Antibodies binding to hla-a2 / wt1

Abstract

This invention generally relates to antibodies binding to HLA-A2 / WT1, including, for example, bispecific antigen-binding molecules for activating T cells. Additionally, this invention relates to polynucleotides encoding such antibodies, and to carriers and host cells containing such polynucleotides. Furthermore, this invention relates to methods for generating antibodies and methods for treating diseases using such antibodies.

Description

[Technical Field] This invention generally relates to antibodies binding to HLA-A2 / WT1, including, for example, bispecific antigen-binding molecules for activating T cells. Additionally, this invention relates to polynucleotides encoding such antibodies, and to carriers and host cells containing such polynucleotides. Furthermore, this invention relates to methods for generating antibodies and methods for treating diseases using such antibodies. [Previous Technology] WT1 (Wilms tumor 1) is an oncogenic transcription factor involved in cell proliferation, differentiation, apoptosis, and organ development, and its expression is extremely low in normal adult tissues (Hinrichs and Restifo, Nat Biotechnol (2013) 31, 999-1008). However, WT1 has been reported to be overexpressed in several types of hematologic malignancies and a wide range of solid tumors (Van Driessche et al., Oncologist (2012) 17, 250-259). WT1 is an intracellular nuclear protein. Intracellular proteins can be degraded in the proteasome, processed on the cell surface by major histocompatibility complex (MHC) I, and presented as T-cell antigenic determinants, and recognized by the T-cell receptor (TCR). Therefore, WT1-derived peptides such as WT1RMF (RMFPNAPYL) and WT1VLD (VLDFAPPGA) are presented on the cell surface in the presence of HLA-A2 and can trigger T cell recognition. Several approaches have been adopted to utilize WT1 as a target for cancer (immunotherapy), including the development of cancer vaccines based on WT1-derived peptides and donor-recipient T cell transfer or WT1-specific T cells. TCR-like antibodies against the HLA-A2 / WT1RMF complex have also been developed, including its bispecific derivatives (Dao et al., Sci Transl Med (2013) 5, 176ra33; WO 2012 / 135854; Dao et al., Nat Biotechnol (2015) 33, 1079-1086; WO 2015 / 070061; WO 2017 / 060201). Bispecific antibodies that bind to both surface antigens on target cells and activating T-cell antigens (such as CD3 on T cells) (also referred to herein as T-cell bispecific antibodies or "TCBs") hold great promise for the treatment of various cancers. The simultaneous binding of such antibodies to both of their targets forces a transient interaction between the target cell and the T cell, leading to cross-linking of the T-cell receptor and subsequent activation of any cytotoxic T cells and subsequent lysis of the target cell. Given their efficacy in killing target cells, the specificity of the target and the targeting antibody is paramount to avoiding hit-and-off-target toxicity in T-cell bispecific antibodies. Intracellular proteins such as WT1 represent attractive targets, but are only accessible by T-cell receptor (TCR)-like antibodies, which bind to the major histocompatibility complex (MHC) that presents peptide antigens derived from intracellular proteins on the cell surface. An inherent problem with TCR-like antibodies is the potential cross-reactivity with their own MHC molecules or with MHC molecules that present peptides other than the desired peptide, which may impair organ or tissue selectivity. [Summary of the Invention] This invention provides novel antibodies, including bispecific antibodies that bind to HLA-A2 / WT1 and have particularly advantageous properties for therapeutic purposes. The inventors have developed a novel antibody with unexpectedly improved properties that binds to HLA-A2 / WT1. Specifically, the antibody binds to HLA-A2 / WT1 with good affinity and excellent specificity. Furthermore, the inventors have developed a bispecific antigen-binding molecule that binds to both HLA-A2 / WT1 and activated T-cell antigens, combining good efficacy and manufacturability with low toxicity and favorable pharmacokinetic properties. In a first-state sample, the present invention provides an antibody binding to HLA-A2 / WT1, wherein the antibody comprises (i) a heavy chain variable region (VH) comprising the heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO: 1, HCDR 2 of SEQ ID NO: 2, and HCDR 3 of SEQ ID NO: 3, and a light chain variable region (VL) comprising the light chain complementarity-determining region (LCDR) 1 of SEQ ID NO: 4, LCDR 2 of SEQ ID NO: 5, and LCDR 3 of SEQ ID NO: 6; (ii) a VH comprising HCDR 1 of SEQ ID NO: 9, HCDR 2 of SEQ ID NO: 10, and HCDR 3 of SEQ ID NO: 11, and a VL comprising LCDR 1 of SEQ ID NO: 12, LCDR 2 of SEQ ID NO: 13, and LCDR 3 of SEQ ID NO: 14; and (iii) a heavy chain variable region comprising HCDR 1 of SEQ ID NO: 17, HCDR 2 of SEQ ID NO: 18, and SEQ ID NO: 6. VH of HCDR 3 (SEQ ID NO: 19) and VL of LCDR 1 (SEQ ID NO: 20), LCDR 2 (SEQ ID NO: 21) and LCDR 3 (SEQ ID NO: 22); (iv) VH of HCDR 1 (SEQ ID NO: 25), HCDR 2 (SEQ ID NO: 26) and HCDR 3 (SEQ ID NO: 27) and VL of LCDR 1 (SEQ ID NO: 28), LCDR 2 (SEQ ID NO: 29) and LCDR 3 (SEQ ID NO: 30); (v) VH of HCDR 1 (SEQ ID NO: 33), HCDR 2 (SEQ ID NO: 34) and HCDR 3 (SEQ ID NO: 35) and VL of LCDR 1 (SEQ ID NO: 36), LCDR 2 (SEQ ID NO: 37) and LCDR 3 (SEQ ID NO: 38); (vi) VH of HCDR 1 (SEQ ID NO: 41), SEQ ID NO: 20, LCDR 1 (SEQ ID NO: 20), LCDR 2 (SEQ ID NO: 21) and LCDR 3 (SEQ ID NO: 22); VH of HCDR 2 of SEQ ID NO: 42 and HCDR 3 of SEQ ID NO: 43, and VL of LCDR 1 of SEQ ID NO: 44, LCDR 2 of SEQ ID NO: 45 and LCDR 3 of SEQ ID NO: 46;(vii) A VH containing HCDR 1 (SEQ ID NO: 49), HCDR 2 (SEQ ID NO: 50), and HCDR 3 (SEQ ID NO: 51), and a VL containing LCDR 1 (SEQ ID NO: 52), LCDR 2 (SEQ ID NO: 53), and LCDR 3 (SEQ ID NO: 54); (viii) A VH containing HCDR 1 (SEQ ID NO: 57), HCDR 2 (SEQ ID NO: 58), and HCDR 3 (SEQ ID NO: 59), and a VL containing LCDR 1 (SEQ ID NO: 60), LCDR 2 (SEQ ID NO: 61), and LCDR 3 (SEQ ID NO: 62); or (ix) A VH containing HCDR 1 (SEQ ID NO: 65), HCDR 2 (SEQ ID NO: 66), and HCDR 3 (SEQ ID NO: 67), and a VL containing LCDR 1 (SEQ ID NO: 68), LCDR 2 (SEQ ID NO: 69), and HCDR 3 (SEQ ID NO: 54). NO: 70 LCDR 3 VL. In one embodiment, the antibody comprises (i) a VH comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 7, and a VL comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 8; (ii) a VH comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 15, and a VL comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 16; and (iii) a VH comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 23, and a VL comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 8. (iv) A VL containing an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 24; and a VH containing an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 31, and a VL containing an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 32; (v) A VH containing an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 39, and a VL containing an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 40; and (vi) A VL containing an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 40; VH containing an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 47, and VL containing an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 48; (vii) VH containing an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 55, and VL containing an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 56;(viii) A VH comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 63, and a VL comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 64; or (ix) A VH comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 71, and a VL comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 72. In one embodiment, the antibody is an IgG antibody, specifically an IgG1 antibody. In one embodiment, the antibody is a full-length antibody. In another embodiment, the antibody is an antibody fragment selected from the group consisting of Fv molecules, scFv molecules, Fab molecules, and F(ab')2 molecules. In one embodiment, the antibody is a multispecific antibody. The present invention also provides a bispecific antigen-binding molecule comprising (a) a first antigen-binding portion bound to a first antigen, wherein the first antigen is HLA-A2 / WT1, and the first antigen-binding portion comprising (i) a heavy chain variable region (VH) comprising the heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO: 1, HCDR 2 of SEQ ID NO: 2, and HCDR 3 of SEQ ID NO: 3, and a light chain variable region (VL) comprising the light chain complementarity-determining region (LCDR) 1 of SEQ ID NO: 4, LCDR 2 of SEQ ID NO: 5, and LCDR 3 of SEQ ID NO: 6; (ii) a VH comprising HCDR 1 of SEQ ID NO: 9, HCDR 2 of SEQ ID NO: 10, and HCDR 3 of SEQ ID NO: 11, and a VL comprising LCDR 1 of SEQ ID NO: 12, LCDR 2 of SEQ ID NO: 13, and LCDR 3 of SEQ ID NO: 14; and (iii) a heavy chain variable region comprising HCDR 1 of SEQ ID NO: 17. 1. VH containing HCDR 2 of SEQ ID NO: 18 and HCDR 3 of SEQ ID NO: 19, and VL containing LCDR 1 of SEQ ID NO: 20, LCDR 2 of SEQ ID NO: 21, and LCDR 3 of SEQ ID NO: 22; (iv) VH containing HCDR 1 of SEQ ID NO: 25, HCDR 2 of SEQ ID NO: 26, and HCDR 3 of SEQ ID NO: 27, and VL containing LCDR 1 of SEQ ID NO: 28, LCDR 2 of SEQ ID NO: 29, and LCDR 3 of SEQ ID NO: 30; (v) VH containing HCDR 1 of SEQ ID NO: 33, HCDR 2 of SEQ ID NO: 34, and HCDR 3 of SEQ ID NO: 35, and VL containing LCDR 1 of SEQ ID NO: 36, LCDR 2 of SEQ ID NO: 37, and LCDR 3 of SEQ ID NO: 38; (vi) VL containing SEQ ID NO: 18, HCDR 2 of SEQ ID NO: 19, and LCDR 3 of SEQ ID NO: 20, LCDR 2 of SEQ ID NO: 21, and LCDR 3 of SEQ ID NO: 22; VH of HCDR 1 of SEQ ID NO: 41, HCDR 2 of SEQ ID NO: 42 and HCDR 3 of SEQ ID NO: 43, and VL of LCDR 1 of SEQ ID NO: 44, LCDR 2 of SEQ ID NO: 45 and LCDR 3 of SEQ ID NO: 46;(vii) A VH containing HCDR 1 (SEQ ID NO: 49), HCDR 2 (SEQ ID NO: 50), and HCDR 3 (SEQ ID NO: 51), and a VL containing LCDR 1 (SEQ ID NO: 52), LCDR 2 (SEQ ID NO: 53), and LCDR 3 (SEQ ID NO: 54); (viii) A VH containing HCDR 1 (SEQ ID NO: 57), HCDR 2 (SEQ ID NO: 58), and HCDR 3 (SEQ ID NO: 59), and a VL containing LCDR 1 (SEQ ID NO: 60), LCDR 2 (SEQ ID NO: 61), and LCDR 3 (SEQ ID NO: 62); or (ix) A VH containing HCDR 1 (SEQ ID NO: 65), HCDR 2 (SEQ ID NO: 66), and HCDR 3 (SEQ ID NO: 67), and a VL containing LCDR 1 (SEQ ID NO: 68), LCDR 2 (SEQ ID NO: 69), and HCDR 3 (SEQ ID NO: 54). NO: 70's LCDR 3 VL; and (b) specific binding to the second antigen-binding site of the second antigen. In one embodiment, the first antigen-binding portion comprises (i) a VH comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 7, and a VL comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 8; (ii) a VH comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 15, and a VL comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 16; and (iii) a VH comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 23, and a VL comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 8. (iv) A VL containing an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 24; and a VH containing an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 31, and a VL containing an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 32; (v) A VH containing an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 39, and a VL containing an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 40; and (vi) A VL containing an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 40; VH containing an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 47, and VL containing an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 48; (vii) VH containing an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 55, and VL containing an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 56;(viii) A VH comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 63, and a VL comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 64; or (ix) A VH comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 71, and a VL comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 72. In one embodiment, the second antigen is CD3, specifically CD3ε. In one embodiment, the second antigen-binding portion comprises a VH comprising HCDR 1 of SEQ ID NO: 115, HCDR 2 of SEQ ID NO: 116, and HCDR 3 of SEQ ID NO: 117, and a VL comprising LCDR 1 of SEQ ID NO: 118, LCDR 2 of SEQ ID NO: 119, and LCDR 3 of SEQ ID NO: 120. In one embodiment, the VH of the second antigen-binding portion comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 121, and the VL of the second antigen-binding portion comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 122. In one embodiment, the first and / or second antigen-binding portion is a Fab molecule. In one embodiment, the second antigen-binding portion is a Fab molecule in which the variable domains VL and VH of the Fab light chain and the constant domains CL and CH1 (specifically, the variable domains VL and VH) are mutually substituted. In one embodiment, the first antigen-binding portion is a Fab molecule in which, in the constant domain CL, the amino acid at position 124 is independently substituted with lysine (K), arginine (R), or histidine (H) (according to Kabat numbering), and the amino acid at position 123 is independently substituted with lysine (K), arginine (R), or histidine (H) (according to Kabat numbering), and in the constant domain CH1, the amino acid at position 147 is independently substituted with glutamic acid (E) or aspartic acid (D) (according to Kabat EU index numbering), and the amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D) (according to Kabat EU index numbering). In one embodiment, the first and second antigen-binding portions are fused together, optionally via a peptide linker. In one embodiment, the first and second antigen-binding portions are each Fab molecules, and (i) the second antigen-binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding portion, or (ii) the first antigen-binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding portion. In one embodiment, the bispecific antigen-binding molecule includes a third antigen-binding portion. In one embodiment, the third antigen portion is identical to the first antigen-binding portion. In one embodiment, the bispecific antigen-binding molecule includes an Fc domain composed of first and second subunits.In one embodiment, the first, second, and (if present) third antigen-binding portions are each Fab molecules; and (i) the second antigen-binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding portion, and the first antigen-binding portion is fused at the C-terminus of the first unit of the Fc domain, or (ii) the first antigen-binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding portion, and the second antigen-binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first unit of the Fc domain; and the third antigen-binding portion (if present) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second unit of the Fc domain. In one embodiment, the Fc domain is an IgG Fc domain, specifically an IgG1 Fc domain. In one embodiment, the Fc domain is a human Fc domain. In one embodiment, amino acid residues in the CH3 domain of the first unit of the Fc domain are replaced with amino acid residues having a larger side chain volume, thereby creating a bulge within the CH3 domain of the first unit. This bulge can be located within a cavity in the CH3 domain of the second unit. Similarly, amino acid residues in the CH3 domain of the second unit of the Fc domain are replaced with amino acid residues having a smaller side chain volume, thereby creating a cavity within the CH3 domain of the second unit. The bulge within the CH3 domain of the first unit can be located within this cavity. In one embodiment, the Fc domain includes one or more amino acid substitutions that weaken binding to the Fc receptor and / or reduce effector function. According to another embodiment of the invention, one or more isolated polynucleotides encoding antibodies or bispecific antigen-binding molecules of the invention are provided. The invention further provides one or more expression vectors comprising the isolated polynucleotides of the invention, and a host cell comprising the isolated polynucleotides or expression vectors of the invention. In some embodiments, the host cell is a eukaryotic cell, specifically a mammalian cell. In another embodiment, a method for generating an antibody bound to HLA-A2 / WT1 is provided, the method comprising the steps of: a) culturing the host cells of the present invention under conditions suitable for antibody expression and b) recovering the antibody. The present invention also covers antibodies bound to HLA-A2 / WT1 generated by the method of the present invention. The present invention further provides a pharmaceutical composition comprising the antibody or bispecific antigen-binding molecule of the present invention and a pharmaceutically acceptable carrier. This invention also covers methods of using the antibodies, bispecific antigen-binding molecules, and pharmaceutical compositions of this invention. In one embodiment, the invention provides an antibody, bispecific antigen-binding molecule, or pharmaceutical composition according to the invention for use as a pharmaceutical agent. In another embodiment, the invention provides an antibody, bispecific antigen-binding molecule, or pharmaceutical composition according to the invention for treating a disease. In one specific embodiment, the disease is cancer. Also provided are uses of the antibody or bispecific antigen-binding molecule according to the invention for manufacturing medicaments for treating diseases; and a method of treating a disease in an individual comprising administering to the individual a therapeutically effective amount of a composition comprising the antibody or bispecific antigen-binding molecule according to the invention in a pharmaceutically acceptable form. In one specific embodiment, the disease is cancer. In any of the above embodiments, the individual is preferably a mammal, specifically a human. [Simplified Explanation of the Diagram] Figure 1. Illustrative configurations of the bispecific antigen-binding molecules of the present invention. (A, D) Schematic illustration of a "1+1 crossMab" molecule. (B, E) Schematic illustration of a "2+1 IgG crossMab" molecule with an alternative crossMab and Fab component order ("reverse"). (C, F) Schematic illustration of a "2+1 IgG crossMab" molecule. (G, K) Schematic illustration of a "1+1 IgG crossMab" molecule with an alternative crossMab and Fab component order ("reverse"). (H, L) Schematic illustration of a "1+1 IgG crossMab" molecule. (I, M) Schematic illustration of a "2+1 IgG crossMab" molecule with two crossMabs. (J, N) Schematic illustration of a "2+1 IgG crossMab" molecule with two crossMabs and an alternative crossMab and Fab component order ("reverse"). (O, S) Schematic illustration of a "Fab-crossMab" molecule. (P, T) Diagram of the "interchangeable Fab-Fab" molecule. (Q, U) Diagram of the "(Fab)2-interchangeable Fab" molecule. (R, V) Diagram of the "interchangeable Fab-(Fab)2" molecule. (W, Y) Diagram of the "Fab-(interchangeable Fab)2" molecule. (X, Z) Diagram of the "(interchangeable Fab)2-Fab" molecule. Black dots: Modifications in the Fc domain that promote heterodimerization, as appropriate. ++, --: Introducing amino acids with opposite charges in the CH1 and CL domains, as appropriate. The interchangeable Fab molecule is depicted as including the exchange of VH and VL regions, but in embodiments where no charge modification is introduced in the CH1 and CL domains, the exchange of CH1 and CL domains may alternatively be included. Figure 2. Schematic illustration of the T-cell bispecific (TCB) antibody molecules prepared in the example. All TCB antibody molecules tested were generated in the form of a charge-modified "reverse 2+1 IgG exchange Fab" (VH / VL exchange in the CD3 conjugate and charge modification in the WT1 conjugate, EE=147E, 213E; RK=123R, 124K). Figure 3. Binding of HLA-A2 / WT1 IgG antibody to T2 cells pulsed with peptide. (A) 1ID06 IgG, (B) 33H09-IgG, (C) 11B09-IgG, (D) 13B04-IgG, (E) 5E11-IgG, (F) 5C01-IgG, (G) 11G06-IgG. Figure 4. Activation of T cells by HLA-A2 / WT1 x CD3 bispecific antibody (TCB) after binding to T2 cells subjected to peptide pulse shock (NFAT reporter assay). (A) 11D06-TCB, (B) 33H09-TCB, (C) 11B09-TCB, (D) 13B04-TCB, (E) ESK1-TCB, (F) 5E11-TCB, (G) 5C01-TCB, (H) DP47GS-TCB. Figure 5. Killing of T2 cells induced by peptide pulse shock mediated by HLA-A2 / WT1 x CD3 bispecific antibody (TCB). (A) 11D06-TCB, (B) 33H09-TCB, (C) 13B04-TCB, (D) 11B09-TCB, (E) 33F05-TCB, (F) 5C01-TCB. Figure 6. Killing of HLA-A2+WT1+ tumor cell lines mediated by HLA-A2 / WT1 x CD3 bispecific antibody (TCB). (A) Overview of cell lines. (BE) Killing of cell lines by (B) 11D06-TCB, (C) 33H09-TCB, (D) 11B09-TCB, (E) 13B04-TCB. (F) Killing of SKM-1 cells by different TCBs. (G) Killing of BJAB cells by different TCBs. Figure 7. Activation of T cells by the HLA-A2 / WT1 x CD3 bispecific antibody (TCB) after binding to the HLA-A2+WT1+ tumor cell line. (A) SKM-1 cells, (B) BJAB cells. Figure 8. Selected HLA-A2 / WT1 x CD3 bispecific antibodies (TCBs) do not bind to off-target peptides. (AC) Binding of (A) 11D06-TCB, (B) 33H09-TCB, (C) ESK1-TCB to peptide-pulsed T2 cells. (DE) Activation of T cells by (D) 11D06-TCB and (E) 33H09-TCB after binding to peptide-pulsed T2 cells. (F) Overview of peptides. Figure 9. Selected HLA-A2 / WT1 x CD3 bispecific antibodies (TCBs) do not bind to additional off-target peptides. (AB) Activation of T cells after binding to peptide-pulsed T2 cells by (A) 11D06-TCB and (B) 33H09-TCB. Testing of 6 indicated off-target peptides and RMF peptides. (CG) Killing of peptide-pulsed T2 cells by (C, E) 11D06-TCB, (D, F) 33H09-TCB and (G) ESK1-TCB. Testing of 19 indicated off-target peptides, as well as RMF and VLD peptides. Figure 10. Mediation of normal bone marrow-derived CD34+ stem cells without killing by selected HLA-A2 / WT1 x CD3 bispecific antibody (TCB). (A) 11D06-TCB, (B) 33H09-TCB. Figure 11. Identification of selected HLA-A2 / WT1 x CD3 bispecific antibodies (TCBs) by alanine scanning of binding residues in RMF peptides. (A) Native RMF peptide, (B) RMF R1Y peptide, (C) RMF R1A peptide, (D) RMF M2A peptide, (E) RMF F3A peptide, (F) RMF P4A peptide, (G) RMF N5A peptide, (H) RMF A6G peptide, (I) RMF P7A peptide, (J) RMF Y8A peptide, (K) RMF L9A peptide. (L) Overview of peptides. (M) FDIC of EC50 relative to the native RMF peptide. (N) Key contact residues. Figure 12. Pharmacokinetic profile of HLA-A2 / WT1 x CD3 bispecific antibodies (11D06-TCB and 33H09-TCB) after a single injection in NSG mice. Figure 13. Efficacy study of HLA-A2 / WT1 x CD3 bispecific antibody ("TCB") in SKM-1 xenografts in humanized mice. (A) Study design. (B) Treatment groups. (C) Tumor growth kinetics (mean) in all treatment groups. (D) Individual tumor growth kinetics in the mediator group. (E) Individual tumor growth kinetics in the 11D06-TCB group. (F) Individual tumor growth kinetics in the 33H09-TCB group. (G) Statistics. Calculations were performed based on day 38 (mediator as control). Tumor growth inhibition (TGI): TGI>100 → tumor regression, TGI=100 → tumor arrest. Treatment-to-control ratio (TCR): TCR=1 → no effect, TCR=0 → complete regression. Figure 14. Overview of the crystal structure of the HLA-A2 / WT1 antibody-pMHC complex. The antibody (Fab fragment) is shown at the top, with the heavy chain in dark gray and the light chain in light gray. Solvent atoms are not shown. (A) 1.98 Å resolution crystal structure of the 5C01 Fab complex with HLA-A02 / VLD pMHC. Fab-pMHC contact area: 476 Ų, peptide contribution: 68 Ų. (B) 2.60 Å resolution crystal structure of the 11D06 Fab complex with HLA-A02 / RMF pMHC. Fab-pMHC contact area: 397 Ų, peptide contribution: 107 Ų. (C) 3.05 Å resolution crystal structure of the ESK1 Fab complex with HLA-A02 / RMF pMHC (published, PDB ID 4WUU). Fab-pMHC contact area: 505 Ų, peptide contribution: 60 Ų. Figure 15. Close-up view of the pMHC binding interface of 5C01 Fab-HLA-A2 / WT1VLD. The major chemical interactions between Fab and pMHC identified using BIOVIA Discovery Studio 4.5 are highlighted. Solvent atoms are not shown. Figure 16. Interface and interaction matrix between 5C01 Fab residues (columns) and HLA-A2 / WT1VLD pMHC residues (rows). N = proximity / nearby, H = H bond, Pi = π interaction, SB = salt bridge. Interface residues are defined as residues that undergo changes in the solvent-accessible surface region in the absence / in the presence of interacting complexes. Figure 17. Close-up view of the pMHC binding interface of 11D06 Fab-HLA-A2 / WT1RMF. The major chemical interactions between Fab and pMHC identified using BIOVIA Discovery Studio 4.5 are highlighted. Solvent atoms are not shown. Figure 18. Interface and interaction matrix between 11D06 Fab residues (columns) and HLA-A2 / WT1RMF pMHC residues (rows). N = proximity, H = H bond, Pi = π interaction, SB = salt bridge. Interface residues are defined as residues that undergo changes in the solvent-accessible surface region in the absence or presence of interacting complexes. Figure 19. Close-up view of the ESK1 Fab-HLA-A2 / WT1RMF pMHC binding interface (PDB ID 4WUU). The major chemical interactions between Fab and pMHC identified using BIOVIA Discovery Studio 4.5 are highlighted. Solvent atoms are not shown. Figure 20. Interface and interaction matrix between ESK1 Fab residues (columns) and HLA-A2 / WT1RMF pMHC residues (rows). N = proximity, H = H bond, Pi = π interaction, SB = salt bridge. Interface residues are defined as residues that undergo changes in the solvent-accessible surface region in the absence or presence of interacting complexes. Figure 21. Killing of HLA-A2+ / WT1+ SKM-1 cells mediated by HLA-A2 / WT1xCD3 bispecific antibodies with different CD3 binding molecules. Figure 22. Killing of T2 cells induced by RMF peptide pulse shock mediated by HLA-A2 / WT1 x CD3 bispecific antibody (TCB). Figure 23. Evaluation of the binding of HLA-A2 / WT1 x CD3 bispecific antibodies (TCBs) Aali-TCB (A), Daniel-TCB (B), ESK1-TCB (C), and 11D06-TCB (D) to off-target peptides. Figure 24. Pharmacokinetic profile of the HLA-A2 / WT1 x CD3 bispecific antibody 11D06-TCB(V9) after a single injection in NSG mice. Figure 25. Efficacy study of the HLA-A2 / WT1 x CD3 bispecific antibody 11D06-TCB(V9) in humanized mice using SKM-1 xenografts. (A) Tumor growth kinetics (mean) in all treatment groups. (B) Individual tumor growth kinetics in the mediator group. (C) Individual tumor growth kinetics in the 11D06-TCB(V9) group. (D) Statistics. Calculations were performed based on day 48 (mediator as control). Tumor growth inhibition (TGI): TGI>100 → tumor regression, TGI=100 → tumor arrest. Treatment-to-control ratio (TCR): TCR=1 → no effect, TCR=0 → complete regression. Figure 26. Activation of T cells by HLA-A2 / WT1 x CD3 bispecific antibody (TCB) after binding to CHO-K1 cells expressing the HLA-A02 / WT1RMF pMHC complex (NFAT reporter assay). Solid line: 11D06-TCB(V9) ("2+1" form). Dashed line: Similar molecules (11D06 and V9 binders) in "1+1 interchangeable monoclonal antibody" form.

Implementation Method

Claims

1. An antibody that binds to HLA-A2 / WT1, wherein the antibody comprises: a heavy chain variable region (VH) comprising the heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO: 1, the HCDR 2 of SEQ ID NO: 2, and the HCDR 3 of SEQ ID NO: 3, and a light chain variable region (VL) comprising the light chain complementarity-determining region (LCDR) 1 of SEQ ID NO: 4, the LCDR 2 of SEQ ID NO: 5, and the LCDR 3 of SEQ ID NO:

6.

2. The antibody of claim 1, wherein the antibody comprises: a VH comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 7, and a VL comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:

8.

3. The antibody as requested in item 1 or 2, wherein the antibody is an IgG antibody.

4. The antibody as requested in item 3, wherein the antibody is an IgG1 antibody.

5. The antibody as requested in item 1 or 2, wherein the antibody is a full-length antibody.

6. The antibody as requested in claim 1 or 2, wherein the antibody is an antibody fragment selected from the group consisting of Ev molecules, scFv molecules, Fab molecules and F(ab')2 molecules.

7. The antibody as requested in item 1 or 2, wherein the antibody is a multispecific antibody.

8. A bispecific antigen-binding molecule comprising: (a) a first antigen-binding portion bound to a first antigen, wherein the first antigen is HLA-A2 / WT1, and the first antigen-binding portion comprising: a heavy chain variable region (VH) comprising the heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO: 1, the HCDR 2 of SEQ ID NO: 2, and the HCDR 3 of SEQ ID NO: 3, and a light chain variable region (VL) comprising the light chain complementarity-determining region (LCDR) 1 of SEQ ID NO: 4, the LCDR 2 of SEQ ID NO: 5, and the LCDR 3 of SEQ ID NO: 6; and (b) a second antigen-binding portion specifically bound to a second antigen.

9. The bispecific antigen-binding molecule of claim 8, wherein the first antigen-binding portion comprises: a VH comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 7, and a VL comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:

8.

10. A bispecific antigen-binding molecule as claimed in claim 8 or 9, wherein the second antigen is CD3.

11. The bispecific antigen-binding molecule of claim 10, wherein the second antigen is CD3ε.

12. The bispecific antigen-binding molecule of claim 10, wherein the second antigen-binding portion comprises: (i) a VH comprising HCDR 1 of SEQ ID NO: 115, HCDR 2 of SEQ ID NO: 116, and HCDR 3 of SEQ ID NO: 117, and a VL comprising LCDR 1 of SEQ ID NO: 118, LCDR 2 of SEQ ID NO: 119, and LCDR 3 of SEQ ID NO: 120; or (ii) a VH comprising HCDR 1 of SEQ ID NO: 130, HCDR 2 of SEQ ID NO: 131, and HCDR 3 of SEQ ID NO: 132, and a VL comprising LCDR 1 of SEQ ID NO: 133, LCDR 2 of SEQ ID NO: 134, and LCDR 3 of SEQ ID NO:

135.

13. The bispecific antigen-binding molecule of claim 12, wherein the second antigen-binding portion comprises: (i) a VH comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 121, and a VL comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 122; or (ii) a VH comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 136, and a VL comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:

137.

14. The bispecific antigen-binding molecule of claim 8 or 9, wherein the first and / or the second antigen-binding portion is a Fab molecule.

15. A bispecific antigen-binding molecule as claimed in claim 8 or 9, wherein the second antigen-binding portion is a Fab molecule in which the variable domains VL and VH or the constant domains CL and CH1 of the Fab light chain and the Fab heavy chain are mutually substituted.

16. The bispecific antigen-binding molecule of claim 15, wherein the Fab light chain is interchanged with the variable domains VL and VH of the Fab heavy chain.

17. A bispecific antigen-binding molecule as claimed in claim 8 or 9, wherein the first antigen-binding portion is a Fab molecule in which, in the constant domain CL, the amino acid at position 124 is independently substituted with lysine (K), arginine (R), or histidine (H) (according to Kabat number), and the amino acid at position 123 is independently substituted with lysine (K), arginine (R), or histidine (H) (according to Kabat number), and in the constant domain CH1, the amino acid at position 147 is independently substituted with glutamic acid (E) or aspartic acid (D) (according to Kabat EU index number), and the amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D) (according to Kabat EU index number).

18. The bispecific antigen-binding molecule of claim 8 or 9, wherein the first and second antigen-binding portions are fused together.

19. The bispecific antigen-binding molecule of claim 18, wherein the first and second antigen-binding portions are fused via a peptide linker.

20. A bispecific antigen-binding molecule as claimed in claim 8 or 9, wherein the first and second antigen-binding portions are each Fab molecules, and wherein (i) the second antigen-binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding portion, or (ii) the first antigen-binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding portion.

21. The bispecific antigen-binding molecule of claim 8 or 9, comprising an Fc domain consisting of a first and a second unit.

22. The bispecific antigen-binding molecule of claim 21, comprising a third antigen-binding portion bound to the first antigen.

23. The bispecific antigen-binding molecule of claim 22, wherein the third antigen-binding portion is identical to the first antigen-binding portion.

24. The bispecific antigen-binding molecule of claim 22, wherein the first antigen-binding portion, the second antigen-binding portion, and the third antigen-binding portion are each Fab molecules; and wherein (i) the second antigen-binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding portion, and the first antigen-binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first unit of the Fc domain, or (ii) the first antigen-binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding portion, and the second antigen-binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first unit of the Fc domain; and wherein the third antigen-binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second unit of the Fc domain.

25. The bispecific antigen-binding molecule of claim 21, wherein the Fc domain is an IgG Fc domain.

26. The bispecific antigen-binding molecule of claim 25, wherein the Fc domain is the IgG1 Fc domain.

27. The bispecific antigen-binding molecule of claim 21, wherein the Fc domain is a human Fc domain.

28. The bispecific antigen-binding molecule of claim 21, wherein the Fc domain is the human IgG1 Fc domain.

29. The bispecific antigen-binding molecule of claim 21, wherein an amino acid residue in the CH3 domain of the first unit of the Fc domain is replaced by an amino acid residue having a larger side chain volume, thereby creating a bulge in the CH3 domain of the first unit, the bulge being localized in a cavity in the CH3 domain of the second unit, and an amino acid residue in the CH3 domain of the second unit of the Fc domain is replaced by an amino acid residue having a smaller side chain volume, thereby creating a cavity in the CH3 domain of the second unit, the bulge in the CH3 domain of the first unit being localized in the cavity.

30. The bispecific antigen-binding molecule of claim 21, wherein in the CH3 domain of the first unit of the Fc domain, the threonine residue at position 366 is replaced by a tryptophan residue (T366W), and in the CH3 domain of the second unit of the Fc domain, the tyrosine residue at position 407 is replaced by a valine residue (Y407V) (according to Kabat EU index number).

31. The bispecific antigen-binding molecule of claim 30, wherein in the second unit of the Fc domain, the threonine residue at position 366 is replaced by a serine residue (T366S) and the leucine residue at position 368 is replaced by an alanine residue (L368A) (according to Kabat EU index number).

32. The bispecific antigen-binding molecule of claim 30, wherein in the first unit of the Fc domain, the serine residue at position 354 is additionally replaced by a cysteine ​​residue (S354C), or the glutamic acid residue at position 356 is additionally replaced by a cysteine ​​residue (E356C), and in the second unit of the Fc domain, the tyrosine residue at position 349 is additionally replaced by a cysteine ​​residue (Y349C) (according to Kabat EU index number).

33. The bispecific antigen-binding molecule of claim 31, wherein in the first unit of the Fc domain, the serine residue at position 354 is additionally replaced by a cysteine ​​residue (S354C), or the glutamic acid residue at position 356 is additionally replaced by a cysteine ​​residue (E356C), and in the second unit of the Fc domain, the tyrosine residue at position 349 is additionally replaced by a cysteine ​​residue (Y349C) (according to Kabat EU index number).

34. The bispecific antigen-binding molecule of claim 21, wherein the Fc domain comprises one or more amino acid substitutions that weaken binding to the Fc receptor and / or reduce effector function.

35. The bispecific antigen-binding molecule of claim 34, wherein the one or more amino acid substitutions are selected from the group consisting of: E233, L234, L235, N297, P331 and P329 (according to the Kabat EU index number).

36. The bispecific antigen-binding molecule of claim 21, wherein each subunit of the Fc domain comprises L234A, L235A and P329G amino acid substitutions (according to Kabat EU index number).

37. The bispecific antigen-binding molecule of claim 8, comprising: (a) first and third antigen-binding portions bound to a first antigen, wherein the first antigen is HLA-A2 / WT1, and wherein each of the first and third antigen-binding portions is a (conventional) Fab molecule comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 7 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 8; (b) a second antigen-binding portion bound to a second antigen, wherein the second antigen is CD3, and wherein the second antigen-binding portion is a Fab molecule in which the variable domains VL and VH of the Fab light chain and the Fab heavy chain are interchanged, comprising a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 136 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 137; (c) human IgG1 composed of the first and second units. Fc domain; wherein (i) in the constant domain of the first and third antigen-binding portions of (a), the amino acid at position 124 is substituted with lysine (K) (according to Kabat number), and the amino acid at position 123 is substituted with arginine (R) (according to Kabat number), and wherein in the constant domain CH1 of the first and third antigen-binding portions, the amino acid at position 147 is substituted with glutamic acid (E) (according to Kabat EU index number), and the amino acid at position 213 is substituted with glutamic acid (E) (according to Kabat number). (EU index number); (ii) the first antigen-binding portion of item (a) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding portion of item (b), and the second antigen-binding portion of item (b) and the third antigen-binding portion of item (a) are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain of item (c); and (iii) the first unit of the Fc domain contains S354C and T3366W amino acid substitutions, and the second unit of the Fc domain contains Y349C, T366S, L368A and Y407V amino acid substitutions, and each subunit of the Fc domain further contains L234A, L235A and P329G amino acid substitutions (according to Kabat EU index number).

38. The bispecific antigen-binding molecule of claim 8 or 37, comprising a polypeptide comprising an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 123, a polypeptide comprising an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 125, a polypeptide comprising an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 139, and a polypeptide comprising an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:

140.

39. One or more isolated polynucleotides encoding an antibody as claimed in any one of claims 1 to 7 or a bispecific antigen-binding molecule as claimed in any one of claims 8 to 38.

40. One or more carriers comprising the polynucleotide as claimed in claim 39.

41. A host cell comprising a polynucleotide as claimed in claim 39 or a vector as claimed in claim 40.

42. A method for producing an antibody that binds to HLA-A2 / WT1, comprising the steps of: a) culturing a host cell as claimed in claim 41 under conditions suitable for expressing the antibody.

43. The method of claim 42 further includes step b) recovering the antibody.

44. An antibody that binds to HLA-A2 / WT1, which is produced by the method of claim 42 or 43.

45. A pharmaceutical composition comprising an antibody as claimed in any one of claims 1 to 7 and 44, or a bispecific antigen-binding molecule as claimed in any one of claims 8 to 38, and a pharmaceutically acceptable carrier.

46. ​​Use of an antibody as claimed in any one of claims 1 to 7 or a bispecific antigen-binding molecule as claimed in any one of claims 8 to 38, for use in the manufacture of a medicament for treating a disease, wherein the disease is cancer.

47. As used in claim 46, wherein the disease is leukemia.

48. For the purposes of claim 47, wherein the disease is acute lymphoblastic leukemia (ALL) or acute myeloid leukemia (AML).

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