include the following: i. the amino acid at position 39 (Kabat numbering) of the VH1 is K and the amino acid at position 38 (Kabat numbering) of the VL1 is D; and ii. the CH1 H1 The amino acid at position 185 (EU numbering) of CL1 is K and the amino acid at position 137 (EU numbering) of CL1 is D; and b) said H2 and said L2 include the following: i. the amino acid at position 39 (Kabat numbering) of the VH2 is D and the amino acid at position 38 (Kabat numbering) of the VL2 is K; ii. the CH2 H1 The amino acid at position 147 (EU numbering) of CL2 is D and the amino acid at position 180 (EU numbering) of CL2 is R; iii. the CH2 H1 The amino acid at position 131 (EU numbering) of CL is C and the amino acid at position 114 (EU numbering) of CL2 is C; and iv. the amino acid at position 220 (EU numbering) in the H2H is S and the amino acid at position 214 (EU numbering) in the CL2 is S.

33. The antibody of claim 1, wherein a) The H1 and L1 include the following: i. the amino acid at position 39 (Kabat numbering) of the VH1 is K and the amino acid at position 38 (Kabat numbering) of the VL1 is D; and ii. the CH1 H1 The amino acid at position 185 (EU numbering) of CL1 is K and the amino acid at position 137 (EU numbering) of CL1 is D; and b) said H2 and said L2 include the following: i. the amino acid at position 39 (Kabat numbering) of the VH2 is D and the amino acid at position 38 (Kabat numbering) of the VL2 is K; ii. the CH2 H1 The amino acid at position 187 (EU numbering) of CL2 is D and the amino acid at position 138 (EU numbering) of CL2 is K; iii. the CH2 H1 The amino acid at position 170 (EU numbering) of CL is C and the amino acid at position 162 (EU numbering) of CL2 is C; and iv. the amino acid at position 220 (EU numbering) in the H2H is S and the amino acid at position 214 (EU numbering) in the CL2 is S.

34. The antibody of claim 1, wherein a) The H1 and L1 include the following: i. the amino acid at position 39 (Kabat numbering) of the VH1 is K and the amino acid at position 38 (Kabat numbering) of the VL1 is D; ii. the CH1 H1 The amino acid at position 185 (EU numbering) of is E, and the amino acid at position 137 (EU numbering) of CL1 is K; and iii. the amino acid at position 179 (EU numbering) of the CL1 is E; and b) said H2 and said L2 include the following: i. the amino acid at position 39 (Kabat numbering) of the VH2 is D and the amino acid at position 38 (Kabat numbering) of the VL2 is K; ii. the CH2 H1 The amino acid at position 187 (EU numbering) of CL2 is D and the amino acid at position 138 (EU numbering) of CL2 is K; iii. the CH2 H1 The amino acid at position 171 (EU numbering) of CL is C and the amino acid at position 162 (EU numbering) of CL2 is C; and iv. the amino acid at position 220 (EU numbering) in the H2H is S and the amino acid at position 214 (EU numbering) in the CL2 is S.

35. The antibody of claim 1, wherein a) The H1 and L1 include the following: i. the amino acid at position 39 (Kabat numbering) of the VH1 is K and the amino acid at position 38 (Kabat numbering) of the VL1 is D; and ii. the CH1 H1 The amino acid at position 185 (EU numbering) of CL1 is K and the amino acid at position 137 (EU numbering) of CL1 is D; and b) said H2 and said L2 include the following: i. the amino acid at position 39 (Kabat numbering) of the VH2 is D and the amino acid at position 38 (Kabat numbering) of the VL2 is K; ii. the CH2 H1 The amino acid at position 187 (EU numbering) of CL2 is D and the amino acid at position 138 (EU numbering) of CL2 is K; iii. the CH2 H1 The amino acid at position 171 (EU numbering) of CL is C and the amino acid at position 162 (EU numbering) of CL2 is C; and iv. the amino acid at position 220 (EU numbering) in the H2H is S and the amino acid at position 214 (EU numbering) in the CL2 is S.

36. The antibody of claim 1, wherein a) The H1 and L1 include the following: i. the amino acid at position 39 (Kabat numbering) of the VH1 is K and the amino acid at position 38 (Kabat numbering) of the VL1 is D; ii. the CH1 H1 The amino acid at position 185 (EU numbering) of CL1 is E and the amino acid at position 137 (EU numbering) of CL1 is K; and iii. the amino acid at position 179 (EU numbering) of the CL1 is E; and b) said H2 and said L2 include the following: i. the amino acid at position 39 (Kabat numbering) of the VH2 is D and the amino acid at position 38 (Kabat numbering) of the VL2 is K; ii. the CH2 H1 The amino acid at position 147 (EU numbering) of CL2 is D and the amino acid at position 180 (EU numbering) of CL2 is R; iii. the CH2 H1 The amino acid at position 171 (EU numbering) of CL is C and the amino acid at position 162 (EU numbering) of CL2 is C; and iv. the amino acid at position 220 (EU numbering) in the H2H is S and the amino acid at position 214 (EU numbering) in the CL2 is S.

37. The antibody of claim 1, wherein a) The H1 and L1 include the following: i. the amino acid at position 39 (Kabat numbering) of the VH1 is K and the amino acid at position 38 (Kabat numbering) of the VL1 is D; and ii. the CH1 H1 The amino acid at position 185 (EU numbering) of CL1 is K and the amino acid at position 137 (EU numbering) of CL1 is D; and b) said H2 and said L2 include the following: i. the amino acid at position 39 (Kabat numbering) of the VH2 is D and the amino acid at position 38 (Kabat numbering) of the VL2 is K; ii. the CH2 H1 The amino acid at position 147 (EU numbering) of CL2 is D and the amino acid at position 180 (EU numbering) of CL2 is R; iii. the CH2 H1 The amino acid at position 171 (EU numbering) of CL is C and the amino acid at position 162 (EU numbering) of CL2 is C; and iv. the amino acid at position 220 (EU numbering) in the H2H is S and the amino acid at position 214 (EU numbering) in the CL2 is S.

38. The antibody of claim 1, wherein a) The H1 and L1 include the following: i. the amino acid at position 39 (Kabat numbering) of the VH1 is K and the amino acid at position 38 (Kabat numbering) of the VL1 is D; ii. the CH1 H1 The amino acid at position 147 (EU numbering) of is K and the amino acid at position 131 (EU numbering) of CL1 is D; iii. The CH1 H1 The amino acid at position 185 (EU numbering) of CL1 is K and the amino acid at position 137 (EU numbering) of CL1 is D; and b) said H2 and said L2 include the following: i. the amino acid at position 39 (Kabat numbering) of the VH2 is D and the amino acid at position 38 (Kabat numbering) of the VL2 is K; ii. the CH2 H1 The amino acid at position 147 (EU numbering) of CL2 is D and the amino acid at position 180 (EU numbering) of CL2 is R; iii. the CH2 H1 The amino acid at position 171 (EU numbering) of CL is C and the amino acid at position 162 (EU numbering) of CL2 is C; and iv. the amino acid at position 220 (EU numbering) in the H2H is S and the amino acid at position 214 (EU numbering) in the CL2 is S.

39. The antibody of claim 1, wherein a) The H1 and L1 include the following: i. the amino acid at position 39 (Kabat numbering) of the VH1 is D and the amino acid at position 38 (Kabat numbering) of the VL1 is K; and ii. the CH1 H1 The amino acid at position 185 (EU numbering) of CL1 is E and the amino acid at position 137 (EU numbering) of CL1 is K; and b) said H2 and said L2 include the following: i. the amino acid at position 39 (Kabat numbering) of the VH2 is K and the amino acid at position 38 (Kabat numbering) of the VL2 is D; ii. the CH2 H1 The amino acid at position 187 (EU numbering) of CL2 is D and the amino acid at position 138 (EU numbering) of CL2 is K; iii. the CH2 H1 The amino acid at position 136 (EU numbering) of CL is C and the amino acid at position 114 (EU numbering) of CL2 is C; and iv. the amino acid at position 220 (EU numbering) in the H2H is S and the amino acid at position 214 (EU numbering) in the CL2 is S.

40. The antibody of claim 1, wherein a) The H1 and L1 include the following: i. the amino acid at position 39 (Kabat numbering) of the VH1 is D and the amino acid at position 38 (Kabat numbering) of the VL1 is K; and ii. the CH1 H1 The amino acid at position 185 (EU numbering) of CL1 is E and the amino acid at position 137 (EU numbering) of CL1 is K; and b) said H2 and said L2 include the following: i. the amino acid at position 39 (Kabat numbering) of the VH2 is K and the amino acid at position 38 (Kabat numbering) of the VL2 is D; ii. the CH2 H1 The amino acid at position 187 (EU numbering) of CL2 is D and the amino acid at position 138 (EU numbering) of CL2 is K; iii. the CH2 H1 The amino acid at position 171 (EU numbering) of CL is C and the amino acid at position 162 (EU numbering) of CL2 is C; and iv. the amino acid at position 220 (EU numbering) in the H2H is S and the amino acid at position 214 (EU numbering) in the CL2 is S.

41. The antibody of claim 1, wherein a) The H1 and L1 include the following: i. the amino acid at position 39 (Kabat numbering) of the VH1 is D and the amino acid at position 38 (Kabat numbering) of the VL1 is K; and ii. the CH1 H1 The amino acid at position 185 (EU numbering) of CL1 is K and the amino acid at position 137 (EU numbering) of CL1 is D; and b) said H2 and said L2 include the following: i. the amino acid at position 39 (Kabat numbering) of the VH2 is K and the amino acid at position 38 (Kabat numbering) of the VL2 is D; ii. the CH2 H1 The amino acid at position 187 (EU numbering) of CL2 is D and the amino acid at position 138 (EU numbering) of CL2 is K; iii. the CH2 H1 The amino acid at position 171 (EU numbering) of CL is C and the amino acid at position 162 (EU numbering) of CL2 is C; and iv. the amino acid at position 220 (EU numbering) in the H2H is S and the amino acid at position 214 (EU numbering) in the CL2 is S.

42. The antibody of claim 1, wherein a) The H1 and L1 include the following: i. the amino acid at position 39 (Kabat numbering) of the VH1 is D and the amino acid at position 38 (Kabat numbering) of the VL1 is K; ii. the CH1 H1 The amino acid at position 147 (EU numbering) of is K and the amino acid at position 131 (EU numbering) of CL1 is D; iii. The CH1 H1 The amino acid at position 185 (EU numbering) of CL1 is E and the amino acid at position 137 (EU numbering) of CL1 is K; and iv. the CH1 H1 The amino acid at position 145 (EU numbering) of CL1 is S and the amino acid at position 180 (EU numbering) of CL1 is E; and b) said H2 and said L2 include the following: i. the amino acid at position 39 (Kabat numbering) of the VH2 is K and the amino acid at position 38 (Kabat numbering) of the VL2 is D; ii. the CH2 H1 The amino acid at position 187 (EU numbering) of CL2 is D and the amino acid at position 138 (EU numbering) of CL2 is K; iii. the amino acid at position 170 (EU numbering) of the VH2 is C and the amino acid at position 162 (EU numbering) of the VL2 is C; and iv. the amino acid at position 220 (EU numbering) in the H2H is S and the amino acid at position 214 (EU numbering) in the CL2 is S.

43. The antibody of claim 1, wherein a) The H1 and L1 include the following: i. the amino acid at position 39 (Kabat numbering) of the VH1 is K and the amino acid at position 38 (Kabat numbering) of the VL1 is D; ii. the CH1 H1 The amino acid at position 147 (EU numbering) of CL1 is D and the amino acid at position 131 (EU numbering) of CL1 is K; and iii. The CH1 H1 The amino acid at position 185 (EU numbering) of CL1 is E and the amino acid at position 137 (EU numbering) of CL1 is D; and b) said H2 and said L2 include the following: i. the amino acid at position 39 (Kabat numbering) of the VH2 is D and the amino acid at position 38 (Kabat numbering) of the VL2 is K; ii. the CH2 H1 The amino acid at position 187 (EU numbering) of CL2 is D and the amino acid at position 137 (EU numbering) of CL2 is K; iii. the amino acid at position 138 (EU numbering) of the CL2 is R; iv. the CH2 H1 The amino acid at position 170 (EU numbering) of CL is C and the amino acid at position 162 (EU numbering) of CL2 is C; and v. The amino acid at position 220 (EU numbering) in H2H is S and the amino acid at position 214 (EU numbering) in CL2 is S.

44. The antibody of claim 1, wherein a) The H1 and L1 include the following: i. the amino acid at position 39 (Kabat numbering) of the VH1 is K and the amino acid at position 38 (Kabat numbering) of the VL1 is D; ii. the CH1 H1 The amino acid at position 147 (EU numbering) of CL1 is D and the amino acid at position 131 (EU numbering) of CL1 is K; and iii. The CH1 H1 The amino acid at position 145 (EU numbering) is S; and b) said H2 and said L2 include the following: i. the amino acid at position 39 (Kabat numbering) of the VH2 is D and the amino acid at position 38 (Kabat numbering) of the VL2 is K; ii. the CH2 H1 The amino acid at position 187 (EU numbering) of CL2 is D and the amino acid at position 138 (EU numbering) of CL2 is K; iii. the CH2 H1 The amino acid at position 170 (EU numbering) of CL is C and the amino acid at position 162 (EU numbering) of CL2 is C; and iv. the amino acid at position 220 (EU numbering) in the H2H is S and the amino acid at position 214 (EU numbering) in the CL2 is S.

45. The antibody of claim 1, wherein a) The H1 and L1 include the following: i. the amino acid at position 39 (Kabat numbering) of the VH1 is D and the amino acid at position 38 (Kabat numbering) of the VL1 is K; ii. the CH1 H1 The amino acid at position 147 (EU numbering) of CL1 is K and the amino acid at position 131 (EU numbering) of CL1 is D; and iii. The CH1 H1 The amino acid at position 185 (EU numbering) of CL1 is E and the amino acid at position 137 (EU numbering) of CL1 is K; and b) said H2 and said L2 include the following: i. the amino acid at position 39 (Kabat numbering) of the VH2 is K and the amino acid at position 38 (Kabat numbering) of the VL2 is D; ii. the CH2 H1 The amino acid at position 147 (EU numbering) of CL2 is D and the amino acid at position 180 (EU numbering) of CL2 is R; iii. the CH2 H1 The amino acid at position 136 (EU numbering) of CL is C and the amino acid at position 114 (EU numbering) of CL2 is C; and iv. the amino acid at position 220 (EU numbering) in the H2H is S and the amino acid at position 214 (EU numbering) in the CL2 is S.

46. ​​The antibody of claim 1, wherein a) The H1 and L1 include the following: i. the amino acid at position 39 (Kabat numbering) of the VH1 is K and the amino acid at position 38 (Kabat numbering) of the VL1 is E; ii. the CH1 H1 The amino acid at position 147 (EU numbering) of CL1 is K and the amino acid at position 131 (EU numbering) of CL1 is D; and iii. The CH1 H1 The amino acid at position 185 (EU numbering) of CL1 is E and the amino acid at position 137 (EU numbering) of CL1 is K; and b) said H2 and said L2 include the following: i. the amino acid at position 39 (Kabat numbering) of the VH2 is D and the amino acid at position 38 (Kabat numbering) of the VL2 is K; ii. the CH2 H1 The amino acid at position 187 (EU numbering) of CL2 is D and the amino acid at position 138 (EU numbering) of CL2 is K; iii. the CH2 H1 The amino acid at position 136 (EU numbering) of CL is C and the amino acid at position 114 (EU numbering) of CL2 is C; and iv. the amino acid at position 220 (EU numbering) in the H2H is S and the amino acid at position 214 (EU numbering) in the CL2 is S.

47. The antibody of claim 1, wherein a) The H1 and L1 include the following: i. the amino acid at position 39 (Kabat numbering) of the VH1 is K and the amino acid at position 38 (Kabat numbering) of the VL1 is D; ii. the CH1 H1 The amino acid at position 147 (EU numbering) of CL1 is D and the amino acid at position 131 (EU numbering) of CL1 is K; and iii. The CH1 H1 The amino acid at position 185 (EU numbering) of CL1 is D and the amino acid at position 137 (EU numbering) of CL1 is K; and b) said H2 and said L2 include the following: i. the amino acid at position 39 (Kabat numbering) of the VH2 is D and the amino acid at position 38 (Kabat numbering) of the VL2 is K; ii. the CH2 H1 The amino acid at position 187 (EU numbering) of CL2 is D and the amino acid at position 138 (EU numbering) of CL2 is K; iii. the CH2 H1 The amino acid at position 171 (EU numbering) of CL is C and the amino acid at position 162 (EU numbering) of CL2 is C; and iv. the amino acid at position 220 (EU numbering) in the H2H is S and the amino acid at position 214 (EU numbering) in the CL2 is S.

48. The antibody of any one of claims 27-47, wherein: i) the amino acid at position 87 (Kabat numbering) of VH1 and / or VH2 is G; and ii) the amino acid at position 45 (Kabat numbering) of VL1 and / or VL2 is W.

49. The antibody of any one of claims 27-48, wherein: i) CH1 H3 has C at position 349, S at position 366, A at position 368, and V at position 407 (EU numbering); and the CH2 H3 C at position 354 and W at position 366 (EU numbering); ii) the CH2 H3 has C at position 349, S at position 366, A at position 368, and V at position 407 (EU numbering); and said CH1 H3 C at position 354 and W at position 366 (EU numbering); iii) the CH1 H3 has C at position 354, S at position 366, A at position 368, and V at position 407 (EU numbering); and the CH2 H3 C at position 349 and W at position 366 (EU numbering); or iv) the CH2 H3 has C at position 354, S at position 366, A at position 368, and V at position 407 (EU numbering); and said CH1 H3 It has a C at position 349 and a W at position 366 (EU numbering).

50. The antibody of any one of claims 27-49, further comprising said CH1 H3 and / or the CH2 H3 the amino acid at position 446 (EU numbering) and / or the amino acid at position 447 (EU numbering); optionally wherein the amino acid at position 446 (EU numbering) is G; and optionally wherein the amino acid at position 447 (EU numbering) is K.

51. The antibody of any one of claims 27-50, wherein: i) H1H and / or H2H have A at positions 234 and 235 (EU numbering); i) said H1H and / or said H2H has A at positions 234, 235 and 237 (EU numbering); or iii) said H1H and / or said H2H has A at position 234 and 235, and G at position 329 (EU numbering).

52. The antibody of any one of claims 27-51, wherein: i) CH1 H3 and / or the CH2 H3 has an A at position 297 (EU numbering); ii) CH1 H3 and / or the CH2 H3 with a G at position 297 (EU numbering); or iii) the CH1 H3 and / or the CH2 H3 There is S at position 297 (EU numbering).

53. The antibody of any one of claims 27-52, wherein the CH1 H3 and / or the CH2 H3 There is S at position 331 (EU numbering).

54. The antibody of any one of claims 27-51, wherein: i) CH1 H2 and / or the CH2 H2 has a C at position 370 (Kabat numbering); and ii) CH1 H2 and / or the CH2 H2 There is a C at position 375 (Kabat numbering).

55. The antibody of any one of claims 27-54, wherein: a) the VH1 comprises the amino acid sequence of SEQ ID NO: 13; and b) the VL1 comprises the amino acid sequence of SEQ ID NO:

22.

56. The antibody of any one of claims 1-55, wherein: a) the VH1 comprises the amino acid sequence of EVQLLESGGGLVQPGGSLKLSCAASGFTFNX1YAMX2WVRX3APGK GLEWVAX4IRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNL KTEDTAVYYCVRHGX5FGNX6X7YVSWFAYWGQGTLVTVSS (SEQ ID NO: 440), wherein X1 of SEQ ID NO:440 is D or T, wherein X2 of SEQ ID NO:440 is D or N, wherein X3 of SEQ ID NO:440 is D or K, wherein X4 of SEQ ID NO:440 is K or R, wherein X5 of SEQ ID NO:440 is D or T, wherein X6 of SEQ ID NO:440 is D or N, and wherein X7 of SEQ ID NO:440 is D or S; and b) the VL1 comprises the amino acid sequence of ELVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQX1KPGQAP RGLIGX2TNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCAL WX3SX4LWVFGGGTKLTVL (SEQ ID NO: 441), wherein X1 of SEQ ID NO:441 is E or D, wherein X2 of SEQ ID NO:441 is D or G, wherein X3 of SEQ ID NO:441 is D or Y, and Wherein X4 of SEQ ID NO:441 is D, N or Q.

57. The antibody of any one of claims 1-56, wherein: a) said VH2 comprises: Complementarity determining region 1 (VH2 CDR1 ); Complementarity determining region 2 (VH2) comprising the amino acid sequence of SEQ ID NO: 430 CDR2 );as well as Complementarity determining region 3 (VH2 CDR3 );as well as b) the VL2 comprises: Complementarity determining region 1 (VL2) comprising the amino acid sequence of SEQ ID NO: 433 CDR1 ); Complementarity determining region 2 (VL2) comprising the amino acid sequence of SEQ ID NO: 434 CDR2 );as well as Complementarity determining region 3 (VL2) comprising the amino acid sequence of SEQ ID NO: 435 CDR3 ).

58. The antibody of any one of claims 1-56, wherein: a) said VH2 comprises: Complementarity determining region 1 (VH2) comprising the amino acid sequence of SEQ ID NO: 5 CDR1 ); Complementarity determining region 2 (VH2) comprising the amino acid sequence of SEQ ID NO: 7 CDR2 );as well as Complementarity determining region 3 (VH2) comprising the amino acid sequence of SEQ ID NO: 9 CDR3 );as well as b) the VL2 comprises: A complementary determining region 1 (VL2) comprising the amino acid sequence of SEQ ID NO: 10 CDR1 ); Complementarity determining region 2 (VL2) comprising the amino acid sequence of SEQ ID NO: 11 CDR2 );as well as Complementarity determining region 3 (VL2) comprising the amino acid sequence of SEQ ID NO: 12 CDR3 ).

59. The antibody of any one of claims 16-58, wherein the CD58 comprises the amino acid sequence of SEQ ID NOs: 49-50.

60. The antibody of any one of claims 16-58, wherein the IL-7 comprises the amino acid sequence of SEQ ID NO:

51.

61. A polynucleotide comprising a nucleic acid sequence encoding the antibody of any one of claims 1-58.

62. A vector comprising the polynucleotide of claim 61.

63. A pharmaceutical composition comprising the antibody of any one of claims 1-60, the polynucleotide of claim 61 or the vector of claim 62, and a pharmaceutically acceptable carrier.

64. The pharmaceutical composition of claim 63, wherein the pharmaceutical composition is packaged in liposomes or lipid nanoparticles.

65. A method of treating cancer in a subject in need thereof, comprising administering a therapeutically effective amount of the pharmaceutical composition of claim 63.

66. A method for treating T cell retargeting in a subject in need thereof, comprising administering a therapeutically effective amount of the pharmaceutical composition of claim 63.

67. A method of treating T cell activation in a subject in need thereof, comprising administering a therapeutically effective amount of the pharmaceutical composition of claim 63.

68. The method of any one of claims 66-67, wherein the subject has cancer.

69. The method of claim 68, wherein the cancer is a ULBP2-positive cancer.

70. The method of claim 68, wherein the cancer is a primary tumor, a metastatic cancer, a multidrug-resistant cancer, a progressive tumor, or a recurrent cancer.

71. The method of claim 68, wherein the cancer is a solid tumor.

72. The method of claim 68, wherein the cancer is bladder cancer, lung cancer, brain cancer, head and neck cancer, breast cancer, skin cancer, melanoma, liver cancer, pancreatic cancer, stomach cancer, colon cancer, rectal cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer, testicular cancer, skin cancer, or esophageal cancer.

73. The method of any one of claims 65-72, further comprising administering a therapeutically effective amount of a ligand or cytokine.

74. The method of claim 73, wherein the ligand is CD48, CD58, CD86, TNFSF9, OX40L, 4-1BBL, GITL, CD70, CD80, MR1, TNFSF4, ICOSL, ICOSLG, MICA, MICB, ULBP1, ULBP2, ULBP3, RAET1G (ULBP5) and RAET1L (ULBP6) or cytokines IL-2, IL-7, IL-10, IL-12, IL-15, IL-18 or IL-21, or agonistic antibodies that bind to receptors for these ligands and cytokines.

75. The method of any one of claims 65-74, wherein the method also prevents T cell exhaustion.

76. A method for expanding a T cell population, comprising contacting the T cell population with the pharmaceutical composition of claim 63.

77. The method of claim 76, wherein contacting of the T cell population occurs in vitro, in vivo, ex vivo, or a combination thereof.

78. The method of any one of claims 76-77, wherein under the same conditions, the expansion of the plurality of T cells is about 2 to about 50 times greater than the expansion of the plurality of T cells without the pharmaceutical composition.

79. A method of expanding a T cell population, comprising contacting the T cell population with the antibody of any one of claims 15-26.

80. The method of claim 79, wherein contacting the T cell population occurs in vitro, in vivo, ex vivo, or a combination thereof.

81. The method of any one of claims 79-80, wherein under the same conditions, the expansion of the plurality of T cells is about 2 to about 50 times greater than the expansion of the plurality of T cells using an antibody without a polypeptide fused to the N-terminus or C-terminus of the H1 and / or H2.

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