Anti-WT1 / HLA antibodies and their uses

Abstract

The present disclosure relates to antibodies or antigen-binding fragments thereof that bind to a complex containing a WT1 peptide and an MHC molecule, as well as related antibody-drug conjugates (ADCs) derived therefrom, chimeric antigen receptors (CARs) derived therefrom, and methods of using them.

Description

[Technical Field]

[0001] Priority claims This application claims priority to PCT / CN2022 / 128430, filed October 28, 2022. The entire contents of the above-mentioned application are incorporated herein by reference.

[0002] The present disclosure relates to antibodies or antigen-binding fragments thereof that bind to a complex containing a WT1 peptide and an MHC molecule, as well as related antibody-drug conjugates (ADCs) derived therefrom, chimeric antigen receptors (CARs) derived therefrom, and methods of using them. [Background technology]

[0003] WT1 (Wilms tumor 1, Wilms tumor protein) is an oncogenic transcription factor involved in cell proliferation, differentiation, apoptosis, and organ development. It is rarely expressed in normal adult tissues. However, WT1 has been reported to be overexpressed in several types of hematological malignancies and a wide range of solid tumors. WT1 is a nuclear protein localized intracellularly. The intracellular protein is degraded in the proteasome and processed and presented on the cell surface as a T cell epitope by major histocompatibility complex (MHC) I, which is recognized by the T cell receptor (TCR). Therefore, WT1-derived peptides can be presented on the cell surface in the context of human leukocyte antigens (HLA), specifically HLA-A2, and can trigger T cell recognition.

[0004] Given the important role of WT1 in tumors, there is a need to develop therapeutic agents that target WT1. Summary of the Invention

[0005] The present disclosure relates to anti-WT1 / HLA antibodies, antigen-binding fragments thereof, antibody-drug conjugates (ADCs) derived therefrom, chimeric antigen receptors (CARs) derived therefrom, and uses thereof.

[0006] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof that binds to a complex comprising a WT1 peptide and an MHC molecule, a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH CDR1 region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of a selected VH CDR1, the VH CDR2 region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of a selected VH CDR2, and the VH CDR3 region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of a selected VH CDR3; a light chain variable region (VL) comprising CDR1, 2, and 3, wherein the VL CDR1 region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of a selected VL CDR1, the VL CDR2 region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of a selected VL CDR2, and the VL CDR3 region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of a selected VL CDR3; The amino acid sequences of the selected VH CDR1, 2, and 3 and the amino acid sequences of the selected VL CDR1, 2, and 3 are one of the following: The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 55 to 57, respectively; The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 4 to 6, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 58 to 60, respectively. The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 7 to 9, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 61 to 63, respectively. The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 10 to 12, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 64 to 66, respectively. The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 13 to 15, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 67 to 69, respectively. The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 16 to 18, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 70 to 72, respectively. The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 19 to 21, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 73 to 75, respectively. The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 22 to 24, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 76 to 78, respectively. The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 25 to 27, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 79 to 81, respectively. The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 28 to 30, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 55 to 57, respectively. The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 31 to 33, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 58 to 60, respectively. The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 34 to 36, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 61 to 63, respectively. The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 37 to 39, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 64 to 66, respectively. The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 40 to 42, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 67 to 69, respectively. The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 43 to 45, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 70 to 72, respectively. The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 46 to 48, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 73 to 75, respectively. The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 49 to 51, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 76 to 78, respectively. The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 52 to 54, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 79 to 81, respectively. The present invention relates to an antibody or an antigen-binding fragment thereof.

[0007] In some embodiments, according to the Kabat definition, VH comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 1 to 3, respectively, and VL comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 55 to 57, respectively.

[0008] In some embodiments, according to the Kabat definition, VH comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 4 to 6, respectively, and VL comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 58 to 60, respectively.

[0009] In some embodiments, according to the Kabat definition, VH comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 7 to 9, respectively, and VL comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 61 to 63, respectively.

[0010] In some embodiments, according to the Kabat definition, VH comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 10 to 12, respectively, and VL comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 64 to 66, respectively.

[0011] In some embodiments, according to the Kabat definition, VH comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 13 to 15, respectively, and VL comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 67 to 69, respectively.

[0012] In some embodiments, according to the Kabat definition, VH comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 16 to 18, respectively, and VL comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 70 to 72, respectively.

[0013] In some embodiments, according to the Kabat definition, VH comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 19 to 21, respectively, and VL comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 73 to 75, respectively.

[0014] In some embodiments, according to the Kabat definition, VH comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 22 to 24, respectively, and VL comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 76 to 78, respectively.

[0015] In some embodiments, according to the Kabat definition, VH comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 25 to 27, respectively, and VL comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 79 to 81, respectively.

[0016] In some embodiments, according to the definition of Chothia, VH comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 28 to 30, respectively, and VL comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 55 to 57, respectively.

[0017] In some embodiments, according to the Chothia definition, VH comprises CDR1, 2, and 3 having the amino acid sequences set forth in SEQ ID NOs: 31 to 33, respectively, and VL comprises CDR1, 2, and 3 having the amino acid sequences set forth in SEQ ID NOs: 58 to 60, respectively.

[0018] In some embodiments, according to the Chothia definition, VH comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 34 to 36, respectively, and VL comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 61 to 63, respectively.

[0019] In some embodiments, according to the Chothia definition, VH comprises CDR1, 2, and 3 having the amino acid sequences set forth in SEQ ID NOs: 37 to 39, respectively, and VL comprises CDR1, 2, and 3 having the amino acid sequences set forth in SEQ ID NOs: 64 to 66, respectively.

[0020] In some embodiments, according to the definition of Chothia, VH comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 40 to 42, respectively, and VL comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 67 to 69, respectively.

[0021] In some embodiments, according to the Chothia definition, VH comprises CDR1, 2, and 3 having the amino acid sequences set forth in SEQ ID NOs: 43 to 45, respectively, and VL comprises CDR1, 2, and 3 having the amino acid sequences set forth in SEQ ID NOs: 70 to 72, respectively.

[0022] In some embodiments, according to the Chothia definition, VH comprises CDR1, 2, and 3 having the amino acid sequences set forth in SEQ ID NOs: 46 to 48, respectively, and VL comprises CDR1, 2, and 3 having the amino acid sequences set forth in SEQ ID NOs: 73 to 75, respectively.

[0023] In some embodiments, according to the definition of Chothia, VH comprises CDR1, 2, and 3 having the amino acid sequences set forth in SEQ ID NOs: 49 to 51, respectively, and VL comprises CDR1, 2, and 3 having the amino acid sequences set forth in SEQ ID NOs: 76 to 78, respectively.

[0024] In some embodiments, according to the definition of Chothia, VH comprises CDR1, 2, and 3 having the amino acid sequences set forth in SEQ ID NOs: 52 to 54, respectively, and VL comprises CDR1, 2, and 3 having the amino acid sequences set forth in SEQ ID NOs: 79 to 81, respectively.

[0025] In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to a complex comprising a human WT1 peptide and an MHC molecule.

[0026] In some embodiments, the WT1 peptide comprises a sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence of SEQ ID NO:100.

[0027] In some embodiments, the MHC is HLA (eg, HLA-A2).

[0028] In some embodiments, the antibody or antigen-binding fragment thereof is a human antibody or humanized antibody or antigen-binding fragment thereof, a single-chain variable fragment (scFv), a single-arm antibody, and / or a multispecific antibody (e.g., a bispecific antibody).

[0029] In some embodiments, the antibody or antigen-binding fragment thereof is a human IgG1 antibody or antigen-binding fragment thereof, a human IgG2 antibody or antigen-binding fragment thereof, or a human IgG4 antibody or antigen-binding fragment thereof.

[0030] In one aspect, the disclosure provides a nucleic acid comprising a polynucleotide encoding a polypeptide, an immunoglobulin heavy chain or a fragment thereof, comprising a heavy chain variable region (VH) including complementarity determining regions (CDRs) 1, 2, and 3 each containing the amino acid sequences set forth in SEQ ID NOs: 1 to 3, wherein the VH binds to a complex containing a WT1 peptide and an MHC molecule when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 91; an immunoglobulin light chain or a fragment thereof comprising a VL comprising complementarity-determining regions (CDRs) 1, 2, and 3 each comprising the amino acid sequences set forth in SEQ ID NOs: 55 to 57, wherein the VL binds to a complex comprising a WT1 peptide and an MHC molecule when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 82; an immunoglobulin heavy chain or a fragment thereof, comprising a heavy chain variable region (VH) including complementarity determining regions (CDRs) 1, 2, and 3 each containing the amino acid sequences set forth in SEQ ID NOs: 4 to 6, wherein the VH binds to a complex containing a WT1 peptide and an MHC molecule when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 92; an immunoglobulin light chain or a fragment thereof comprising a VL comprising complementarity-determining regions (CDRs) 1, 2, and 3 each comprising the amino acid sequences set forth in SEQ ID NOs: 58 to 60, wherein the VL binds to a complex comprising a WT1 peptide and an MHC molecule when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 83; an immunoglobulin heavy chain or a fragment thereof, comprising a heavy chain variable region (VH) including complementarity determining regions (CDRs) 1, 2, and 3 each containing the amino acid sequences set forth in SEQ ID NOs: 7 to 9, wherein the VH binds to a complex containing a WT1 peptide and an MHC molecule when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 93; an immunoglobulin light chain or a fragment thereof comprising a VL comprising complementarity-determining regions (CDRs) 1, 2, and 3 each comprising the amino acid sequences set forth in SEQ ID NOs: 61 to 63, wherein the VL binds to a complex comprising a WT1 peptide and an MHC molecule when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 84; an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising complementarity-determining regions (CDRs) 1, 2, and 3 each comprising the amino acid sequence set forth in SEQ ID NOs: 10 to 12, wherein the VH binds to a complex comprising a WT1 peptide and an MHC molecule when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 94; an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDR1, 2, and 3 each comprising the amino acid sequence set forth in SEQ ID NO: 64 to 66, wherein the VL binds to a complex comprising a WT1 peptide and an MHC molecule when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 85; an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising complementarity-determining regions (CDRs) 1, 2, and 3 each comprising the amino acid sequence set forth in SEQ ID NOs: 13 to 15, wherein the VH binds to a complex comprising a WT1 peptide and an MHC molecule when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 95; an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDR1, 2, and 3 each comprising the amino acid sequences set forth in SEQ ID NOs: 67 to 69, wherein the VL binds to a complex comprising a WT1 peptide and an MHC molecule when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 86; an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising complementarity-determining regions (CDRs) 1, 2, and 3 each comprising the amino acid sequence set forth in SEQ ID NOs: 16 to 18, wherein the VH binds to a complex comprising a WT1 peptide and an MHC molecule when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 96; an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDR1, 2, and 3 each comprising the amino acid sequence set forth in SEQ ID NO: 70 to 72, wherein the VL binds to a complex comprising a WT1 peptide and an MHC molecule when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 87; an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising complementarity-determining regions (CDRs) 1, 2, and 3 each comprising the amino acid sequence set forth in SEQ ID NOs: 19 to 21, wherein the VH binds to a complex comprising a WT1 peptide and an MHC molecule when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 97; an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDR1, 2, and 3 each comprising the amino acid sequence set forth in SEQ ID NO: 73 to 75, wherein the VL binds to a complex comprising a WT1 peptide and an MHC molecule when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 88; an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising complementarity-determining regions (CDRs) 1, 2, and 3 each comprising the amino acid sequence set forth in SEQ ID NOs: 22 to 24, wherein the VH binds to a complex comprising a WT1 peptide and an MHC molecule when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 98; an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDR1, 2, and 3 each comprising the amino acid sequence set forth in SEQ ID NO: 76 to 78, wherein the VL binds to a complex comprising a WT1 peptide and an MHC molecule when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 89; an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising complementarity-determining regions (CDRs) 1, 2, and 3 each comprising the amino acid sequence set forth in SEQ ID NOs: 25 to 27, wherein the VH binds to a complex comprising a WT1 peptide and an MHC molecule when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 99; an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDR1, 2, and 3 each comprising the amino acid sequence set forth in SEQ ID NO: 79 to 81, wherein the VL binds to a complex comprising a WT1 peptide and an MHC molecule when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 90; an immunoglobulin heavy chain or a fragment thereof, comprising a heavy chain variable region (VH) including complementarity determining regions (CDRs) 1, 2, and 3 each comprising the amino acid sequences set forth in SEQ ID NOs: 28 to 30, wherein the VH binds to a complex comprising a WT1 peptide and an MHC molecule when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 91; an immunoglobulin light chain or a fragment thereof comprising a VL comprising complementarity-determining regions (CDRs) 1, 2, and 3 each comprising the amino acid sequences set forth in SEQ ID NOs: 55 to 57, wherein the VL binds to a complex comprising a WT1 peptide and an MHC molecule when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 82; an immunoglobulin heavy chain or a fragment thereof, comprising a heavy chain variable region (VH) including complementarity determining regions (CDRs) 1, 2, and 3 each comprising the amino acid sequences set forth in SEQ ID NOs: 31 to 33, wherein the VH binds to a complex comprising a WT1 peptide and an MHC molecule when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 92; an immunoglobulin light chain or a fragment thereof comprising a VL comprising complementarity-determining regions (CDRs) 1, 2, and 3 each comprising the amino acid sequences set forth in SEQ ID NOs: 58 to 60, wherein the VL binds to a complex comprising a WT1 peptide and an MHC molecule when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 83; an immunoglobulin heavy chain or a fragment thereof, comprising a heavy chain variable region (VH) including complementarity determining regions (CDRs) 1, 2, and 3 each containing the amino acid sequences set forth in SEQ ID NOs: 34 to 36, wherein the VH binds to a complex containing a WT1 peptide and an MHC molecule when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 93; an immunoglobulin light chain or a fragment thereof comprising a VL comprising complementarity-determining regions (CDRs) 1, 2, and 3 each comprising the amino acid sequences set forth in SEQ ID NOs: 61 to 63, wherein the VL binds to a complex comprising a WT1 peptide and an MHC molecule when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 84; an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising complementarity-determining regions (CDRs) 1, 2, and 3 each comprising the amino acid sequence set forth in SEQ ID NOs: 37 to 39, wherein the VH binds to a complex comprising a WT1 peptide and an MHC molecule when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 94; an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDR1, 2, and 3 each comprising the amino acid sequence set forth in SEQ ID NO: 64 to 66, wherein the VL binds to a complex comprising a WT1 peptide and an MHC molecule when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 85; an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising complementarity-determining regions (CDRs) 1, 2, and 3 each comprising the amino acid sequence set forth in SEQ ID NOs: 40 to 42, wherein the VH binds to a complex comprising a WT1 peptide and an MHC molecule when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 95; an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDR1, 2, and 3 each comprising the amino acid sequences set forth in SEQ ID NOs: 67 to 69, wherein the VL binds to a complex comprising a WT1 peptide and an MHC molecule when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 86; an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising complementarity-determining regions (CDRs) 1, 2, and 3 each comprising the amino acid sequence set forth in SEQ ID NOs: 43 to 45, wherein the VH binds to an MHC complex containing a WT1 peptide when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 96; an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDR1, 2, and 3 each comprising the amino acid sequence set forth in SEQ ID NO: 70 to 72, wherein the VL binds to a complex comprising a WT1 peptide and an MHC molecule when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 87; an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising complementarity-determining regions (CDRs) 1, 2, and 3 each comprising the amino acid sequences set forth in SEQ ID NOs: 46 to 48, wherein the VH binds to a complex comprising a WT1 peptide and an MHC molecule when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 97; an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDR1, 2, and 3 each comprising the amino acid sequence set forth in SEQ ID NO: 73 to 75, wherein the VL binds to a complex comprising a WT1 peptide and an MHC molecule when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 88; an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising complementarity-determining regions (CDRs) 1, 2, and 3 each comprising the amino acid sequence set forth in SEQ ID NOs: 49 to 51, wherein the VH binds to a complex comprising a WT1 peptide and an MHC molecule when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 98; an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDR1, 2, and 3 each comprising the amino acid sequence set forth in SEQ ID NO: 76 to 78, wherein the VL binds to a complex comprising a WT1 peptide and an MHC molecule when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 89; an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising complementarity-determining regions (CDRs) 1, 2, and 3 each comprising the amino acid sequence set forth in SEQ ID NOs: 52 to 54, wherein the VH binds to a complex comprising a WT1 peptide and an MHC molecule when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 99; an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDR1, 2, and 3 each comprising the amino acid sequence set forth in SEQ ID NO: 79 to 81, wherein the VL binds to a complex comprising a WT1 peptide and an MHC molecule when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 90; The present invention relates to nucleic acids, including:

[0031] In some embodiments, the VH, when paired with the VL, specifically binds to a complex comprising the WT1 peptide and an MHC molecule, or the VL, when paired with the VH, specifically binds to a complex comprising the WT1 peptide and an MHC molecule.

[0032] In some embodiments, the immunoglobulin heavy chain or fragment thereof is a human immunoglobulin heavy chain or fragment thereof (e.g., a human IgG1 heavy chain or fragment thereof, a human IgG2 heavy chain or fragment thereof, or a human IgG4 heavy chain or fragment thereof), and the immunoglobulin light chain or fragment thereof is a human immunoglobulin light chain or fragment thereof.

[0033] In some embodiments, the nucleic acid encodes a single-chain variable fragment (scFv), a single-arm antibody, a multispecific antibody (e.g., a bispecific antibody), or a chimeric antigen receptor (CAR).

[0034] In some embodiments, the nucleic acid is cDNA.

[0035] In one aspect, the disclosure relates to a vector comprising one or more of the nucleic acids described herein.

[0036] In one aspect, the present disclosure relates to a vector comprising two of the nucleic acids described herein, wherein the vector encodes a VL region and a VH region, respectively, that bind to a complex comprising a WT1 peptide and an MHC molecule.

[0037] In one aspect, the present disclosure relates to a pair of vectors, each vector comprising one of the nucleic acids described herein, and the pair of vectors collectively encode a VL region and a VH region, respectively, that bind to a complex comprising a WT1 peptide and an MHC molecule.

[0038] In one aspect, the disclosure relates to a cell comprising a vector described herein or a pair of vectors described herein.

[0039] In some embodiments, the cells are CHO cells.

[0040] In one aspect, the disclosure relates to a cell comprising one or more of the nucleic acids described herein.

[0041] In one aspect, the disclosure relates to a cell comprising two of the nucleic acids described herein.

[0042] In some embodiments, the two nucleic acids comprehensively encode a VL region and a VH region, respectively, that bind to a complex containing a human WT1 peptide and an MHC molecule.

[0043] In one aspect, the disclosure relates to a method of producing an antibody or antigen-binding fragment thereof, comprising culturing a cell described herein under conditions sufficient for the cell to produce the antibody or antigen-binding fragment, and recovering the antibody or antigen-binding fragment produced by the cell.

[0044] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof that binds to a complex comprising a WT1 peptide and an MHC molecule, The present invention relates to an antibody or antigen-binding fragment thereof comprising: a heavy chain variable region (VH) comprising an amino acid sequence that is at least 90% identical to a selected VH sequence; and a light chain variable region (VL) comprising an amino acid sequence that is at least 90% identical to a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following: The selected VH sequence is SEQ ID NO: 82 and the selected VL sequence is SEQ ID NO: 91. The selected VH sequence is SEQ ID NO: 83 and the selected VL sequence is SEQ ID NO: 92. The selected VH sequence is SEQ ID NO: 84 and the selected VL sequence is SEQ ID NO: 93. The selected VH sequence is SEQ ID NO: 85 and the selected VL sequence is SEQ ID NO: 94. The selected VH sequence is SEQ ID NO: 86 and the selected VL sequence is SEQ ID NO: 95. The selected VH sequence is SEQ ID NO: 87 and the selected VL sequence is SEQ ID NO: 96. The selected VH sequence is SEQ ID NO: 88 and the selected VL sequence is SEQ ID NO: 97. the selected VH sequence is SEQ ID NO: 89 and the selected VL sequence is SEQ ID NO: 98; and The selected VH sequence is SEQ ID NO:90 and the selected VL sequence is SEQ ID NO:99.

[0045] In some embodiments, the VH comprises the sequence of SEQ ID NO:82 and the VL comprises the sequence of SEQ ID NO:91.

[0046] In some embodiments, the VH comprises the sequence of SEQ ID NO:83 and the VL comprises the sequence of SEQ ID NO:92.

[0047] In some embodiments, the VH comprises the sequence of SEQ ID NO:84 and the VL comprises the sequence of SEQ ID NO:93.

[0048] In some embodiments, the VH comprises the sequence of SEQ ID NO:85 and the VL comprises the sequence of SEQ ID NO:94.

[0049] In some embodiments, the VH comprises the sequence of SEQ ID NO:86 and the VL comprises the sequence of SEQ ID NO:95.

[0050] In some embodiments, the VH comprises the sequence of SEQ ID NO:87 and the VL comprises the sequence of SEQ ID NO:96.

[0051] In some embodiments, the VH comprises the sequence of SEQ ID NO:88 and the VL comprises the sequence of SEQ ID NO:97.

[0052] In some embodiments, the VH comprises the sequence of SEQ ID NO:89 and the VL comprises the sequence of SEQ ID NO:98.

[0053] In some embodiments, the VH comprises the sequence of SEQ ID NO:90 and the VL comprises the sequence of SEQ ID NO:99.

[0054] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof that binds to a complex comprising a WT1 peptide and an MHC molecule, The present invention relates to an antibody or antigen-binding fragment thereof comprising: a heavy chain variable region (VH) comprising VH CDR1, VH CDR2, and VH CDR3 identical to the VH CDR1, VH CDR2, and VH CDR3 of a selected VH sequence; and a light chain variable region (VL) comprising VL CDR1, VL CDR2, and VL CDR3 identical to the VL CDR1, VL CDR2, and VL CDR3 of a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following: The selected VH sequence is SEQ ID NO: 82 and the selected VL sequence is SEQ ID NO: 91. The selected VH sequence is SEQ ID NO: 83 and the selected VL sequence is SEQ ID NO: 92. The selected VH sequence is SEQ ID NO: 84 and the selected VL sequence is SEQ ID NO: 93. The selected VH sequence is SEQ ID NO: 85 and the selected VL sequence is SEQ ID NO: 94. The selected VH sequence is SEQ ID NO: 86 and the selected VL sequence is SEQ ID NO: 95. The selected VH sequence is SEQ ID NO: 87 and the selected VL sequence is SEQ ID NO: 96. The selected VH sequence is SEQ ID NO: 88 and the selected VL sequence is SEQ ID NO: 97. the selected VH sequence is SEQ ID NO: 89 and the selected VL sequence is SEQ ID NO: 98; and The selected VH sequence is SEQ ID NO:90 and the selected VL sequence is SEQ ID NO:99.

[0055] In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to a complex comprising a WT1 peptide and an MHC molecule.

[0056] In some embodiments, the WT1 peptide comprises a sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence of SEQ ID NO:100.

[0057] In some embodiments, the MHC is HLA (eg, HLA-A2).

[0058] In some embodiments, the antibody or antigen-binding fragment thereof is a human antibody or humanized antibody or antigen-binding fragment thereof, a single-chain variable fragment (scFv), a single-arm antibody, and / or a multispecific antibody (e.g., a bispecific antibody).

[0059] In some embodiments, the antibody or antigen-binding fragment thereof is a human IgG1 antibody or antigen-binding fragment thereof, a human IgG2 antibody or antigen-binding fragment thereof, or a human IgG4 antibody or antigen-binding fragment thereof.

[0060] In one aspect, the present disclosure relates to antibodies, or antigen-binding fragments thereof, that cross-compete with the antibodies, or antigen-binding fragments thereof, described herein.

[0061] In some embodiments, the antibody or antigen-binding fragment thereof comprises a fragment crystallizable region (Fc region).

[0062] In one aspect, the present disclosure provides a method for manufacturing a semiconductor device comprising: (1) a first functional portion comprising an antigen-binding fragment thereof as described herein; and (2) a second functional portion comprising a T cell binding molecule; The present invention relates to a protein construct that binds to a complex containing a WT1 peptide and an MHC molecule, comprising:

[0063] In some embodiments, the T cell binding molecule (e.g., scFv or VHH) targets human CD3.

[0064] In some embodiments, the WT1 peptide comprises a sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence of SEQ ID NO:100.

[0065] In some embodiments, the MHC molecule is HLA (eg, HLA-A2).

[0066] In some embodiments, the first functional moiety and the second functional moiety are connected via a linker.

[0067] In one aspect, the present disclosure provides a method for manufacturing a semiconductor device comprising: (1) a first functional portion comprising an antibody or antigen-binding fragment thereof described herein; and (2) a second functional portion comprising a T cell binding molecule, and (3) a third functional portion comprising a single-chain human crystallizable fragment; The present invention relates to a protein construct comprising:

[0068] In some embodiments, the T cell binding molecule is an scFv or VHH that targets human CD3.

[0069] In some embodiments, the first functional portion, the second functional portion, and the third functional portion are connected via one or more linkers.

[0070] In some embodiments, the WT1 peptide comprises a sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence of SEQ ID NO:100.

[0071] In some embodiments, the MHC molecule is HLA (eg, HLA-A2).

[0072] In one aspect, the present disclosure relates to an antibody drug conjugate comprising an antibody or antigen-binding fragment thereof described herein, or a protein construct described herein, covalently attached to a therapeutic agent.

[0073] In some embodiments, the therapeutic agent is a cytotoxic or cytostatic agent.

[0074] In one aspect, the present disclosure relates to a recombinant receptor comprising an antigen-binding fragment thereof described herein.

[0075] In some embodiments, the recombinant receptor further comprises a transmembrane region and an intracellular signaling domain.

[0076] In some embodiments, the recombinant receptor is a chimeric antigen receptor (“CAR”).

[0077] In some embodiments, the recombinant receptor further comprises a hinge region.

[0078] In some embodiments, the transmembrane region comprises the transmembrane region of CD4, CD8, and / or CD28, or a portion thereof.

[0079] In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling sequence of an immune effector cell.

[0080] In some embodiments, the intracellular signaling domain is or comprises a functional signaling domain of CD3 zeta.

[0081] In some embodiments, the intracellular signaling domain further comprises a costimulatory signal domain.

[0082] In some embodiments, the costimulatory signal domain is selected from the group consisting of an MHC class I molecule, a TNF receptor protein, an immunoglobulin-like protein, a cytokine receptor, an integrin, a signaling lymphocytic activation molecule (SLAM protein), an activating NK cell receptor, BTLA, a Toll ligand receptor, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1, CD11a / CD18, 4-1BB (C D137), B7-H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD8 beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6 , CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRTAM, The protein comprises a functional signaling domain derived from a protein selected from the group consisting of Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, and CD83 ligand.

[0083] In some embodiments, the costimulatory signal domain comprises the intracellular signaling domain of 4-1BB and / or CD28.

[0084] In some embodiments, the recombinant receptor comprises a signal peptide.

[0085] In some embodiments, the recombinant receptor is a chimeric T cell receptor (“cTCR”).

[0086] In one aspect, the present disclosure relates to polynucleotides encoding the recombinant receptors described herein.

[0087] In one aspect, the present disclosure relates to a vector comprising a polynucleotide described herein.

[0088] In some embodiments, the vector is a viral vector.

[0089] In one aspect, the present disclosure relates to recombinant cells expressing the recombinant receptors described herein.

[0090] In some embodiments, the recombinant cell is an immune cell.

[0091] In some embodiments, the immune cells are NK cells or T cells.

[0092] In some embodiments, the recombinant cell is a T cell.

[0093] In some embodiments, the T cells are selected from the group consisting of cytotoxic T cells, helper T cells, natural killer T (NK-T) cells, and γδ T cells.

[0094] In one aspect, the disclosure relates to a method for producing a recombinant cell comprising introducing a vector described herein into a cell, either in vitro or ex vivo.

[0095] In some embodiments, the vector is a viral vector and the introduction is by transduction.

[0096] In one aspect, the present disclosure relates to a method of treating a subject having cancer, the method comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof described herein, a protein construct described herein, a recombinant cell described herein, or a composition comprising an antibody drug conjugate described herein.

[0097] In some embodiments, the subject has a solid tumor.

[0098] In some embodiments, the cancer is leukemia, breast cancer, ovarian cancer, glioblastoma, or soft tissue sarcoma.

[0099] In some embodiments, the subject is further treated with an effective amount of an anti-4-1BB antibody, an anti-OX40 antibody, an anti-CD40 antibody, an anti-PD-1 antibody, an anti-CTLA4 antibody, or an anti-PD-L1 antibody.

[0100] In one aspect, the present disclosure relates to a method of reducing tumor growth rate, comprising contacting tumor cells with a therapeutically effective amount of an antibody or antigen-binding fragment thereof described herein, a protein construct described herein, a recombinant cell described herein, or a composition comprising an antibody drug conjugate described herein.

[0101] In one aspect, the present disclosure relates to a method of killing tumor cells, the method comprising contacting the tumor cells with a therapeutically effective amount of an antibody or antigen-binding fragment thereof described herein, a protein construct described herein, a recombinant cell described herein, or a composition comprising an antibody drug conjugate described herein.

[0102] In one aspect, the present disclosure relates to a method of increasing an immune response in a subject, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof described herein, a protein construct described herein, a recombinant cell described herein, or a composition comprising an antibody drug conjugate described herein.

[0103] In one aspect, the present disclosure relates to a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof described herein, a protein construct described herein, a recombinant cell described herein, or an antibody drug conjugate described herein, and a pharmaceutically acceptable carrier.

[0104] In some embodiments, the antibody drug conjugates described herein have a drug-to-antibody ratio (DAR) of about 4.

[0105] In some embodiments, the antibodies or antigen-binding fragments thereof described herein bind to positions 2 and / or 9 of a WT1 peptide in a complex comprising the WT1 peptide and an MHC molecule.

[0106] In some embodiments, the antibodies or antigen-binding fragments thereof described herein bind to positions 1, 3, 4, 5, and / or 8 of a WT1 peptide in a complex comprising the WT1 peptide and an MHC molecule.

[0107] In one aspect, the present disclosure relates to an antibody or antigen-binding fragment thereof that binds to a complex comprising a WT1 peptide and an MHC molecule, wherein the antibody or antigen-binding fragment thereof is (1) an amino acid residue corresponding to position 2 of the WT1 peptide in a complex containing the WT1 peptide and an MHC molecule; (2) an amino acid residue corresponding to position 9 of the WT1 peptide in a complex containing the WT1 peptide and an MHC molecule; (3) an amino acid residue corresponding to position 1 of the WT1 peptide in a complex containing the WT1 peptide and an MHC molecule; (4) an amino acid residue corresponding to position 3 of the WT1 peptide in a complex containing the WT1 peptide and an MHC molecule; (5) an amino acid residue corresponding to position 4 of the WT1 peptide in a complex containing the WT1 peptide and an MHC molecule; (6) an amino acid residue corresponding to position 5 of the WT1 peptide in a complex containing the WT1 peptide and an MHC molecule, and / or (7) an amino acid residue corresponding to position 8 of the WT1 peptide in a complex containing the WT1 peptide and an MHC molecule; The WT1 peptide has the sequence shown in SEQ ID NO:100.

[0108] In one aspect, the present disclosure relates to an antibody or antigen-binding fragment thereof that binds to a complex comprising a WT1 peptide and an MHC molecule, wherein the antibody or antigen-binding fragment thereof specifically binds to an epitope within the WT1 peptide, the epitope being one or more of the following: (1) an amino acid residue corresponding to R1 in SEQ ID NO: 100; (2) amino acid residues corresponding to F3 of SEQ ID NO: 100; (3) an amino acid residue corresponding to P4 of SEQ ID NO: 100; (4) an amino acid residue corresponding to N5 of SEQ ID NO: 100, and (5) Amino acid residue corresponding to Y8 of SEQ ID NO: 100.

[0109] As used herein, the term "cancer" refers to cells capable of autonomous proliferation. Examples of such cells include cells with an abnormal state or condition characterized by rapid proliferation of cell growth. The term is intended to include cancerous growths, e.g., tumors, oncogenic processes, metastatic tissues, and malignantly transformed cells, tissues, or organs, regardless of the type of tissue change or stage of invasiveness. Also included are malignant tumors of various organ systems, e.g., respiratory, cardiovascular, renal, reproductive, hematological, nervous system, liver, gastrointestinal, and endocrine systems, as well as most colon cancers, renal cell carcinoma, prostate and / or testicular tumors, non-small cell lung cancer, and small intestine cancer. "Naturally occurring" cancers include any cancer that is not experimentally induced by implanting cancer cells into a subject, such as naturally occurring cancers, cancers caused by exposing a patient to a carcinogen, cancers caused by the insertion of a transgenic oncogene or the knockout of a tumor suppressor gene, and cancers caused by infectious diseases, e.g., viral infections. The term "carcinoma" is art-recognized and refers to a malignant tumor of epithelial or endocrine tissue. The term also includes carcinosarcomas, which include malignant tumors composed of carcinomatous and sarcomatous tissue. "Adenocarcinoma" refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures. The term "sarcoma" is art-recognized and refers to a malignant tumor of mesenchymal origin. The term "hematopoietic neoplastic disorder" includes disorders involving hyperplastic / neoplastic cells of hematopoietic origin. Hematopoietic neoplastic disorders can arise from the myeloid, lymphoid, or erythroid lineages, or their precursor cells.

[0110] As used herein, the term "antibody" refers to any antigen-binding molecule that contains at least one (e.g., 1, 2, 3, 4, 5, or 6) complementarity-determining region (CDR) (e.g., any of the three CDRs from an immunoglobulin light chain or any of the three CDRs from an immunoglobulin heavy chain) and is capable of specifically binding to an epitope. Non-limiting examples of antibodies include monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), single-chain antibodies, and single-chain variable domain (V) antibodies. HH) Antibodies include chimeric antibodies, human antibodies, and humanized antibodies. In some embodiments, the antibody can contain the Fc region of a human antibody. The term antibody also includes derivatives such as bispecific antibodies, single-chain antibodies, diabodies, linear antibodies, and multispecific antibodies formed from antibody fragments.

[0111] As used herein, the term "antigen-binding fragment" refers to a portion of a full-length antibody, which portion of the antibody is capable of specifically binding to an antigen. In some embodiments, the antigen-binding fragment contains at least one variable domain (e.g., a heavy chain variable domain or a light chain variable domain). Non-limiting examples of antibody fragments include, for example, Fab, Fab', F(ab')2, and Fv fragments.

[0112] As used herein, the term "anti-WT1 / HLA antibody" means an antibody capable of specifically binding to an MHC complex containing a WT1 peptide and an HLA-A2 molecule (which may also be referred to as a complex containing a WT1 peptide and an MHC molecule).

[0113] As used herein, the term "human antibody" means an antibody encoded by endogenous nucleic acid present in a human (e.g., a rearranged human immunoglobulin heavy or light chain locus). In some embodiments, a human antibody is recovered from a human or produced in human cell culture (e.g., in human hybridoma cells). In some embodiments, a human antibody is produced in a non-human cell (e.g., a mouse or hamster cell line). In some embodiments, a human antibody is produced in a bacterial cell or yeast cell. In some embodiments, a human antibody is produced in a transgenic non-human animal (e.g., a cow) containing unrearranged or rearranged human immunoglobulin loci (e.g., a heavy or light chain human immunoglobulin locus).

[0114] As used herein, the term "chimeric antibody" refers to an antibody that contains sequences present in at least two different antibodies (e.g., antibodies from two different mammalian species, such as human and murine antibodies). A non-limiting example of a chimeric antibody is an antibody that contains variable domain sequences (e.g., all or part of the light and / or heavy chain variable domain sequences) of a non-human (e.g., murine) antibody and the constant domain of a human antibody. Further examples of chimeric antibodies are described herein and are well known in the art.

[0115] As used herein, the term "humanized antibody" refers to a non-human antibody that contains minimal sequence derived from non-human (e.g., murine) immunoglobulin and contains sequence derived from human immunoglobulin. In a non-limiting example, a humanized antibody is a human antibody (recipient antibody) in which residues from hypervariable (e.g., CDR) regions of the recipient antibody are replaced by residues from hypervariable (e.g., CDR) regions of a non-human antibody (e.g., donor antibody), such as a mouse, rat, or rabbit antibody, having the desired specificity, affinity, and capacity. In some embodiments, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human (e.g., murine) immunoglobulin residues. In some embodiments, humanized antibodies can contain residues that are not found in the recipient antibody or the donor antibody. These modifications can be made to further refine antibody performance. In some embodiments, a humanized antibody contains nearly all of at least one, and typically two, variable domains, with all or substantially all of the hypervariable loops (CDRs) corresponding to those of a non-human (e.g., murine) immunoglobulin and all or substantially all of the framework regions being those of a human immunoglobulin sequence. A humanized antibody can also contain at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. Humanized antibodies can be produced using molecular biology techniques well known in the art. Non-limiting examples of methods for generating humanized antibodies are described herein.

[0116] As used herein, the term "single-chain antibody" means a single polypeptide containing at least two immunoglobulin variable domains (e.g., the variable domains of a mammalian immunoglobulin heavy or light chain) that are capable of specifically binding to an antigen. Non-limiting examples of single-chain antibodies are described herein.

[0117] As used herein, the terms "subject" and "patient" are used interchangeably throughout the specification and refer to an animal, human, or non-human to whom treatment according to the methods of the invention is provided. Veterinary and non-veterinary uses are contemplated by the present invention. A human patient can be an adult human or a juvenile human (e.g., a human under the age of 18). In addition to humans, patients include, but are not limited to, mice, rats, hamsters, guinea pigs, rabbits, ferrets, cats, dogs, and primates. Examples include non-human primates (e.g., monkeys, chimpanzees, gorillas, etc.), rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits), lagomorphs, swine (e.g., pigs, minipigs), equines, canines, felines, bovines, and other domestic, livestock, and zoo animals.

[0118] As used herein, the phrases "specifically bind" and "specifically bind" when referring to an antibody mean that the antibody interacts with the target molecule, preferably more than other molecules, because the interaction depends on the presence of a particular structure (i.e., an antigenic determinant or epitope) on the target molecule; in other words, the reagent generally recognizes and binds to a molecule containing a particular structure rather than to the entire molecule. An antibody that specifically binds to a target molecule can also be referred to as a target-specific antibody. For example, an antibody that specifically binds to the WT1 / HLA-A2 complex can be referred to as a WT1 / HLA-A2-specific antibody, an anti-WT1 / HLA antibody, or an anti-WT1 / HLA complex antibody.

[0119] As used herein, the term "bispecific antibody" refers to an antibody that binds to two different epitopes, which can be on the same antigen or on different antigens.

[0120] As used herein, the term "multispecific antibody" refers to an antibody that binds to two or more different epitopes. The epitopes can be on the same antigen or on different antigens. A multispecific antibody can be, for example, a bispecific antibody or a trispecific antibody. In some embodiments, a multispecific antibody binds to 2, 3, 4, 5, or 6 different epitopes.

[0121] As used herein, "chimeric antigen receptor" or "CAR" refers to a fusion protein comprising an extracellular domain capable of binding to an antigen and an intracellular region comprising one or more intracellular signaling domains derived from a signal transduction protein. The extracellular domain can be any proteinaceous molecule or portion thereof capable of specifically binding to a predetermined antigen. In some embodiments, the extracellular domain comprises an antibody or antigen-binding fragment thereof. In some embodiments, the intracellular signaling domain can be any oligopeptide or polypeptide domain known to function in transducing a signal that results in the activation or inhibition of a biological process within the cell, for example, activation of immune cells such as T cells or NK cells.

[0122] As used herein, the terms "polypeptide," "peptide," and "protein" are used interchangeably to refer to polymers of amino acids of any length, of at least two amino acids.

[0123] As used herein, the terms "polynucleotide," "nucleic acid molecule," and "nucleic acid sequence" are used interchangeably herein and refer to polymers of nucleotides of any length, of at least two nucleotides, including, but not limited to, DNA, RNA, DNA / RNA hybrids, and modifications thereof.

[0124] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials for use in the present invention are described herein; other suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0125] Other features and advantages of the invention will become apparent from the following detailed description and drawings, and from the claims. [Brief explanation of the drawings]

[0126] [Figure 1-1] FIG. 1A shows an exemplary antibody structure for a bispecific antibody, FIG. 1B shows an exemplary antibody structure for a bispecific antibody, and FIG. 1C shows an exemplary antibody structure for a bispecific antibody. [Figure 1-2] FIG. 1-D shows an exemplary structure of the construct. [Figure 2] The CDR sequences of the heavy and light chain variable regions of anti-WT1 / HLA antibodies are listed below according to the Kabat definition. [Figure 3] The CDR sequences of the heavy chain variable region and light chain variable region of anti-WT1 / HLA antibodies are listed below based on the definition of Chothia. [Figure 4-1]Figures 4A-4J show the binding between T2 cells pulsed with alanine-substituted peptides (25 μM), including ESK1 analogs (Figure 4A), 7B9-IgG1-SI (Figure 4B), P01264-IgG1-SI (Figure 4C), 1E9-IgG1-SI (Figure 4D), P01218-IgG1-SI (Figure 4E), P01199-IgG1-SI (Figure 4F), 1G7-IgG1-SI (Figure 4G), 3E6-IgG1-SI (Figure 4H), 3A6-IgG1-SI (Figure 4I), and 4F10-IgG1-SI (Figure 4J), and anti-WT1 / HLA antibodies, as measured by flow cytometry. [Figure 4-2] Same as above [Figure 4-3] Same as above [Figure 4-4] Same as above [Figure 4-5] Same as above [Figure 5-1] Panels A to D show the cytotoxicity induced in CD3+ T cells by anti-WT1 / CD3 bispecific antibodies, including 1G7-CD3 (Fig. 5A), 7B9-CD3 (Fig. 5B), P01199-CD3 (Fig. 5C), and P01218-CD3 (Fig. 5D), against WT1- and HLA-A2-positive tumor cells. [Figure 5-2] Same as above [Figure 6] Shown is the release of human IFN-γ cytokine at 24 h as measured by ELISA. [Figure 7] 1 shows the mean tumor volumes in various groups of B-NDG mice subcutaneously injected with HCT116-WT1 cells and treated with PBS or anti-WT1 / CD3 bispecific antibody. [Figure 8] 1 shows the mean tumor volume in various groups of B-NDG mice subcutaneously injected with HCT116-WT1 cells and treated with saline, mock T cells, or CAR-T cells (1G7-CAR-T(VH-VL), 3E6-CAR-T(VH-VL), P01264-CAR-T(VH-VL), ESK1-CAR-T(VH-VL), 1G7-CAR-T(VL-VH), 3E6-CAR-T(VL-VH), P01264-CAR-T(VL-VH)). [Figure 9-1] Selected amino acid sequences discussed in this disclosure are listed below. [Figure 9-2] Same as above [Figure 9-3] Same as above [Figure 10] 1 shows the mean tumor volume in various groups of B-NDG mice subcutaneously injected with HCT116-WT1 cells and treated with saline, mock T cells, or CAR-T cells (1E9-CAR-T(VL-VH), 3A6-CAR-T(VL-VH), 4F10-CAR-T(VL-VH), P01199-CAR-T(VL-VH), P01218-CAR-T(VL-VH)). [Figure 11] Figure 1 shows mouse survival rates in various groups of B-NDG mice intravenously injected with THP-1-luc cells and treated with saline, mock T cells, or CAR-T cells (1E9-CAR-T(VL-VH), 3A6-CAR-T(VL-VH), 4F10-CAR-T(VL-VH), P01199-CAR-T(VL-VH), P01218-CAR-T(VL-VH)). [Figure 12] Figure 1 shows the cytotoxicity of CD3+ T cells against THP-1 or OVCAR3 cells induced by the anti-WT1 / CD3 bispecific antibodies 1E9-CD3, 1G7-CD3, and P01218-CD3. An RG6007 analog was used as a positive control. [Figure 13] Figure 1 shows the cytotoxicity of CD3+ T cells against THP-1 or OVCAR3 cells induced by the anti-WT1 / CD3 bispecific antibodies 3E6-CD3, 7B9-CD3, and P01199-CD3. An RG6007 analog was used as a positive control. [Figure 14] Figure 1 shows the cytotoxicity of CD3+ T cells against THP-1 cells or OVCAR3 cells induced by the anti-WT1 / CD3 bispecific antibodies 1E9-CD3 (VHH), 1G7-CD3 (VHH), 3A6-CD3 (VHH), 3E6-CD3 (VHH), 4F10-CD3 (VHH), 7B9-CD3 (VHH), P01199-CD3 (VHH), P01218-CD3 (VHH), and P01264-CD3 (VHH). [Figure 15]Panels A to B show the binding measured by flow cytometry between T2 cells pulsed with WT1RMF, MED13L peptide, or PIGQ peptide and 1G7-IgG1-SI (FIG. 15A) or RG6007-WT1 (FIG. 15B). DETAILED DESCRIPTION OF THE INVENTION

[0127] The present disclosure provides examples of antibodies and antigen-binding fragments thereof that bind to MHC complexes containing WT1 peptides.

[0128] WT1 The WT1 (Wilms tumor 1) gene, encoding a protein consisting of four zinc finger domains at the C-terminus and glutamine- and proline-rich domains at the N-terminus, plays important roles in cell proliferation, differentiation, apoptosis, organ development, and the maintenance of several adult tissues. Although recognized as a classic tumor suppressor gene in Wilms tumor, increasing evidence indicates that wild-type WT1 is expressed in various tumors arising from various tissues that do not normally express WT1. WT1 may be able to inhibit cell apoptosis by transcriptional activation and / or upregulation of proto-oncogenes. Recent studies have also revealed that WT1 is a key regulator of downstream sequences of KRAS signaling and is involved in the apoptotic response to cytotoxic stress, further demonstrating its oncogenic effects. Furthermore, WT1 can also promote invasion, migration, and metastasis, promote angiogenesis, and confer drug resistance to cancer cells. WT1 is overexpressed in many cancer cells. In particular, WT1 is highly expressed in various adult epithelial tumors and some leukemias, which has led to considerable efforts being made in the pursuit of immunotherapies targeting WT1 epitopes.

[0129] A detailed review of WT1 and its functions can be found in Qi, Xiao-wei, et al. "Wilms' tumor 1 (WT1) expression and prognosis in solid cancer patients: a systematic review and meta-analysis." Scientific reports 5.1 (2015): 1-9 and Hastie, Nicholas D. "Wilms' tumor 1 (WT1) in development, homeostasis and disease." Development 144.16 (2017): 2862-2872, each of which is incorporated by reference in its entirety.

[0130] The present disclosure provides "TCR-like" antibodies that target the WT1 peptide-MHC complex. The development of these TCR-like antibodies as therapeutic agents can improve therapeutic efficacy. Thus, in one aspect, the present disclosure provides methods for treating disorders associated with WT1. In some embodiments, the disorder is a disorder in which WT1 is overexpressed. In some embodiments, the disorder is cancer.

[0131] Among these peptides presented by cells, the WT1(126-134) peptide (WT1 RMF , RMFPNAPYL, SEQ ID NO: 100) is a peptide restricted by HLA-A2. In some embodiments, the antibodies or antigen-binding fragments described herein specifically bind to the WT1 / HLA-A2 complex.

[0132] Antibodies and antigen-binding fragments The present disclosure provides anti-WT1 / HLA antibodies and antigen-binding fragments thereof. Generally, antibodies (also called immunoglobulins) are composed of two classes of polypeptide chains: light chains and heavy chains. Non-limiting antibodies of the present disclosure can be intact four immunoglobulin chain antibodies, including two heavy chains and two light chains. The heavy chains of the antibodies can be of any isotype, including IgM, IgG, IgE, IgA, or IgD, or subisotypes, including IgG1, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgE1, IgE2, etc. The light chains can be κ or λ light chains. The antibody can include two identical copies of the light chains and two identical copies of the heavy chains, each containing one variable domain (or variable region, V H ), and heavy chains containing multiple constant domains (or constant regions) linked together via disulfide bonds within their constant domains to form the "stem" of the antibody. L Each light chain, containing a constant domain (or constant region) and one light chain, binds to one heavy chain via disulfide bonds. The variable region of each light chain aligns with the variable region of the heavy chain to which it is bound. The variable regions of both the light and heavy chains contain three hypervariable regions sandwiched between more conserved framework regions (FRs).

[0133] The hypervariable regions, known as complementarity-determining regions (CDRs), form the loops that comprise the principal antigen-binding surface of an antibody. The four framework regions largely conform to a β-sheet structure, and the CDRs form connecting loops that, in some cases, form part of the β-sheet structure. The CDRs of each chain are held in close proximity by the framework regions and, together with the CDRs of the other chain, contribute to the formation of the antigen-binding region.

[0134] Methods for identifying CDR regions of antibodies by analyzing their amino acid sequences are well known, and several definitions of CDRs are commonly used: the Kabat definition is based on sequence variability, and the Chothia definition is based on the location of structural loop regions. These methods and definitions are described, for example, by Martin, “Protein sequence and structure analysis of antibody variable domains,” Antibody engineering, Springer Berlin Heidelberg, 2001.422-439, Abhinandan, et al. Kabat, EA (1970) J.Exp.Med.132:211-250, Martin et al., Methods Enzymol.203:121-53(1991), Morea et al., Biophys Chem.68(1-3):9-16(Oct.1997), Morea et al., J Mol Biol.275(2):269-94(Jan.1998), Chothia et al., Nature 342(6252):877-83 (Dec. 1989), Ponomarenko and Bourne, BMC Structural Biology 7:64 (2007), each of which is incorporated by reference in its entirety.

[0135] CDRs are important for recognizing the epitope of an antigen. As used herein, "epitope" refers to the smallest portion of a target molecule that can be specifically bound by the antigen-binding domain of an antibody. The minimum size of an epitope can be approximately 3, 4, 5, 6, or 7 amino acids, but these amino acids do not need to be in a contiguous linear sequence in the primary structure of the antigen, as the epitope may depend on the three-dimensional structure of the antigen based on the secondary and tertiary structure of the antigen.

[0136] In some embodiments, antibodies are intact immunoglobulin molecules (e.g., IgG1, IgG2a, IgG2b, IgG3, IgM, IgD, IgE, IgA). The IgG subclasses (IgG1, IgG2, IgG3, and IgG4) are highly conserved and differ in their constant regions, particularly the hinge and upper CH2 domains. The sequences and differences between IgG subclasses are well known in the art and are described, for example, in Vidarsson, et al., "IgG subclasses and allotypes: from structure to effector functions," Frontiers in Immunology 5 (2014), Irani, et al., "Molecular properties of human IgG subclasses and their implications for designing therapeutic monoclonal antibodies against infectious diseases," Molecular Immunology 67.2 (2015):171-182, and Shakib, Farouk, ed., The human IgG subclasses: molecular analysis of structure, function and regulation. Elsevier, 2016, each of which is incorporated herein by reference in its entirety.

[0137] An antibody can also be an immunoglobulin molecule from any species (e.g., human, rodent, mouse, camelid). Antibodies disclosed herein include, but are not limited to, polyclonal, monoclonal, monospecific, multispecific antibodies, and chimeric antibodies comprising an immunoglobulin binding domain fused to another polypeptide. The term "antigen-binding domain" or "antigen-binding fragment" refers to any portion of an antibody that retains the specific binding activity of the intact antibody, i.e., any portion of an antibody that is capable of specifically binding to an epitope on the intact antibody's target molecule. This includes, for example, Fab, Fab', F(ab')2, and variants of these fragments. Thus, in some embodiments, an antibody or antigen-binding fragment thereof can be, for example, an scFv, Fv, Fd, dAb, diabody, bispecific scFv, diabody, linear antibody, single-chain antibody molecule, multispecific antibody formed from antibody fragments, and any polypeptide comprising a binding domain that is, or is homologous to, an antibody-binding domain. Non-limiting examples of antigen-binding domains include, for example, the heavy and / or light chain CDRs of an intact antibody, the heavy and / or light chain variable regions of an intact antibody, the full-length heavy or light chain of an intact antibody, or individual CDRs derived from either the heavy or light chain of an intact antibody.

[0138] In some embodiments, the antigen-binding fragment can form part of a chimeric antigen receptor (CAR). In some embodiments, the chimeric antigen receptor is a fusion of a single-chain variable fragment (scFv) described herein fused to the CD3ζ transmembrane and endodomain. In some embodiments, the chimeric antigen receptor also comprises intracellular signaling domains from various costimulatory protein receptors (e.g., CD28, 41BB, ICOS). In some embodiments, the chimeric antigen receptor comprises multiple signaling domains, e.g., CD3z-CD28-41BB or CD3z-CD28-OX40, for increased potency. Thus, in one aspect, the present disclosure further provides a cell (e.g., a T cell) expressing a chimeric antigen receptor described herein.

[0139] In some embodiments, an scFv has one heavy chain variable domain and one light chain variable domain, hi some embodiments, an scFv has two heavy chain variable domains and two light chain variable domains.

[0140] In some embodiments, the sequences of the antibodies or antigen-binding fragments thereof described herein (e.g., CDR or VH / VL sequences) can be used to generate bispecific antibodies that target the WT1 / HLA-A2 complex and an additional antigen (e.g., human CD3).

[0141] Anti-WT1 / HLA antibodies and antigen-binding fragments The present disclosure provides antibodies and antigen-binding fragments thereof that specifically bind to an MHC complex containing a WT1 peptide. In some embodiments, the MHC molecule is HLA (e.g., HLA-A2). The antibodies and antigen-binding fragments described herein are capable of binding to an MHC complex containing a WT1 peptide. In some embodiments, these antibodies are capable of increasing an immune response.

[0142] The present disclosure provides, for example, anti-WT1 / HLA antibodies 1E9, 1G7, 3A6, 3E6, 4F10, 7B9, P01199, P01218, and P01264, chimeric antibodies thereof, and human or humanized antibodies thereof.

[0143] CDR sequences for 1E9 and antibodies derived from 1E9 (e.g., human or humanized antibodies) include the heavy chain variable domain CDR sequences set forth in SEQ ID NOS: 1 to 3 and the light chain variable domain CDR sequences set forth in SEQ ID NOS: 55 to 57, as defined by the Kabat definition. CDRs can also be defined by the Chothia definition, in which the heavy chain variable domain CDR sequences are set forth in SEQ ID NOS: 28 to 30 and the light chain variable domain CDR sequences are set forth in SEQ ID NOS: 55 to 57.

[0144] Similarly, CDR sequences for 1G7 and antibodies derived from 1G7 include the heavy chain variable domain CDR sequences shown in SEQ ID NOS: 4 to 6 and the light chain variable domain CDR sequences shown in SEQ ID NOS: 58 to 60, as defined by Kabat. According to Chothia's definition, the heavy chain variable domain CDR sequences are shown in SEQ ID NOS: 31 to 33, and the light chain variable domain CDR sequences are shown in SEQ ID NOS: 58 to 60.

[0145] CDR sequences for 3A6 and antibodies derived from 3A6 include the heavy chain variable domain CDR sequences shown in SEQ ID NOS: 7 to 9 and the light chain variable domain CDR sequences shown in SEQ ID NOS: 61 to 63, as defined by Kabat. According to Chothia's definition, the heavy chain variable domain CDR sequences are shown in SEQ ID NOS: 34 to 36, and the light chain variable domain CDR sequences are shown in SEQ ID NOS: 61 to 63.

[0146] CDR sequences for 3E6 and antibodies derived from 3E6 include, as defined by Kabat, heavy chain variable domain CDR sequences shown in SEQ ID NOS: 10 to 12 and light chain variable domain CDR sequences shown in SEQ ID NOS: 64 to 66. According to Chothia's definition, heavy chain variable domain CDR sequences shown in SEQ ID NOS: 37 to 39 and light chain variable domain CDR sequences shown in SEQ ID NOS: 64 to 66.

[0147] CDR sequences for 4F10 and antibodies derived from 4F10 include, as defined by Kabat, heavy chain variable domain CDR sequences shown in SEQ ID NOS: 13 to 15 and light chain variable domain CDR sequences shown in SEQ ID NOS: 67 to 69. According to Chothia's definition, heavy chain variable domain CDR sequences shown in SEQ ID NOS: 40 to 42 and light chain variable domain CDR sequences shown in SEQ ID NOS: 67 to 69.

[0148] CDR sequences for 7B9 and antibodies derived from 7B9 include, as defined by Kabat, heavy chain variable domain CDR sequences shown in SEQ ID NOS: 16 to 18 and light chain variable domain CDR sequences shown in SEQ ID NOS: 70 to 72. According to Chothia's definition, heavy chain variable domain CDR sequences shown in SEQ ID NOS: 43 to 45 and light chain variable domain CDR sequences shown in SEQ ID NOS: 70 to 72.

[0149] CDR sequences for P01199 and antibodies derived from P01199 include the heavy chain variable domain CDR sequences shown in SEQ ID NOs: 19 to 21 and the light chain variable domain CDR sequences shown in SEQ ID NOs: 73 to 75, as defined by Kabat. According to Chothia's definition, the heavy chain variable domain CDR sequences are shown in SEQ ID NOs: 46 to 48, and the light chain variable domain CDR sequences are shown in SEQ ID NOs: 73 to 75.

[0150] CDR sequences for P01218 and antibodies derived from P01218 include the heavy chain variable domain CDR sequences shown in SEQ ID NOs: 22 to 24 and the light chain variable domain CDR sequences shown in SEQ ID NOs: 76 to 78, as defined by Kabat. According to Chothia's definition, the heavy chain variable domain CDR sequences are shown in SEQ ID NOs: 49 to 51, and the light chain variable domain CDR sequences are shown in SEQ ID NOs: 76 to 78.

[0151] CDR sequences for P01264 and antibodies derived from P01264 include, as defined by the Kabat definition, heavy chain variable domain CDR sequences shown in SEQ ID NOS: 25 to 27 and light chain variable domain CDR sequences shown in SEQ ID NOS: 79 to 81. According to the Chothia definition, heavy chain variable domain CDR sequences shown in SEQ ID NOS: 52 to 54 and light chain variable domain CDR sequences shown in SEQ ID NOS: 79 to 81.

[0152] The amino acid sequence for the heavy chain variable region of the 1E9 antibody is shown in SEQ ID NO: 82. The amino acid sequence for the light chain variable region of the 1E9 antibody is shown in SEQ ID NO: 91.

[0153] The amino acid sequence for the heavy chain variable region of the 1G7 antibody is shown in SEQ ID NO: 83. The amino acid sequence for the light chain variable region of the 1G7 antibody is shown in SEQ ID NO: 92.

[0154] The amino acid sequence for the heavy chain variable region of the 3A6 antibody is shown in SEQ ID NO: 84. The amino acid sequence for the light chain variable region of the 3A6 antibody is shown in SEQ ID NO: 93.

[0155] The amino acid sequence for the heavy chain variable region of the 3E6 antibody is shown in SEQ ID NO: 85. The amino acid sequence for the light chain variable region of the 3E6 antibody is shown in SEQ ID NO: 94.

[0156] The amino acid sequence for the heavy chain variable region of the 4F10 antibody is shown in SEQ ID NO: 86. The amino acid sequence for the light chain variable region of the 4F10 antibody is shown in SEQ ID NO: 95.

[0157] The amino acid sequence for the heavy chain variable region of the 7B9 antibody is shown in SEQ ID NO: 87. The amino acid sequence for the light chain variable region of the 7B9 antibody is shown in SEQ ID NO: 96.

[0158] The amino acid sequence for the heavy chain variable region of the P01199 antibody is shown in SEQ ID NO: 88. The amino acid sequence for the light chain variable region of the P01199 antibody is shown in SEQ ID NO: 97.

[0159] The amino acid sequence for the heavy chain variable region of the P01218 antibody is shown in SEQ ID NO: 89. The amino acid sequence for the light chain variable region of the P01218 antibody is shown in SEQ ID NO: 98.

[0160] The amino acid sequence for the heavy chain variable region of the P01264 antibody is shown in SEQ ID NO: 90. The amino acid sequence for the light chain variable region of the P01264 antibody is shown in SEQ ID NO: 99.

[0161] Also provided are amino acid sequences for the heavy and light chain variable regions of the modified antibodies. In some embodiments, the heavy chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 82-90. In some embodiments, the light chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 91-99. The heavy chain variable region sequence can be paired with the corresponding light chain variable region sequence, and both bind to the WT1 / HLA-A2 complex.

[0162] Percent humanization refers to the percent identity of a heavy or light chain variable region sequence compared to human antibody sequences in the International Immunogenetics Database (IMGT). In some embodiments, the percent humanization is greater than 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95%. Detailed descriptions of how to determine the percent humanization and how to determine the top hits are well known in the art and are described, for example, in Jones, et al. "The INNs and outs of antibody nonproprietary names," MAbs. Vol. 8. No. 1. Taylor & Francis, 2016, the entire contents of which are incorporated herein by reference. A high percent humanization often has various advantages, such as being safer and more effective in humans, more likely to be tolerated by human subjects, and / or less likely to have side effects. In some embodiments, the variable regions are fully human, e.g., derived from human heavy chain immunoglobulin locus sequences (e.g., a combination of human IGHV, human IGHD, and human IGHJ genes) and / or human kappa chain immunoglobulin locus sequences (e.g., a combination of human IGKV and human IGKJ genes).

[0163] Furthermore, in some embodiments, the antibodies or antigen-binding fragments described herein may also contain one, two, or three heavy chain variable region CDRs selected from the group consisting of SEQ ID NOs: 1 to 3, SEQ ID NOs: 4 to 6, SEQ ID NOs: 7 to 9, SEQ ID NOs: 10 to 12, SEQ ID NOs: 13 to 15, SEQ ID NOs: 16 to 18, SEQ ID NOs: 19 to 21, SEQ ID NOs: 22 to 24, SEQ ID NOs: 25 to 27, SEQ ID NOs: 28 to 30, SEQ ID NOs: 31 to 33, SEQ ID NOs: 34 to 36, SEQ ID NOs: 37 to 39, SEQ ID NOs: 40 to 42, SEQ ID NOs: 43 to 45, SEQ ID NOs: 46 to 48, SEQ ID NOs: 49 to 51, and SEQ ID NOs: 52 to 54, and / or one, two, or three light chain variable region CDRs selected from the group consisting of SEQ ID NOs: 55 to 57, SEQ ID NOs: 58 to 60, SEQ ID NOs: 61 to 63, SEQ ID NOs: 64 to 66, SEQ ID NOs: 67 to 69, SEQ ID NOs: 70 to 72, SEQ ID NOs: 73 to 75, SEQ ID NOs: 76 to 78, and SEQ ID NOs: 79 to 81.

[0164] In some embodiments, the antibody may have a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3, wherein the CDR1 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to the amino acid sequence of a selected VH CDR1; the CDR2 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to the amino acid sequence of a selected VH CDR2; and the CDR3 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to the amino acid sequence of a selected VH CDR3. In some embodiments, the antibody can have a light chain variable region (VL) comprising CDR1, 2, and 3, wherein the CDR1 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to the amino acid sequence of a selected VL CDR1, the CDR2 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to the amino acid sequence of a selected VL CDR2, and the CDR3 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to the amino acid sequence of a selected VL CDR3. The amino acid sequences of selected VH CDR1, 2, and 3 and selected VL CDR1, 2, and 3 are shown in Figure 2 (Kabat CDRs) and Figure 3 (Chothia CDRs).

[0165] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 1 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 2 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; or SEQ ID NO: 3 with 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0166] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 4 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 5 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; or SEQ ID NO: 6 with 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0167] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 7 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 8 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; or SEQ ID NO: 9 with 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0168] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 10 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 11 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; or SEQ ID NO: 12 with 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0169] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 13 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 14 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; or SEQ ID NO: 15 with 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0170] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 16 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 17 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; or SEQ ID NO: 18 with 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0171] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 19 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 20 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; or SEQ ID NO: 21 with 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0172] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 22 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 23 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; or SEQ ID NO: 24 with 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0173] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 25 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 26 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; or SEQ ID NO: 27 with 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0174] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 28 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 29 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; or SEQ ID NO: 30 with 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0175] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 31 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 32 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; or SEQ ID NO: 33 with 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0176] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 34 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 35 with zero, one, or two amino acid insertions, deletions, or substitutions; or SEQ ID NO: 36 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0177] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 37 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 38 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; or SEQ ID NO: 39 with 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0178] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 40 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 41 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; or SEQ ID NO: 42 with 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0179] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 43 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 44 with zero, one, or two amino acid insertions, deletions, or substitutions; or SEQ ID NO: 45 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0180] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 46 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 47 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; or SEQ ID NO: 48 with 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0181] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 49 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 50 with zero, one, or two amino acid insertions, deletions, or substitutions; or SEQ ID NO: 51 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0182] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 52 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 53 with zero, one, or two amino acid insertions, deletions, or substitutions; or SEQ ID NO: 54 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0183] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 55 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 56 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; or SEQ ID NO: 57 with 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0184] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 58 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 59 with zero, one, or two amino acid insertions, deletions, or substitutions; or SEQ ID NO: 60 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0185] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 61 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 62 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; or SEQ ID NO: 63 with 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0186] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 64 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 65 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; or SEQ ID NO: 66 with 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0187] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 67 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 68 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; or SEQ ID NO: 69 with 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0188] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 70 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 71 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; or SEQ ID NO: 72 with 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0189] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 73 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 74 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; or SEQ ID NO: 75 with 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0190] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 76 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 77 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; or SEQ ID NO: 78 with 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0191] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 79 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 80 with zero, one, or two amino acid insertions, deletions, or substitutions; or SEQ ID NO: 81 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0192] Insertions, deletions, and substitutions can occur within the CDR sequences or at either or both ends of the CDR sequences. In some embodiments, CDRs are determined based on the Kabat definition. In some embodiments, CDRs are determined based on the Chothia definition. In some embodiments, CDRs are determined based on a combination of the Kabat and Chothia definitions.

[0193] The present disclosure also provides antibodies or antigen-binding fragments thereof that bind to the WT1 / HLA-A2 complex. The antibodies or antigen-binding fragments thereof contain a heavy chain variable region (VH) comprising, or consisting of, an amino acid sequence at least 80%, 85%, 90%, or 95% identical to a selected VH sequence, and a light chain variable region (VL) comprising, or consisting of, an amino acid sequence at least 80%, 85%, 90%, or 95% identical to a selected VL sequence. In some embodiments, the selected VH sequence is SEQ ID NO: 82 and the selected VL sequence is SEQ ID NO: 91. In some embodiments, the selected VH sequence is SEQ ID NO: 83 and the selected VL sequence is SEQ ID NO: 92. In some embodiments, the selected VH sequence is SEQ ID NO: 84 and the selected VL sequence is SEQ ID NO: 93. In some embodiments, the selected VH sequence is SEQ ID NO: 85 and the selected VL sequence is SEQ ID NO: 94. In some embodiments, the selected VH sequence is SEQ ID NO: 86 and the selected VL sequence is SEQ ID NO: 95. In some embodiments, the selected VH sequence is SEQ ID NO: 87 and the selected VL sequence is SEQ ID NO: 96. In some embodiments, the selected VH sequence is SEQ ID NO: 88 and the selected VL sequence is SEQ ID NO: 97. In some embodiments, the selected VH sequence is SEQ ID NO: 89 and the selected VL sequence is SEQ ID NO: 98. In some embodiments, the selected VH sequence is SEQ ID NO: 90 and the selected VL sequence is SEQ ID NO: 99.

[0194] The present disclosure also provides antibodies or antigen-binding fragments thereof that can compete with the antibodies described herein. In some aspects, the antibodies or antigen-binding fragments can bind to the same epitope as the antibodies described herein.

[0195] The present disclosure also provides antibodies or antigen-binding fragments thereof that cross-compete with any of the antibodies or antigen-binding fragments described herein. Cross-competition assays are well known in the art and are described, for example, in Moore et al., "Antibody cross-competition analysis of the human immunodeficiency virus type 1 gp120 exterior envelope glycoprotein," Journal of Virology 70.3 (1996):1863-1872, the entire contents of which are incorporated herein by reference. In one aspect, the present disclosure also provides antibodies or antigen-binding fragments thereof that bind to the same epitope or region as any of the antibodies or antigen-binding fragments described herein. Epitope binding assays are well known in the art and are described, for example, in Estep et al., "High throughput solution-based measurement of antibody-antigen affinity and epitope binning," MAbs. Vol. 5. No. 2. Taylor & Francis, 2013, the entire contents of which are incorporated herein by reference.

[0196] To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison (e.g., gaps may be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences may be ignored). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that need to be introduced to optimally align the two sequences. For example, sequence comparison and percent identity determination between two sequences can be performed using the Blossum62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0197] The present disclosure also provides a nucleic acid comprising a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or an immunoglobulin light chain. The immunoglobulin heavy chain or immunoglobulin light chain comprises the CDRs shown in Figure 2 or Figure 3, or has the sequence shown in Figure 9. When a polypeptide is paired with a corresponding polypeptide (e.g., a corresponding heavy chain variable region or a corresponding light chain variable region), the paired polypeptide binds to the WT1 / HLA-A2 complex.

[0198] Anti-WT1 / HLA antibodies and antigen-binding fragments can also be antibody variants (including derivatives and conjugates) of antibodies or antibody fragments, as well as multispecific (e.g., bispecific) antibodies or antibody fragments. Additional antibodies provided herein include polyclonal, monoclonal, multispecific (multimeric, e.g., bispecific), human, chimeric (e.g., human-mouse chimeras), single-chain, and intracellularly produced antibodies (i.e., intrabodies), and antigen-binding fragments thereof. The antibodies or antigen-binding fragments thereof can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. In some embodiments, the antibody or antigen-binding fragment thereof is an IgG antibody or antigen-binding fragment thereof.

[0199] Antibody fragments are suitable for use in the provided methods, so long as they retain the desired affinity and specificity of the full-length antibody. Thus, antibody fragments that bind to the WT1 / HLA-A2 complex retain the ability to bind to the WT1 / HLA-A2 complex. Fv ​​fragments are antibody fragments that contain the complete antigen recognition and binding site. This region consists of a dimer of one heavy-chain variable domain and one light-chain variable domain in tight association, which can be essentially covalent, for example, in scFv. In this configuration, the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six CDRs, or a subset thereof, confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three CDRs specific for a given antigen) can have the ability to recognize and bind to an antigen, although usually with lower affinity than the entire binding site.

[0200] Single-chain Fv or (scFv) antibody fragments comprise the VH and VL domains (or regions) of an antibody, wherein these domains are present in a single polypeptide chain. Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding.

[0201] Fab fragments contain the variable and constant domains of the light chain and the variable and first constant domain (CH1) of the heavy chain. F(ab')2 antibody fragments comprise a pair of Fab fragments, generally covalently linked near their carboxy termini by hinge cysteines between them. Other chemical linkages of antibody fragments are well known in the art.

[0202] Diabodies are small antibody fragments that contain two antigen-binding sites, a VH connected to a VL (VH and VL) in the same polypeptide chain. By using a linker that is too short to allow pairing between the two domains on the same chain, the domains can be paired with the complementary domains of another chain and create two antigen-binding sites.

[0203] Linear antibodies comprise a pair of tandem Fd segments (VH-CH1-VH-CH1) that, together with complementary light chain polypeptides, form a pair of antigen-binding regions. Linear antibodies may be bispecific or monospecific.

[0204] A single-armed antibody can have a heavy chain and a light chain, and a heavy chain fragment containing the CH2 and CH3 domains of IgG. In some embodiments, a single-armed antibody is an antibody that has only one of the two antigen-binding arms of a typical antibody. In some embodiments, a single-armed antibody comprises an antigen-binding arm (e.g., VH+CH1 and VL+CL) and an Fc.

[0205] The antibodies and antibody fragments of the disclosure can be modified in the Fc region to confer desired effector function or serum half-life, hi some embodiments, the Fc region can be modified to silence or reduce complement-dependent cytotoxicity (CDC) or antibody-dependent cellular cytotoxicity (ADCC).

[0206] Antibody multimerization can be achieved by natural antibody aggregation or by chemical or recombinant conjugation techniques well known in the art. For example, a percentage of purified antibody preparations (e.g., purified IgG1 molecules) naturally form protein aggregates containing antibody homodimers and other higher order antibody multimers.

[0207] Alternatively, antibody homodimers can be formed by chemical coupling techniques well known in the art. For example, antibody multimers can be formed using heterobifunctional cross-linkers, including, but not limited to, SMCC (succinimidyl 4-(maleimidomethyl)cyclohexane-1-carboxylate) and SATA (N-succinimidyl S-acetylthioacetate). An exemplary procedure for forming antibody homodimers is described in Ghetie et al. (Proc. Natl. Acad. Sci. USA 94:7509-7514, 1997). Antibody homodimers can be converted to Fab'2 homodimers by pepsin digestion. Another method for forming antibody homodimers is to use the autophilic T15 peptide, as described in Zhao et al. (J. Immunol. 25:396-404, 2002).

[0208] In some embodiments, multispecific antibodies are bispecific antibodies. Bispecific antibodies can be produced by engineering the interface between a pair of antibody molecules to maximize the percentage of heterodimers recovered from recombinant cell culture. For example, the interface can comprise at least a portion of the CH3 domain of the antibody constant domain. In this method, one or more small amino acid side chains from the interface of a first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). By replacing the large amino acid side chains with smaller ones (e.g., alanine or threonine), a compensatory "cavity" of identical or similar size to the large side chain is created at the interface of the second antibody molecule. This provides a mechanism for increasing the yield of heterodimers over other unwanted end-products, such as homodimers. This method is described, for example, in WO 96 / 27011, incorporated by reference in its entirety.

[0209] Bispecific antibodies include cross-linked or "heteroconjugate" antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin, the other to biotin. Heteroconjugate antibodies can be made using any convenient cross-linking method. Suitable cross-linking agents and techniques are well known in the art and are disclosed in U.S. Pat. No. 4,676,980, incorporated herein by reference in its entirety.

[0210] Methods for generating bispecific antibodies from antibody fragments are also well known in the art. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al. (Science 229:81, 1985) describe a procedure in which intact antibodies are proteolytically cleaved to generate F(ab')2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The resulting Fab' fragments are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab'TNB derivatives is then reconverted to the Fab' thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of another Fab'TNB derivative to form the bispecific antibody.

[0211] In some embodiments, the present disclosure relates to a bispecific antibody comprising: (1) a first functional portion comprising an antibody or antigen-binding fragment described herein; (2) a second functional portion comprising a T cell binding molecule; and (3) a third functional portion comprising a single-chain human crystallizable fragment. In some embodiments, the T cell binding molecule targets CD3. In some embodiments, the T cell binding molecule is an anti-CD3 scFv. In some embodiments, the antibody or antigen-binding fragment described herein is an scFv. In some embodiments, the C-terminus of the antibody or antigen-binding fragment described herein is linked to the N-terminus of the anti-CD3 scFv via a peptide linker, and the C-terminus of the anti-CD3 scFv is linked to the single-chain human crystallizable fragment via a peptide linker. Figure 1B shows an exemplary structure of a bispecific antibody. In some embodiments, the T cell binding molecule is an anti-CD3 VHH. In some embodiments, an antibody or antigen-binding fragment described herein is attached to a human crystallizable fragment via a hinge region, and an anti-CD3 antibody or antigen-binding fragment thereof is attached to the same human crystallizable fragment via a hinge region. Figure 1C shows an exemplary structure of a bispecific antibody.

[0212] In some embodiments, provided herein are multispecific antibodies (e.g., bispecific antibodies) having an anti-WT1 arm and an anti-CD3 arm. In some embodiments, the anti-WT1 arm comprises a heavy chain (e.g., any heavy chain having a VH described herein) and a light chain (e.g., any light chain having a VL described herein). In some embodiments, the anti-CD3 arm comprises the heavy chain variable region (VHH) of an anti-CD3 heavy chain antibody. In some embodiments, the VHH comprises or consists of an amino acid sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 113. In some embodiments, the bispecific antibodies described herein have the schematic structure shown in FIG. 1C.

[0213] Any of the antibodies or antigen-binding fragments described herein can be conjugated to a stabilizing molecule (e.g., a molecule that increases the half-life of the antibody or antigen-binding fragment thereof in a subject or in solution). Non-limiting examples of stabilizing molecules include polymers (e.g., polyethylene glycol) or proteins (e.g., serum albumins, such as human serum albumin). Conjugation of a stabilizing molecule can increase the half-life or prolong the biological activity of the antibody or antigen-binding fragment in vitro (e.g., when stored in tissue culture medium or as a pharmaceutical composition) or in vivo (e.g., in humans).

[0214] In some embodiments, the antibodies or antigen-binding fragments described herein can be conjugated to a therapeutic agent. The antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof can be covalently or non-covalently bound to the therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic or cytostatic agent (e.g., cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracene, maytansinoids (such as DM-1 and DM-4), dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin, and cyclophosphamide, and analogs).

[0215] In some embodiments, the WT1 peptide comprises a sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence of SEQ ID NO:100.

[0216] In some embodiments, the antibodies or antigen-binding fragments described herein do not bind to MHC molecules in the absence of a WT1 peptide.

[0217] In some embodiments, the antibodies or antigen-binding fragments described herein do not bind to off-target peptides (sequence-similar peptides derived from the proteins MED13L and PIGQ) as measured by flow cytometry.

[0218] Antibody Drug Conjugates (ADCs) The antibodies, antigen-binding fragments thereof, or antigen-binding protein constructs (e.g., bispecific antibodies) described herein can be conjugated to a therapeutic agent (drug). The therapeutic agent can be covalently or non-covalently bound to the antibody or antigen-binding fragment, or antigen-binding protein construct (e.g., bispecific antibody). In some embodiments, bispecific antibodies share a common light chain.

[0219] In some embodiments, the therapeutic agent is a cytotoxic or cytostatic agent (e.g., monomethyl auristatin E, monomethyl auristatin F, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracene, maytansinoids (such as DM-1 and DM-4), dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin, and cyclophosphamide, and analogs). Useful classes of cytotoxic, cytostatic, or immunoregulatory agents include, for example, antitubulin agents, DNA minor groove binding agents, DNA replication inhibitors, and alkylating agents.

[0220] In some embodiments, therapeutic agents may include, but are not limited to, cytotoxic agents (e.g., chemotherapeutic agents, immunotherapeutic agents, etc.), antiviral agents, or antibacterial agents. In some embodiments, conjugable therapeutic agents may be selected from, but are not limited to, MMAE (monomethylauristatin E), MMAD (monomethylauristatin D), or MMAF (monomethylauristatin F).

[0221] In some embodiments, the cytotoxic agent is a camptothecin compound, an analog, or a derivative thereof (e.g., compounds CPT-1, CPT-2, CPT-3, CPT-4 in patent application PCT / CN2023 / 093976).

[0222] In some embodiments, the therapeutic agent is an auristatin, such as auristatin E (also known in the art as a derivative of dolastatin-10), or a derivative thereof. The auristatin can be, for example, an ester formed between auristatin E and a keto acid. For example, auristatin E can be reacted with paraacetylbenzoic acid or benzoylvaleric acid to produce AEB and AEVB, respectively. Other exemplary auristatins include AFP, MMAF, and MMAE. The synthesis and structures of exemplary auristatins are described in U.S. Patent Publication No. 2003-0083263, International Patent Publication No. WO 04 / 010957, International Patent Publication No. WO 04 / 010958, and U.S. Pat. Appl. KOKAI Publication No. 2004 / 010959. 02 / 088172, as well as U.S. Patent Nos. 7,498,298; 6,884,869; 6,323,315; 6,239,104; 6,034,065; 5,780,588; 5,665,860; 5,663,149; 5,635,483; 5,599,902; 5,554,725; 5,530,097; 5, Nos. 5,504,191; 5,410,024; 5,138,036; 5,076,973; 4,986,988; 4,978,744; 4,879,278; 4,816,444; and 4,486,414, each of which is incorporated by reference herein in its entirety for all purposes.

[0223] Auristatins have been shown to interfere with microtubule dynamics, as well as nuclear and cell division, and have been shown to have anti-cancer activity. Auristatins can bind to tubulin and exert cytotoxic or cytostatic effects in cancer cells. Many different assays known in the art exist that can be used to determine whether an auristatin or the resulting antibody-drug conjugate exerts a cytostatic or cytotoxic effect in the desired cells.

[0224] In some embodiments, the therapeutic agent is a chemotherapeutic agent. Examples of chemotherapeutic agents include thiotepa and cyclosphosphamide (CYTOXAN). TMalkylating agents such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneimines and methylamelamines such as altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; chlorambucil, chlornaphazine, colofsfamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobembine, phenethylamine ... Nitrogen mustards such as nesterin, prednimustine, trofosfamide, and uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; aclacinomycin, actinomycin, anthramycin, azaserine, bleomycin, cactinomycin, calicheamicin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, and epirubicin Antibiotics such as bicin, esorubicin, idarubicin, marcelomycin, mitomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfilomycin, puromycin, chelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, and trimetrexate; fludarabine, 6-mercaptopurine, Purine analogues such as thiamiprine and thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridinene, doxifluridine, enocitabine, floxuridine, and 5-FU; androgens such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone; antiadrenal agents such as aminoglutethimide, mitotein, and trilostane; folic acid supplements such as folinic acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid; amsacrine;Bestravsil; Bisantrene; Edatrexate; Defofamine; Demecolcine; Diaziquone; Elfomitine; Elliptinium acetate; Etoglucide; Gallium nitrate; Hydroxyurea; Lentinan; Lonidamine; Mitoguazone; Mitoxantrone; Mopidamol; Nitracrine; Pentostatin; Fenamet; Pirarubicin; Podophyllic acid; 2-Ethylhydrazide; Procarbazine; PSK 7; razoxane; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2''-trichlorotriethylamine; urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; taxanes, such as paclitaxel (TAXOL®, Bristol-Myers Squibb) Squibb Oncology, Princeton, NJ), doxetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France), chlorambucil, gemcitabine, 6-thioguanine, platinum analogs such as cisplatin or carboplatin, vinblastine, platinum, etoposide (VP-16), ifosfamide, mitomycin C, mitoxantrone, vincristine, vinorelbine, navelbine, novantrone, teniposide, daunomycin, aminopterin, xeloda, ibandronate, CPT-11, the topoisomerase inhibitor RFS 2000, difluoromethylornithine (DMFO); retinoic acid; esperamicin; capecitabine; and pharmaceutically acceptable salts, acids, or derivatives of any of the above. This definition includes, for example, tamoxifen, raloxifene, aromatase-inhibiting 4(5)imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston); and antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin;Also included are antihormonal agents that regulate or inhibit hormone action in tumors, such as antiestrogens, including pharmaceutically acceptable salts, acids, or derivatives of any of the above. A detailed description of chemotherapeutic agents can be found, for example, in US20180193477A1, which is incorporated by reference in its entirety.

[0225] In some embodiments, the antigen-binding construct is coupled to the drug via a cleavable linker, such as an SPBD linker or a maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (VC) linker. In some embodiments, the antigen-binding construct is coupled to the drug via a non-cleavable linker, such as an MCC linker formed with SMCC or sulfo-SMCC. Selection of an appropriate linker for a given ADC can be readily made by one of skill in the art, taking into account relevant factors such as the binding site to the antigen-binding construct, any structural constraints of the drug, and the hydrophobicity of the drug. Many specific linker-toxin combinations have been described and can, in certain embodiments, be used with the antigen-binding constructs described herein to prepare ADCs. Examples include, but are not limited to, cleavable peptide-based linkers with auristatins such as MMAE and MMAF, camptothecins such as SN-38, duocarmycins, and PBD dimers; non-cleavable MC-based linkers with auristatins MMAF and MMAE; acid-labile hydrazone-based linkers with calicheamicin and doxorubicin; disulfide-based linkers with maytansinoids such as DM1 and DM4; and bismaleimide trioxyethylene glycol (BMPEO)-based linkers with the maytansinoid DM1. Some of these therapeutic agents and linkers are described, for example, in Peters & Brown, (2015) Biosci. Rep. e00225, Dosio et al., (2014) Recent Patents on Anti-Cancer Drug Discovery 9:35-65, U.S. Patent Publication No. U2015 / 0374847, and US20180193477A1, which are incorporated herein by reference in their entireties.

[0226] Depending on the desired drug and the selected linker, those skilled in the art can select a suitable method for coupling them to each other. For example, several conventional coupling methods, such as amine coupling, can be used to form the desired drug-linker conjugate, which still contains a reactive group for covalently conjugating the antibody. In some embodiments, a drug-maleimide conjugate (i.e., a maleimide-linked drug) can be used for the payloads having reactive groups in the present disclosure. The most common reactive group that can be linked to thiol groups in ADC preparation is maleimide. In addition, organic bromides and iodides are also frequently used.

[0227] ADCs can be prepared by one of several art-known routes using organic chemistry reactions, conditions, and reagents well known to those skilled in the art (see, for example, Bioconjugate Techniques (G.T. Hermanson, 2013, Academic Press)). For example, conjugation can be achieved by (1) reacting a nucleophilic or electrophilic group on an antibody with a bivalent linker reagent to form an antibody-linker intermediate Ab-L, followed by covalent reaction with an activated drug moiety D; or (2) covalently reacting a nucleophilic or electrophilic group on a drug moiety with a linker reagent, followed by covalent reaction with a nucleophilic or electrophilic group on the antibody to form a drug-linker intermediate DL. Conjugation methods (1) and (2) can be used with a variety of antibodies, drug moieties, and linkers to prepare the ADCs described herein. The various linkers, linker components, and toxins prepared are either commercially available or can be prepared using standard synthetic organic chemistry techniques, as described, for example, in March's Advanced Organic Chemistry (Smith & March, 2006, Sixth Ed., Wiley), Toki et al., (2002) J. Org. Chem. 67:1866-1872, Frisch et al., (1997) Bioconj. Chem. 7:180-186; Bioconjugate Techniques (GTHermanson, 2013, Academic Press), US20210379193A1, and US20180193477A1, which are incorporated by reference in their entireties. Additionally, numerous preformed drug-linkers suitable for reaction with a selected antigen-binding construct are also commercially available; for example, linker-toxins including DM1, DM4, MMAE, MMAF, or duocarmycin SA are available from Creative BioLabs (Shirley, NY).

[0228] Some specific examples of methods for preparing ADCs are known in the art and are described in U.S. Patent No. 8,624,003 (Pot method), U.S. Patent No. 8,163,888 (one-step), and U.S. Patent No. 5,208,020 (two-step method), as well as U.S. Patent No. 20180193477A1, which are incorporated by reference herein in their entireties. Other methods are known in the art and include those described in Antibody-Drug Conjugates: Methods in Molecular Biology, 2013, Ducry (Ed.), Springer.

[0229] Drug loading is expressed by the number of drug moieties per antibody molecule in an ADC. For some antibody-drug conjugates, drug loading can be limited by the number of binding sites on the antibody. For example, when the linkage is a cysteine ​​thiol, as in certain exemplary embodiments described herein, drug loading can range from 0 to 8 drug moieties per antibody. In certain embodiments, high drug loading, e.g., p≧5, can cause aggregation, insolubility, toxicity, or cell permeability of certain antibody-drug conjugates. In certain embodiments, the average drug loading for an antibody-drug conjugate ranges from 1 to about 8, from about 2 to about 6, or from about 3 to about 5. In fact, it has been shown that the optimal ratio of drug moieties per antibody for certain antibody-drug conjugates can be about 4. In some embodiments, the drug-to-antibody ratio (DAR) is about or at least 1, 2, 3, 4, 5, 6, 7, or 8. In some embodiments, the average DAR in the composition is from about 1 to about 2, from about 2 to about 3, from about 3 to about 4, from about 3 to about 5, from about 4 to about 5, from about 5 to about 6, from about 6 to about 7, or from about 7 to about 8.

[0230] Antibody and ADC properties In some embodiments, the antibody (or antigen-binding fragment thereof), or ADC derived therefrom, is -1 Less than 0.01s -1 Less than 0.001s -1 Less than 0.0001s-1 Less than 0.00001s -1 Less than 0.000001s -1 Less than or 0.0000001s -1 In some embodiments, the antibody specifically binds to the WT1 / HLA-A2 complex with a koff of less than 0.01 s. -1 Super, 0.001s -1 Super, 0.0001s -1 Super, 0.00001s -1 Super, 0.000001s -1 Super, 0.0000001s -1 Greater than or 0.00000001s -1 It's super.

[0231] In some embodiments, the kinetic association rate (k) is 1×10 2 / Ms super, 1×10 3 / Ms super, 1×10 4 / Ms super, 1×10 5 / Ms or more than 1 × 10 6 In some embodiments, the kinetic association rate (k) is greater than 1×10 5 / Ms less than 1 × 10 6 / Ms or less than 1 x 10 7 / Ms is less than.

[0232] Affinity can be estimated from the quotient of the kinetic rate constants (K = k / k). In some embodiments, K is greater than or equal to 1 x 10 -6 Less than M, 1 x 10 -7 Less than M, 1 x 10 -8 Less than M, 1 x 10 -9 Less than M, 1 x 10 -10 Less than M, 1 x 10 -11 Less than M, 1 x 10 -12 Less than M, 1 x 10 -13 Less than M or 1 x 10 -14 In some embodiments, the KD is less than 50 nM, 30 nM, 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM. In some embodiments, the KD is less than 1 x 10 -7Super M, 1×10 -8 Super M, 1×10 -9 Super M, 1×10 -10 Super M, 1×10 -11 Super M, 1×10 -12 Super M, 1×10 -13 Super M, 1×10 -14 It's over M.

[0233] Common techniques for measuring the affinity of an antibody for an antigen include, for example, ELISA, RIA, and surface plasmon resonance (SPR).

[0234] In some embodiments, WT1 RMF The binding activity of the anti-WT1 / HLA antibodies to T2 cells (ATCC, catalog number: CRL-1992) pulsed with WT1 / HLA antibodies was examined by flow cytometry. In some embodiments, the EC50 is less than 0.1 nM, 0.2 nM, 0.4 nM, 0.8 nM, 1 nM, 1.2 nM, 1.4 nM, 1.8 nM, 2 nM, 2.2 nM, 2.4 nM, 2.8 nM, 3 nM, 3.2 nM, 3.4 nM, 3.8 nM, or 4 nM.

[0235] The ESK1 analogue is WT1, developed by Eureka Therapeutics, Inc. RMF It is a fully human TCR-like monoclonal IgG1 antibody (VH SEQ ID NO: 111, VL SEQ ID NO: 112) that targets the HLA-A1 / HLA-A2 complex.

[0236] In some embodiments, WT1 RMF The key targeting site (SEQ ID NO: 100) of the anti-WT1 / HLA antibody above is different from that of the antibody ESK1 analog.

[0237] In some embodiments, the anti-WT1 / HLA antibody is RMF WT1 in the / HLA-A2 complex RMF It binds to positions 2 and 9 of the peptide.

[0238] In some embodiments, the anti-WT1 / HLA antibody is RMFWT1 in the / HLA-A2 complex RMF It binds to positions 1, 3, and 5 of the peptide.

[0239] In some embodiments, the anti-WT1 / HLA antibody is RMF WT1 in the / HLA-A2 complex RMF It binds to position 1 of the peptide.

[0240] In some embodiments, the anti-WT1 / HLA antibody is RMF WT1 in the / HLA-A2 complex RMF It binds to positions 1, 3, 4, and 8 of the peptide.

[0241] In some embodiments, the anti-WT1 / HLA antibody is RMF WT1 in the / HLA-A2 complex RMF It binds to positions 1, 4, and 8 of the peptide.

[0242] In some embodiments, the anti-WT1 / HLA antibody is RMF WT1 in the / HLA-A2 complex RMF It binds to positions 1, 3, and 4 of the peptide.

[0243] In some embodiments, the anti-WT1 / HLA antibody is RMF WT1 in the / HLA-A2 complex RMF It binds to positions 3, 4, 5, and 8 of the peptide.

[0244] In some embodiments, the anti-WT1 / HLA antibody RMF Binding to WT1 and potential off-target peptides (sequence-similar peptides derived from the proteins MED13L and PIGQ) was verified by flow cytometry. RMFThe percentage of positive cells for pulsed T2 cells is greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99%. In some embodiments, the percentage of positive cells for T2 cells, MED13L-pulsed T2 cells, or PIGI-pulsed T2 cells is less than 1%, less than 2%, less than 3%, less than 4%, less than 5%, less than 6%, less than 7%, less than 8%, less than 9%, or less than 10%.

[0245] In some embodiments, anti-WT1 / CD3 bispecific antibodies (1G7-CD3, 7B9-CD3, P01199-CD3, P01218-CD3, P01264-CD3) at various concentrations (0.00001 μg / mL, 0.001 μg / mL, 0.1 μg / mL, 10 μg / mL) were co-incubated with purified CD3 T cells and target cells (HCT116-WT1 cells) at a 10:1 ratio (E:T) for 24 hours. In some embodiments, IFN-γ cytokine release is greater than 10 pg / mL, greater than 25 pg / mL, greater than 50 pg / mL, greater than 100 pg / mL, greater than 250 pg / mL, greater than 500 pg / mL, greater than 1000 pg / mL, greater than 2500 pg / mL, greater than 5000 pg / mL, or greater than 7000 pg / mL.

[0246] In some embodiments, thermal stability is measured. An antibody or antigen-binding fragment described herein, or an ADC derived therefrom, can have a Tm of greater than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95°C. In some embodiments, the Tm is less than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95°C.

[0247] In some embodiments, the ADCs described herein have an average drug-to-antibody ratio (DAR) as measured by HPLC that is greater than 3, greater than 3.2, greater than 3.4, greater than 3.6, greater than 3.8, greater than 4, greater than 4.2, greater than 4.4, or greater than 4.6. In some embodiments, the ADCs described herein have an average DAR as measured by HPLC that is less than 3, less than 3.2, less than 3.4, less than 3.6, less than 3.8, less than 4, less than 4.2, less than 4.4, or less than 4.6.

[0248] In some embodiments, the antibodies or antigen-binding fragments thereof, ADCs derived therefrom, or CAR-T cells described herein have a tumor growth inhibition percentage (TGI%) of more than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%. In some embodiments, the antibodies or antigen-binding fragments thereof described herein, ADCs derived therefrom, or CAR-T cells derived therefrom have a tumor growth inhibition percentage of less than 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%. TGI% can be measured, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days after the start of treatment, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after the start of treatment. As used herein, tumor growth inhibition percentage (TGI%) is calculated using the following formula: TGI(%)=[1-(Ti-T0) / (Vi-V0)]×100 Ti is the mean tumor volume in the treatment group on day i. T0 is the mean tumor volume in the treatment group on day 0. Vi is the mean tumor volume in the control group on day i. V0 is the mean tumor volume in the control group on day 0.

[0249] In some embodiments, the antibodies or antigen-binding fragments described herein, or ADCs derived therefrom, can bind to tumor cells presenting WT1 peptides via MHC molecules (e.g., HLA or HLA-A2). In some embodiments, the antibodies or antigen-binding fragments described herein, or ADCs derived therefrom, can induce complement-dependent cytotoxicity (CDC) and / or antibody-dependent cellular cytotoxicity (ADCC) to kill tumor cells.

[0250] In some embodiments, the antibodies or antigen-binding fragments described herein, or ADCs derived therefrom, comprise a functional Fc region. In some embodiments, the effector function of the functional Fc region is antibody-dependent cellular cytotoxicity (ADCC). In some embodiments, the effector function of the functional Fc region is phagocytosis. In some embodiments, the effector function of the functional Fc region is ADCC and phagocytosis.

[0251] In some embodiments, the antibodies or antigen-binding fragments described herein, or ADCs derived therefrom, are capable of inducing complement-dependent cytotoxicity (CDC).

[0252] In some embodiments, the Fc region is human IgG1, human IgG2, human IgG3, or human IgG4. In some embodiments, the antibody is a human IgG1 antibody, optionally with an SI mutation, a LALA mutation, an N297A mutation, a YTE mutation, and / or a FLAA mutation. In some embodiments, the antibody is a human IgG4 antibody, optionally with an SI mutation, a LALA mutation, an N297A mutation, a YTE mutation, and / or a FLAA mutation.

[0253] In some embodiments, the antibodies or antigen-binding fragments described herein, or ADCs derived therefrom, do not have a functional Fc region. For example, the antibodies or antigen-binding fragments are Fab, Fab', F(ab')2, and Fv fragments. In some embodiments, the Fc region has a LALA mutation (L234A and L235A mutations according to EU numbering) or a LALA-PG mutation (L234A, L235A, P329G mutations according to EU numbering). In some embodiments, the Fc region has a FLAA mutation (F234A and L235A mutations according to EU numbering). In some embodiments, the Fc region has an SI mutation (S239D and I332E mutations according to EU numbering). In some embodiments, the Fc region has an N297A mutation according to EU numbering. In some embodiments, the Fc has YTE mutations (M252Y, S254T, and T256E according to EU numbering).

[0254] Method for producing anti-WT1 / HLA antibodies Synthetic and purified WT1 / MHC complexes can be used as immunogens to generate antibodies using standard techniques for polyclonal and monoclonal antibody preparation. In some embodiments, MHC-I VH / VL mice (details of MHC-I VH / VL mice can be found in PCT / CN2022 / 081924) are transfected with WT1 (126-134) peptide (WT1), which is specifically presented by human HLA-A2. RMF , RMFPNAPYL, SEQ ID NO: 100).

[0255] Polyclonal antibodies can be raised in animals by multiple injections (e.g., subcutaneous or intraperitoneal) of the antigenic peptide or protein. In some embodiments, the antigenic peptide or protein is injected with at least one adjuvant. In some embodiments, the antigenic peptide or protein can be conjugated to an agent that is immunogenic in the species being immunized. Animals can be injected with the antigenic peptide or protein more than once (e.g., two, three, or four times).

[0256] An immunogen is typically used to prepare antibodies by immunizing a suitable subject (e.g., a human or transgenic animal expressing at least one human immunoglobulin locus). An appropriate immunogenic preparation can contain, for example, a recombinantly expressed or chemically synthesized polypeptide. The preparation can further include an adjuvant, such as Freund's complete or incomplete adjuvant, or a similar immunostimulant.

[0257] Variants of the antibodies or antigen-binding fragments described herein can be prepared by introducing appropriate nucleotide changes into DNA encoding a human, humanized, or chimeric antibody, or an antibody or antigen-binding fragment thereof described herein, or by peptide synthesis. Such variants include, for example, deletion, insertion, or substitution of residues within the amino acid sequence that makes up the antigen-binding site or domain of the antibody. Within such a population of variants, some antibodies or antigen-binding fragments may have increased affinity for their target, e.g., the WT1 / HLA-A2 complex. Any combination of deletion, insertion, and / or a combination can be achieved in antibodies or antigen-binding fragments thereof with increased binding affinity for their target. Amino acid changes introduced into the antibody or antigen-binding fragment, such as changing the number (e.g., increasing or decreasing) of glycosylation sites, changing the type of glycosylation site (e.g., changing the amino acid sequence so that different sugars are attached by enzymes present in cells), or introducing new glycosylation sites, can alter the antibody or antigen-binding fragment, or introduce new post-translational modifications into the antibody or antigen-binding fragment.

[0258] The antibodies disclosed herein can be derived from any species of animal, including mammals. Non-limiting examples of natural antibodies include antibodies derived from humans, primates, such as monkeys and apes, cows, pigs, horses, sheep, camelids (e.g., camels and llamas), chickens, goats, and rodents (e.g., rats, mice, hamsters, and rabbits), including transgenic rodents that have been genetically engineered to produce human antibodies.

[0259] Human and humanized antibodies include antibodies having variable and constant regions derived from (or having the same amino acid sequences derived from) human germline immunoglobulin sequences. Human antibodies can include, for example, amino acid residues within the CDRs that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).

[0260] Humanized antibodies typically have a human framework (FR) into which nonhuman CDRs have been grafted. Thus, humanized antibodies have one or more amino acid sequences introduced into a human from a nonhuman source. These nonhuman amino acid residues are often referred to as "import" residues, which typically come from an "import" variable domain. Humanization can essentially be performed, for example, by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. These methods are described, for example, in Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); and Verhoeyen et al., Science, 239:1534-1536 (1988), each of which is incorporated herein by reference in its entirety. Thus, a "humanized" antibody is a chimeric antibody in which significantly less than an intact human V domain has been substituted by the corresponding sequence from a nonhuman species. In practice, humanized antibodies are typically murine antibodies in which some CDR residues and some FR residues are substituted by residues from analogous sites in human antibodies.

[0261] Furthermore, it is important to humanize antibodies while retaining high specificity and affinity for the antigen and other favorable biological properties. To achieve this goal, humanized antibodies can be prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available that can illustrate and display probable three-dimensional conformations of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., analysis of residues that influence the ability of the candidate immunoglobulin to bind to its antigen. In this way, FR residues can be selected and combined from the recipient and import sequences to achieve desired antibody characteristics, such as increased affinity for the target antigen.

[0262] Typically, an amino acid sequence variant of a human, humanized, or chimeric anti-WT1 / HLA antibody contains an amino acid sequence that has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% percent identity with the sequence present in the light chain or heavy chain of the original antibody.

[0263] In some embodiments, mice with a humanized heavy chain immunoglobulin locus and a humanized kappa chain immunoglobulin locus (e.g., RenMab TM The heavy chain immunoglobulin locus is a region on a chromosome that contains the gene for the heavy chain of an antibody. The locus can include, for example, the human IGHV (variable) gene, the human IGHD (diversity) gene, the human IGHJ (joining) gene, and the mouse heavy chain constant domain gene. The kappa chain immunoglobulin locus is a region on a chromosome that contains the gene encoding the light chain (kappa chain) of an antibody. Examples of the kappa chain immunoglobulin locus include the human IGKV (variable) gene, the human IGKJ (joining) gene, and the mouse light chain constant domain gene. RenMab TMA detailed description of the mice can be found in PCT / CN2020 / 075698 or US20200390073A1, which are incorporated by reference in their entirety.

[0264] In some embodiments, mice with a humanized heavy chain immunoglobulin locus and a humanized kappa chain immunoglobulin locus (e.g., RenLite TM The heavy chain immunoglobulin locus is a region on a chromosome that contains genes for the heavy chain of an antibody. The locus can include, for example, the human IGHV (variable) gene, the human IGHD (diversity) gene, the human IGHJ (joining) gene, and the mouse heavy chain constant domain gene. The kappa chain immunoglobulin locus is a region on a chromosome that contains genes encoding the common light chain. Examples of kappa chain immunoglobulin loci include the human IGKV (variable) gene, the human IGKJ (joining) gene, and the mouse light chain constant domain gene. RenLite TM A detailed description of the mice can be found in PCT / CN2021 / 097652, which is incorporated by reference herein in its entirety.

[0265] Antibodies generated by mice have a fully human VH, a fully human VL, and a mouse constant region. In some embodiments, the human VH and human VL bind to a human IgG constant region (e.g., IgG1, IgG2, IgG3, and IgG4).

[0266] Identity or homology to the original sequence is typically the percentage of amino acid residues present in the candidate sequence that are identical to sequences present in a human, humanized, or chimeric anti-WT1 / HLA antibody or fragment, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering conservative substitutions as part of the sequence identity.

[0267] Further modifications can be made to the anti-WT1 / HLA antibody or antigen-binding fragment. For example, cysteine ​​residues can be introduced into the Fc region to allow interchain disulfide bond formation within this region. The homodimeric antibody thus generated may have some increased in vitro and / or in vivo half-life. For example, heterobifunctional cross-linkers, as described by Wolff et al. (Cancer Res. 53:2560-2565, 1993), can also be used to prepare homodimeric antibodies with increased in vitro and / or in vivo half-lives. Alternatively, antibodies can be engineered to have dual Fc regions (see, for example, Stevenson et al., Anti-Cancer Drug Design 3:219-230, 1989).

[0268] In some embodiments, covalent modifications can be made to anti-WT1 / HLA antibodies or antigen-binding fragments thereof. These covalent modifications can be made by chemical or enzymatic synthesis, or by enzymatic or chemical cleavage. Other types of covalent modifications of antibodies or antibody fragments are introduced into the molecule by reacting targeted amino acid residues of the antibody or fragment with organic derivatizing agents capable of reacting with selected side chains or the N- or C-terminal residues.

[0269] In some embodiments, antibody variants are provided that have carbohydrate structures lacking fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibodies may be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose in the glycan at Asn297 relative to the sum of all glycans (e.g., complex, hybrid, and high-mannose structures) attached to Asn297, as measured, for example, by MALDI-TOF mass spectrometry as described in WO 2008 / 077546. Asn297 refers to the asparagine residue located at position 297 (position 314 in the EU numbering of Fc region residues or Kabat numbering) within the Fc region; however, Asn297 may also be located approximately ±3 amino acids upstream or downstream from position 297, i.e., between positions 294 and 300, due to minor sequence variation in antibodies. Such fucosylation variants may have improved ADCC function. In some embodiments, the Fc region of the antibody is further engineered to replace asparagine at position 297 with alanine (N297A) to reduce glycan heterogeneity.

[0270] In some embodiments, to promote production efficiency by avoiding Fab-arm exchange, the Fc region of the antibody is further modified by replacing the serine at position 228 (EU numbering) of IgG4 with proline (S228P). A detailed description of the S228 mutation is provided, for example, in Silva et al. "The S228P mutation prevents in vivo and in vitro IgG4 Fab-arm exchange as demonstrated using a combination of novel quantitative immunoassays and physiological matrix preparation," Journal of Biological Chemistry 290.9(2015):5462-5469, which is incorporated by reference in its entirety.

[0271] Chimeric antigen receptor (CAR) Chimeric antigen receptors (CARs) combine many aspects of conventional T cell activation into a single protein. They link an extracellular antigen recognition domain to an intracellular signaling domain, activating T cells upon antigen binding. CARs typically consist of four regions: an antigen-binding domain, an extracellular hinge region, a transmembrane domain, and an intracellular T cell signaling domain.

[0272] The antigen-binding domain is the ectodomain portion of the receptor, exposed to the outside of the cell. It is a potential target molecule and is responsible for targeting CAR-T cells to any cells expressing the corresponding molecule. The antigen-binding domain is usually derived from the variable region of a monoclonal antibody, which is bound together as a single-chain variable fragment (scFv). An scFv is a chimeric protein formed by an immunoglobulin light chain (VL) and heavy chain (VH) connected by a short linker peptide. The linker between the two chains consists of hydrophilic residues, with a stretch of glycine and serine for flexibility, and a stretch of glutamate and lysine for added solubility. In some embodiments, the antigen-binding domain specifically binds to a tumor-associated antigen, e.g., BCMA, CD19, CD22, CD30, CD33, CD56, CD123 (also known as IL-3R), CEA, an EBV-associated antigen (e.g., LMP2), EGFR, GD2, GPC3, HER2, an HPV-associated antigen (e.g., E6), a MAGE antigen, mesothelin, MUC-1, NY-ESO-1, PSCA, PSMA, ROR1, WT1, or claudin 18.2. In some embodiments, the antigen-binding domain specifically binds to a WT1 / MHC complex (e.g., a WT1 / HLA-A2 complex). In some embodiments, the antigen-binding domain does not bind to an MHC molecule.

[0273] The hinge, also known as the spacer, is a small structural domain located between the antigen-binding domain and the outer membrane of the cell. An ideal hinge increases the flexibility of the scFv receptor head and reduces the spatial constraints between the CAR and its target antigen, facilitating antigen binding and synapse formation between the CAR-T cell and the target cell. Hinge sequences are often based on membrane-proximal regions from immune molecules, including, for example, IgG, CD8, and CD28.

[0274] The transmembrane domain is a structural element consisting of a hydrophobic alpha helix that spans the cell membrane. It anchors the CAR to the plasma membrane and bridges the extracellular hinge and antigen-binding domain to the intracellular signaling region. This domain is essential for the stability of the receptor as a whole. Generally, the transmembrane domain from the most membrane-proximal component of the endodomain is used, but different transmembrane domains confer different receptor stability. The CD28 transmembrane domain is known to result in highly expressed and stable receptors.

[0275] The intracellular T cell signaling domain lies within the endodomain of the intracellular receptor. After antigen binding to the external antigen-binding domain, CAR receptors cluster with each other and transmit an activation signal. The internal cytoplasmic tail of the receptor then perpetuates the signaling within the T cell. Normal T cell activation depends on phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) present in the cytoplasmic domain of CD3 zeta. To mimic this process, the cytoplasmic domain of CD3 zeta is commonly used as the primary CAR endodomain component. In addition to CD3 signaling, T cells also require costimulatory molecules to persist after activation. For this reason, CAR receptor endodomains usually also contain one or more chimeric domains derived from costimulatory proteins. The signaling domains of a wide variety of costimulatory molecules, including CD28, CD27, CD134 (OX40), and CD137 (4-1BB), have been successfully tested.

[0276] Various CAR molecules and vectors expressing these CAR molecules can be used in the methods described herein. In some embodiments, the CAR molecule specifically binds to a tumor-associated antigen, such as the WT1 / HLA-A2 complex.

[0277] Exemplary structures of antigen receptors, including hinges, transmembrane domains, and intracellular T cell signaling domains, as well as methods for recombinantly introducing such receptors into cells, are described, for example, in Chandran et al., "T cell receptor-based cancer immunotherapy: Emerging efficacy and pathways of resistance." Immunological reviews 290.1 ​​(2019): 127-147; Cartellieri, Marc, et al., "Chimeric antigen receptor-engineered T cells for immunotherapy of cancer." BioMed Research International 2010 (2010); and PCT Publication Nos. WO2017173256A1, US2002 / 131960, US2013 / 287748, US2013 / 0149337, US6,451,995, US7,446,190, and US8,252,592, which are incorporated by reference herein in their entireties.

[0278] The present disclosure provides a chimeric antigen receptor (CAR) or a fragment thereof that specifically binds to the WT1 / HLA-A2 complex. The CAR or a fragment thereof described herein is capable of binding to the WT1 / HLA-A2 complex.

[0279] The present disclosure provides a CAR or fragment thereof comprising (a) an extracellular antigen-binding domain that specifically recognizes the WT1 / HLA-A2 complex, (b) a transmembrane domain, and (c) an intracellular signaling region. In some embodiments, the antigen-binding domain comprises a heavy chain variable domain (VH) and a light chain variable domain (VL). In some embodiments, the VH and VL of a CAR or fragment thereof described herein are identical to the VH and VL of any of the antibodies or antigen-binding fragments described herein.

[0280] In some embodiments, single-chain variable fragments (scFv) of anti-WT1 / HLA antibodies (1G7-scFv, 3E6-scFv, P01264-scFv, and ESK1-scFv) were used to generate WT1-targeting CARs.

[0281] In some embodiments, the CAR comprises an anti-WT1 / HLA antigen-binding domain (e.g., 1G7-scFv, 3E6-scFv, P01264-scFv, or ESK1-scFv). In some embodiments, the CAR has the structure shown in Figure ID. In some embodiments, the CAR comprises the following elements linked in tandem: (1) a CD8α signal peptide, (2) an anti-WT1 / HLA antigen-binding domain (e.g., 1G7-scFv, 3E6-scFv, P01264-scFv, or ESK1-scFv), (3) a CD8α hinge region, (4) a CD8TM transmembrane domain, (5) a 4-1BB intracellular domain, and (6) a CD3ζ intracellular domain.

[0282] Recombinant cells The present disclosure provides recombinant cells (e.g., immune cells, T cells, NK cells, tumor-infiltrating lymphocytes) that express CARs and / or various proteins described herein. These recombinant cells can be used to treat various disorders or diseases (e.g., WT1-associated cancers) described herein.

[0283] In various embodiments, the engineered cells can be obtained from, for example, humans and non-human animals. In various embodiments, the engineered cells can be obtained from bacteria, fungi, humans, rats, mice, rabbits, monkeys, pigs, or any other species. Preferably, the cells are from humans, rats, or mice. In some embodiments, the cells are mouse lymphocytes and engineered (e.g., transduced) to express a CAR or an antigen-binding fragment thereof. In some embodiments, the cells are obtained from humans. In various embodiments, the engineered cells are blood cells. Preferably, the cells are leukocytes (e.g., T cells), lymphocytes, or any other suitable blood cell type. In some embodiments, the cells are peripheral blood cells. In some embodiments, the cells are tumor-infiltrating lymphocytes (TILs). In some embodiments, the cells are T cells, B cells, or NK cells. In some embodiments, the cells are human peripheral blood mononuclear cells (PBMCs). In some embodiments, the human PBMCs are CD3+ cells. In some embodiments, the human PBMCs are CD8+ cells.

[0284] In some embodiments, the cells are T cells. In some embodiments, the T cells can express a cell surface receptor that recognizes a specific antigen moiety on the surface of a target cell. The cell surface receptor can be a wild-type or recombinant T cell receptor (TCR), a chimeric antigen receptor (CAR), or any other surface receptor capable of recognizing an antigen moiety associated with a target cell. T cells can be obtained by methods well known in the art, such as in vitro culture of T cells isolated from a patient (e.g., tumor-infiltrating lymphocytes). Genetically modified T cells can be obtained by transducing T cells (e.g., isolated from the patient's peripheral blood) with a viral vector. In some embodiments, the T cells are CD4+ T cells, CD8+ T cells, or regulatory T cells. In some embodiments, the T cells are T helper type 1 T cells and T helper type 2 T cells. In some embodiments, the T cells expressing the receptor are αβ-T cells. In alternative embodiments, the T cells expressing the receptor are γδ-T cells. In some embodiments, the T cells are central memory T cells. In some embodiments, the T cells are effector memory T cells. In some embodiments, the T cells are naive T cells.

[0285] In some embodiments, the cells are NK cells. In some embodiments, preparation of recombinant cells includes one or more culture and / or preparation steps. Cells for introducing a binding molecule, e.g., a CAR, can be isolated from a sample, such as a biological sample, e.g., obtained from or derived from a subject. In some embodiments, the subject from which the cells are isolated is a subject with a disease or condition, or a subject in need of or to whom cell therapy will be administered. In some embodiments, the subject is a human in need of a particular therapeutic intervention, such as adoptive cell therapy, from which the cells are isolated, treated, and / or modified.

[0286] In some embodiments, the cells are stem cells, such as pluripotent and multipotent stem cells, including induced pluripotent stem cells (iPSCs). The cells can be primary cells, such as those isolated directly from a subject and / or isolated and frozen from a subject. In some embodiments, the stem cells are cultured with additional differentiation factors to obtain a desired cell type (e.g., T cells).

[0287] Various cell types can be obtained using appropriate isolation methods. Isolation methods include the separation of different cell types based on the intracellular expression or presence of one or more specific molecules, such as surface markers, e.g., surface proteins, intracellular markers, or nucleic acids. In some embodiments, any known separation method based on such markers can be used. In some embodiments, the separation is affinity- or immunoaffinity-based. For example, separation in some aspects involves the separation of cells and cell populations based on the cellular expression or expression level of one or more markers, typically cell surface markers, e.g., by incubation with an antibody or binding partner that specifically binds to such markers, followed by the separation of cells bound to the antibody or binding partner from those cells that are not bound to the antibody or binding partner, typically by a washing step.

[0288] Such separation steps can be based on positive selection, where cells that bind to the reagent are retained for further use, and / or negative selection, where cells that do not bind to the antibody or binding partner are retained. In some examples, both fractions are retained for further use. In some embodiments, negative selection can be particularly useful when antibodies that specifically identify cell types in a heterogeneous population are not available, and separation is best performed based on markers expressed by cells outside the desired population.

[0289] Also provided are methods, nucleic acids, compositions, and kits for expressing binding molecules and for producing genetically modified cells that express such binding molecules. Genetic modification generally involves introducing a nucleic acid encoding a therapeutic molecule, e.g., a CAR, e.g., a TCR-like CAR, a polypeptide, a fusion protein, into cells, such as by retroviral transduction, transfection, or transformation. In some embodiments, gene transfer is achieved by first stimulating the cells, e.g., in combination with a stimulus that induces a response such as proliferation, survival, and / or activation, e.g., as measured by expression of a cytokine or activation marker, and then transducing the activated cells and expanding them in culture to numbers sufficient for clinical use.

[0290] In some embodiments, the recombinant nucleic acid is delivered to cells using a recombinant infectious viral particle, such as a vector derived from Simian Virus 40 (SV40), adenovirus, or adeno-associated virus (AAV). In some embodiments, the recombinant nucleic acid is delivered to T cells using a retroviral vector, such as a recombinant lentiviral vector or a gammaretroviral vector. In some embodiments, the retroviral vector has a long terminal repeat (LTR), such as a retroviral vector derived from Moloney murine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), murine embryonic stem cell virus (MESV), murine stem cell virus (MSCV), or spleen focus-forming virus (SFFV). Most retroviral vectors are derived from murine retroviruses. In some embodiments, retroviruses include those derived from any avian or mammalian cell source. Retroviruses are typically amphotropic, meaning they can infect host cells of several species, including humans. In some embodiments, the vector is a lentiviral vector. In some embodiments, the recombinant nucleic acid is delivered to T cells by electroporation. In some embodiments, the recombinant nucleic acid is transferred to T cells by transfection. Other methods for introducing and expressing genetic material in immune cells include calcium phosphate transfection, protoplast fusion, cationic liposome-mediated transfection, tungsten particle-assisted particle bombardment, and strontium phosphate DNA co-precipitation. Many of these methods are described in WO2019195486, the entire contents of which are incorporated herein by reference.

[0291] Also provided are populations of recombinant cells, compositions containing such cells, and / or compositions enriched for such cells, such as in cells expressing a binding molecule that represent at least 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the total cells in a particular type of composition or cell, such as T cells, CD8+, or CD4+ cells.

[0292] In some embodiments, the recombinant cells (e.g., CAR-T cells) are co-cultured with target cells (e.g., antigen-presenting cells) for at least or about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 18 hours, 1 day, 2 days, 3 days, or more to activate the recombinant cells (e.g., CAR-T cells). In some embodiments, the target cells are Jurkat cells.

[0293] In some embodiments, IL-12 and modified IL-12 can be expressed by recombinant cells. For example, a fusion protein comprising a modified IL-12 described herein can be expressed on the cell surface of a recombinant cell, e.g., when the fusion protein is a membrane-tethered protein. In some cases, a fusion protein comprising a modified IL-12 described herein can be expressed and secreted, e.g., when the fusion protein is a soluble protein. Expression of IL-12 in recombinant cells provides several additional benefits. For example, it can increase IFN-γ production from NK and T cells, which is the most potent mediator of IL-12's effects; it can activate and stimulate the proliferation and cytotoxicity of NK cells, CD8+, and CD4+ T cells; it can shift the differentiation of CD4+ Th0 cells to a Th1 phenotype; it can increase antibody-dependent cellular cytotoxicity (ADCC) against tumor cells; and it can induce IgG and suppress IgE production from B cells, e.g., by at least or about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or 20-fold.

[0294] In some embodiments, co-culture with target cells can increase cytokine (e.g., IFNγ) secretion by recombinant cells by at least or about 1-fold, 2-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 500-fold, 1000-fold, 2000-fold, 5000-fold, 10000-fold, or more, compared to cytokine secretion levels of recombinant cells not co-cultured.

[0295] In some embodiments, the cells are human PBMCs and have been engineered (e.g., transduced) to express a CAR, or an antigen-binding fragment thereof.

[0296] In some embodiments, when the recombinant cells are co-cultured with target cells (e.g., cells expressing WT1), the recombinant cells can increase cytokine (e.g., IFNγ) expression or secretion by at least or about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or more. In some embodiments, when the recombinant cells are co-cultured with target cells (e.g., cells expressing WT1), the activated T cell population increases by at least or about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or more. In some embodiments, the activation state of T cells can be measured by CD69 expression levels.

[0297] Recombinant vector The present disclosure also provides recombinant vectors (e.g., expression vectors) comprising the isolated polynucleotides disclosed herein (e.g., polynucleotides encoding the polypeptides disclosed herein), host cells into which the recombinant vectors have been introduced (i.e., such that the host cell contains the polynucleotides and / or vectors comprising the polynucleotides), and the production of recombinant antibody polypeptides or fragments thereof by recombinant techniques.

[0298] As used herein, a "vector" is any construct capable of delivering one or more polynucleotides of interest to a host cell when the vector is introduced into the host cell. An "expression vector" is capable of delivering and expressing one or more polynucleotides of interest as encoded polypeptides in a host cell into which the expression vector is introduced. Thus, in an expression vector, a polynucleotide of interest is positioned for expression in the vector by being operably linked to control elements, such as a promoter, enhancer, and / or polyA tail, at, near, or adjacent to the integration site of the polynucleotide of interest, either within the vector or in the genome of the host cell, such that the polynucleotide of interest is translated in a host cell into which the expression vector is introduced.

[0299] Vectors can be introduced into host cells by methods well known in the art, such as electroporation, chemical transfection (e.g., DEAE-dextran), transformation, transfection, and infection and / or transduction (e.g., by recombinant viruses). Thus, non-limiting examples of vectors include viral vectors (which can be used to generate recombinant viruses), naked DNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors associated with cationic condensing agents.

[0300] In some embodiments, a polynucleotide disclosed herein (e.g., a polynucleotide encoding a polypeptide disclosed herein) is introduced using a viral expression system (e.g., variola or other poxvirus, retrovirus, or adenovirus), which may involve the use of a non-pathogenic (defective), replication-competent virus, or may employ a replication-defective virus, in which case viral propagation generally occurs only in complementary viral packaging cells. For example, Fisher-Hoch et al., 1989, Proc.Natl.Acad.Sci.USA 86:317-321, Flexner et al., 1989, Ann.NYAcad Sci.569:86-103, Flexner et al., 1990, Vaccine, 8:17-21, U.S. Pat. Nos. 4,603,112, 4,769,330, and 5,017,487, WO 89 / 01973, U.S. Pat. 91 / 02805, Berkner-Biotechniques, 6:616-627, 1988, Rosenfeld et al., 1991, Suitable systems are disclosed in Science, 252:431-434; Kolls et al., 1994, Proc. Natl. Acad. Sci. USA, 91:215-219; Kass-Eisler et al., 1993, Proc. Natl. Acad. Sci. USA, 90:11498-11502; Guzman et al., 1993, Circulation, 88:2838-2848; and Guzman et al., 1993, Cir. Res., 73:1202-1207. Techniques for incorporating DNA into such expression systems are well known to those skilled in the art. The DNA may also be "naked," as described, for example, in Ulmer et al., 1993, Science, 259:1745-1749 and Cohen, 1993, Science, 259:1691-1692.Uptake of naked DNA can be increased by coating the DNA onto biodegradable beads that are efficiently transported into cells.

[0301] For expression, a DNA insert containing an antibody-encoding or polypeptide-encoding polynucleotide disclosed herein can be operably linked to a suitable promoter (e.g., a heterologous promoter), such as the phage lambda PL promoter, the E. coli lac, trp, and tac promoters, the SV40 early and late promoters, and promoters of retroviral long terminal repeats, to name a few. Other suitable promoters are known to those of skill in the art. The expression construct can further contain sites for transcription initiation and termination, and, within the transcribed region, a ribosome binding site for translation. The coding portion of the mature transcripts expressed by the construct can include a translation initiation codon at the beginning and a termination codon (UAA, UGA, or UAG) positioned approximately at the end of the polypeptide to be translated.

[0302] As indicated, the expression vector can include at least one selectable marker. Such markers include dihydrofolate reductase or neomycin resistance genes for eukaryotic cell culture, and tetracycline or ampicillin resistance genes for culture in E. coli and other bacteria. Representative examples of suitable hosts include, but are not limited to, bacterial cells such as E. coli, Streptomyces, and Salmonella typhimurium cells, fungal cells such as yeast cells, insect cells such as Drosophila melanogaster S2 and Spodoptera litura Sf9 cells, animal cells such as CHO, COS, Bowes melanoma, and HK 293 cells, and plant cells. Appropriate culture media and conditions for the host cells described herein are well known in the art.

[0303] Non-limiting vectors for use in bacteria include pQE70, pQE60, and pQE-9 available from Qiagen, pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A available from Stratagene, and ptrc99a, pKK223-3, pKK233-3, pDR540, and pRIT5 available from Pharmacia. Non-limiting eukaryotic vectors include pWLNEO, pSV2CAT, pOG44, pXT1, and pSG available from Stratagene, and pSVK3, pBPV, pMSG, and pSVL available from Pharmacia. Other suitable vectors will be readily apparent to those of skill in the art.

[0304] Non-limiting bacterial promoters suitable for use include the E. coli lacI and lacZ promoters, the T3 and T7 promoters, the gpt promoter, the lambda PR and PL promoters, and the trp promoter. Suitable eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, promoters of retroviral LTRs such as those of Rous sarcoma virus (RSV), and metallothionein promoters such as the mouse metallothionein-I promoter.

[0305] In the yeast Saccharomyces cerevisiae, several vectors containing constitutive or inducible promoters, such as alpha factor, alcohol oxidase, and PGH, may be used. For reviews, see Ausubel et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, and Grant et al., Methods Enzymol., 153:516-544 (1997).

[0306] Introduction of the construct into the host cell can be accomplished by calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other methods described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986), which is incorporated herein by reference in its entirety.

[0307] Transcription of DNA encoding the antibodies of the present disclosure in more eukaryotic organisms can be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about 10 to 300 bp, that serve to increase transcriptional activity of a promoter in a given host cell type. Examples of enhancers include the SV40 enhancer, which is located on the late side of the replication origin at base pairs 100 to 270, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

[0308] For secretion of the translated protein into the lumen of the endoplasmic reticulum, the periplasmic space or the extracellular environment, appropriate secretion signals can be incorporated into the expressed polypeptide. The signals can be endogenous to the polypeptide or they can be heterologous signals.

[0309] Polypeptides (e.g., antibodies) can be expressed in modified forms, such as fusion proteins (e.g., GST fusions) or with histidine tags, and can contain not only secretion signals but also additional heterologous functional regions. For example, a region of additional amino acids, particularly charged amino acids, can be added to the N-terminus of a polypeptide to improve stability and durability in host cells during purification or during subsequent handling and storage. Peptide moieties can also be added to polypeptides to facilitate purification. Such regions can be removed before final preparation of the polypeptide. The addition of peptide moieties to polypeptides to, inter alia, cause secretion or excretion, improve stability, and facilitate purification is well known and routine in the art.

[0310] Methods for preparing recombinant cells The present disclosure provides methods or processes for preparing, manufacturing, and / or using recombinant cells to treat pathological diseases or conditions.

[0311] Cells for introducing a protein described herein, e.g., a CAR, can be isolated from a sample, such as a biological sample, e.g., obtained from or derived from a subject. In some embodiments, the subject from which the cells are isolated is a subject with a disease or condition, or a subject in need of or to whom cell therapy is to be administered. In some embodiments, the subject is a human in need of a particular therapeutic intervention, such as adoptive cell therapy, from which cells are isolated, treated, and / or modified.

[0312] Thus, the cells, in some embodiments, are primary cells, e.g., primary human cells. Samples include tissues, fluids, and other samples taken directly from a subject, as well as samples obtained after one or more processing steps, such as separation, centrifugation, genetic modification (e.g., transduction with a viral vector), washing, and / or incubation. Biological samples can be samples obtained directly from a biological source or samples that have been processed. Biological samples include, but are not limited to, bodily fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine, and sweat, tissue and organ samples (including processed samples derived therefrom).

[0313] In some aspects, the sample from which cells are derived or isolated is a blood or blood-derived sample, or an apheresis or leukapheresis product, or is derived therefrom. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), white blood cells, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut-associated lymphoid tissue, mucosa-associated lymphoid tissue, spleen, other lymphoid tissue, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsils, or other organs, and / or cells derived therefrom. Samples include samples from autologous and allogeneic sources in the context of cell therapy, e.g., adoptive cell therapy.

[0314] In some embodiments, the cells are derived from a cell line, e.g., a T cell line. In some embodiments, the cells are derived from a heterologous source, e.g., a mouse, rat, or non-human primate. In some embodiments, the cells are isolated from a mouse lymph node.

[0315] In some embodiments, blood cells collected from a subject are washed, e.g., the plasma fraction is removed, and the cells are placed in an appropriate buffer or medium for subsequent processing steps. In some embodiments, the cells are washed with phosphate buffered saline (PBS). In some embodiments, the wash solution lacks calcium and / or magnesium, and / or many or all divalent cations. In some aspects, the washing step is accomplished by a semi-automated "flow-through" centrifuge. In some aspects, the washing step is accomplished by tangential flow filtration (TFF). In some embodiments, the cells are washed after washing, e.g., by removing Ca. 2+ / Mg 2+ The cells are resuspended in various biocompatible buffers, such as PBS-free. In certain embodiments, the components of the blood cell sample are removed and the cells are resuspended directly in culture medium. In some embodiments, methods include density-based cell separation methods, such as preparing white blood cells from peripheral blood by lysing red blood cells and centrifugation through a Percoll or Ficoll gradient.

[0316] In some embodiments, the method includes one or more of the following steps: isolating T cells from the patient's blood; transducing a population of T cells with a viral vector comprising a nucleic acid construct encoding a genetically engineered antigen receptor; expanding the transduced cells in vitro; and / or injecting the expanded cells into the patient, wherein the engineered T cells seek out and destroy antigen-positive tumor cells. In some embodiments, the nucleic acid construct further comprises a sequence encoding an inhibitory protein. The method further includes transfecting the T cells with a viral vector containing the nucleic acid construct.

[0317] In some embodiments, the method involves introducing any of the vectors described herein into cells in vitro or ex vivo. In some embodiments, the vector is a viral vector, and the introducing is performed by transduction. In some embodiments, the cells are transduced for at least or about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 18 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, or more. In some embodiments, the method further involves introducing one or more agents into the cells, each of the one or more agents independently capable of genetic disruption of the T cell receptor alpha constant (TRAC) gene and / or the T cell receptor beta constant (TRBC) gene. In some embodiments, the one or more agents is an inhibitory nucleic acid (e.g., siRNA). In some embodiments, the one or more agents is a fusion protein comprising a DNA-targeting protein and a nuclease or an RNA-guided nuclease (e.g., a clustered regularly interspaced short palindromic nucleic acid (CRISPR)-associated nuclease).

[0318] Transfection of T cells can be achieved using any standard method, such as calcium phosphate, electroporation, liposome-mediated transfer, microinjection, biolistic particle delivery systems, or any other method known to those of skill in the art. In some embodiments, transfection of T cells is performed using calcium phosphate methods.

[0319] The present disclosure provides a method for producing personalized anti-tumor immunotherapy. Genetically modified T cells can be produced from a patient's blood cells. These modified T cells are then reinfused into the patient as a cell therapy product.

[0320] Treatment method The antibodies, antigen-binding fragments thereof, or recombinant cells of the present disclosure can be used for a variety of therapeutic purposes.

[0321] In one aspect, the present disclosure provides methods of treating cancer in a subject, reducing the rate of growth of tumor volume in a subject over time, reducing the risk of developing metastases, or reducing the risk of developing further metastases in a subject. In some embodiments, treatment can halt, slow, prevent, or inhibit the progression of cancer. In some embodiments, treatment can result in a reduction in the number, severity, and / or duration of one or more symptoms of cancer in a subject.

[0322] In one aspect, the disclosure features a method that includes administering a therapeutically effective amount of a recombinant cell expressing a CAR to a subject in need thereof (e.g., a subject having, or identified or diagnosed as having, cancer).

[0323] In one aspect, the disclosure relates to a method comprising administering a therapeutically effective amount of an antibody, antigen-binding fragment thereof, or recombinant cell disclosed herein to a subject in need thereof (e.g., a subject having, or identified or diagnosed as having, cancer, e.g., breast cancer (e.g., triple-negative breast cancer), carcinoid cancer, cervical cancer, endometrial cancer, glioma, head and neck cancer, liver cancer, lung cancer, small cell lung cancer, lymphoma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, colorectal cancer, gastric cancer, testicular cancer, thyroid cancer, bladder cancer, urethral cancer, or a hematological malignancy). In some embodiments, the cancer is unresectable or metastatic melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), bladder cancer, or metastatic hormone-refractory prostate cancer. In some embodiments, the cancer is NSCLC, ovarian cancer, melanoma, colorectal cancer, breast cancer, hematological malignancies, head and neck cancer, gastrointestinal cancer, bladder cancer, or bone cancer. In some embodiments, the subject has a solid tumor. In some embodiments, the cancer is squamous cell carcinoma of the head and neck (SCCHN), renal cell carcinoma (RCC), triple-negative breast cancer (TNBC), or colon cancer. In some embodiments, the subject has Hodgkin's lymphoma. In some embodiments, the subject has triple-negative breast cancer (TNBC), gastric cancer, urothelial carcinoma, Merkel cell carcinoma, or head and neck cancer. In some embodiments, the cancer is melanoma, pancreatic cancer, mesothelioma, hematological malignancies, particularly non-Hodgkin's lymphoma, lymphoma, chronic lymphocytic leukemia, or an aggressive solid tumor. In some embodiments, the cancer is leukemia, breast cancer, ovarian cancer, glioblastoma, or soft tissue sarcoma.

[0324] In some embodiments, the compositions and methods disclosed herein can be used to treat patients at risk for cancer. Patients with cancer can be identified by a variety of methods known in the art.

[0325] As used herein, "effective amount" means an amount or dosage sufficient to bring about a beneficial or desired result, including halting, slowing, preventing, or inhibiting the progression of a disease, e.g., an autoimmune disease or cancer. The effective amount will vary depending on, for example, the age and weight of the subject to whom the antibody, antigen-binding fragment, polynucleotide encoding the antibody, vector comprising the polynucleotide, and / or composition is administered, the severity of the symptoms, and the route of administration, and thus, dosing can be determined on an individual basis.

[0326] An effective amount can be administered in one or more administrations. For example, an effective amount of an antibody or antigen-binding fragment is an amount sufficient to palliate, arrest, stabilize, reverse, inhibit, slow, and / or delay the progression of an autoimmune disease or cancer in a patient, or to palliate, arrest, stabilize, reverse, slow, and / or delay the proliferation of cells (e.g., biopsy cells, any of the cancer cells described herein, or cell lines (e.g., cancer cell lines)) in vitro. As understood in the art, an effective amount of an antibody or antigen-binding fragment may vary depending on other factors, such as, inter alia, the patient's medical history, as well as the type (and / or dosage) of antibody used.

[0327] Effective amounts and schedules for administering the antibodies, antibody-encoding polynucleotides, and / or compositions disclosed herein can be determined empirically, and making such determinations is within the skill of one in the art. One of skill in the art will understand that the dosage required to be administered will vary depending, for example, on the mammal receiving the antibodies, antibody-encoding polynucleotides, and / or compositions disclosed herein, the route of administration, the particular type of antibody, antibody-encoding polynucleotide, antigen-binding fragment, and / or composition disclosed herein used, and other agents administered to the mammal. Guidance for selecting appropriate doses for antibodies or antigen-binding fragments can be found in literature on the therapeutic use of antibodies and antigen-binding fragments, e.g., Handbook of Monoclonal Antibodies, Ferrone et al., eds., Noges Publications, Park Ridge, NJ, 1985, ch. 22 and pp. 303-357; Smith et al., Antibodies in Human Diagnosis and Therapy, Haber et al., eds., Raven Press, New York, 1977, pp. 365-389.

[0328] A typical daily dose of an effective amount of an antibody or ADC is 0.01 mg / kg to 100 mg / kg. In some embodiments, the dose can be less than 100 mg / kg, 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.5 mg / kg, or 0.1 mg / kg. In some embodiments, the dose can be greater than 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.5 mg / kg, 0.1 mg / kg, 0.05 mg / kg, or 0.01 mg / kg. In some embodiments, the dose is about 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.9 mg / kg, 0.8 mg / kg, 0.7 mg / kg, 0.6 mg / kg, 0.5 mg / kg, 0.4 mg / kg, 0.3 mg / kg, 0.2 mg / kg, or 0.1 mg / kg.

[0329] In any of the methods described herein, at least one antibody, antigen-binding fragment thereof, recombinant cell, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding fragments, or pharmaceutical compositions described herein), and optionally at least one additional therapeutic agent, can be administered to a subject at least once a week (e.g., once a week, twice a week, three times a week, four times a week, once a day, twice a day, or three times a day). In some embodiments, at least two different antibodies and / or antigen-binding fragments are administered in the same composition (e.g., a liquid composition). In some embodiments, at least one antibody or antigen-binding fragment and at least one additional therapeutic agent are administered in the same composition (e.g., a liquid composition). In some embodiments, at least one antibody or antigen-binding fragment and at least one additional therapeutic agent are administered in two different compositions (e.g., a liquid composition containing at least one antibody or antigen-binding fragment and a solid oral composition containing at least one additional therapeutic agent). In some embodiments, at least one additional therapeutic agent is administered as a pill, tablet, or capsule. In some embodiments, the at least one additional therapeutic agent is administered in a sustained release oral formulation.

[0330] In some embodiments, one or more additional therapeutic agents can be administered to a subject before or after administration of at least one antibody, antigen-binding antibody fragment, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding antibody fragments, or pharmaceutical compositions described herein). In some embodiments, the one or more additional therapeutic agents and at least one antibody, antigen-binding antibody fragment, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding antibody fragments, or pharmaceutical compositions described herein) are administered to a subject such that there is overlap in the period of biological activity of the one or more additional therapeutic agents with the period of biological activity of the at least one antibody or antigen-binding fragment (e.g., any of the antibodies or antigen-binding fragments described herein) in the subject.

[0331] In some embodiments, a subject can be administered at least one antibody, antigen-binding antibody fragment, recombinant cell, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding antibody fragments, recombinant cells, or pharmaceutical compositions described herein) over an extended period of time (e.g., over a period of at least 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 2 years, 3 years, 4 years, or 5 years). A skilled medical professional can determine the length of treatment period using any of the methods described herein to diagnose or track the effectiveness of the treatment (e.g., to monitor at least one symptom of cancer). As described herein, a skilled medical professional can also vary (e.g., increase or decrease) the identity and number of antibodies or antigen-binding antibody fragments (and / or one or more additional therapeutic agents) administered to a subject, and can adjust (e.g., increase or decrease) the dosage or frequency of administration of at least one antibody or antigen-binding antibody fragment (and / or one or more additional therapeutic agents) to a subject based on an evaluation of the effectiveness of the treatment (e.g., using any of the methods described herein and well known in the art).

[0332] In some embodiments, one or more additional therapeutic agents can be administered to the subject. The additional therapeutic agents can include one or more inhibitors selected from the group consisting of inhibitors of B-Raf, EGFR inhibitors, inhibitors of MEK, inhibitors of ERK, inhibitors of K-Ras, inhibitors of c-Met, inhibitors of anaplastic lymphoma kinase (ALK), inhibitors of phosphatidylinositol 3-kinase (PI3K), inhibitors of Akt, inhibitors of mTOR, dual PI3K / mTOR inhibitors, inhibitors of Bruton's tyrosine kinase (BTK), and inhibitors of isocitrate dehydrogenase 1 (IDH1) and / or isocitrate dehydrogenase 2 (IDH2). In some embodiments, the additional therapeutic agent is an inhibitor of indoleamine 2,3-dioxygenase-1 (IDO1) (e.g., epacadostat).

[0333] In some embodiments, the additional therapeutic agent can comprise one or more inhibitors selected from the group consisting of inhibitors of LSD1, inhibitors of MDM2, inhibitors of BCL2, inhibitors of CHK1, inhibitors of the activated hedgehog signaling pathway, and agents that selectively degrade the estrogen receptor.

[0334] In some embodiments, the additional therapeutic agent is trabectedin, nab-paclitaxel, trebananib, pazopanib, cediranib, palbociclib, everolimus, fluoropyrimidine, IFL, regorafenib, leolysin, Alimta, Dicaida, Sutent, temsirolimus, axitinib, everolimus, sorafenib, Votrient, pazopanib, IMA-901, AGS-003 , cabozantinib, vinflunine, Hsp90 inhibitors, Ad-GM-CSF, temozolomide, IL-2, IFNa, vinblastine, thalomid, dacarbazine, cyclophosphamide, lenalidomide, azacitidine, lenalidomide, bortezomib, amrubicin, carfilzomib, pralatrexate, and enzastaurin.

[0335] In some embodiments, the additional therapeutic agent can comprise one or more therapeutic agents selected from the group consisting of an adjuvant, a TLR agonist, tumor necrosis factor (TNF) alpha, IL-1, HMGB1, an IL-10 antagonist, an IL-4 antagonist, an IL-13 antagonist, an IL-17 antagonist, an HVEM antagonist, an ICOS agonist, a CX3CL1 targeted therapy, a CXCL9 targeted therapy, a CXCL10 targeted therapy, a CCL5 targeted therapy, an LFA-1 agonist, an ICAM1 agonist, and a HER2 agonist.

[0336] In some embodiments, carboplatin, nab-paclitaxel, paclitaxel, cisplatin, pemetrexed, gemcitabine, FOLFOX, or FOLFIRI is administered to the subject.

[0337] In some embodiments, the additional therapeutic agent is an anti-OX40 antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-LAG-3 antibody, an anti-TIGIT antibody, an anti-BTLA antibody, an anti-CTLA-4 antibody, an anti-ICOS antibody, an anti-CD27 antibody, an anti-OX40 antibody, an anti-4-1BB antibody, an anti-CD40 antibody, and / or an anti-GITR antibody.

[0338] In one aspect, the present disclosure provides a combination therapy. In some embodiments, an anti-WT1 / HLA antibody or antigen-binding fragment thereof (e.g., any antibody described herein) can be administered together with an immunomodulatory agent (e.g., lenalidomide).

[0339] Pharmaceutical Compositions and Routes of Administration Also provided herein are pharmaceutical compositions containing at least one (e.g., 1, 2, 3, or 4) of the antibodies or antigen-binding fragments described herein. Two or more (e.g., 2, 3, or 4) of any of the antibodies or antigen-binding fragments described herein can be present in the pharmaceutical composition, in any combination. Pharmaceutical compositions can be formulated in any manner known in the art.

[0340] Pharmaceutical compositions are formulated to be compatible with their intended route of administration (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal). Compositions may contain a sterile diluent (e.g., sterile water or saline), fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents, antibacterial or antifungal agents (e.g., benzyl alcohol, methylparaben, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.), antioxidants (e.g., ascorbic acid or sodium bisulfite), chelating agents (e.g., ethylenediaminetetraacetic acid), buffers (e.g., acetate, citrate, or phosphate), and isotonic agents (e.g., sugars (e.g., dextrose), polyalcohols (e.g., mannitol or sorbitol), or salts (e.g., sodium chloride)), or any combination thereof. Liposomal suspensions can also be used as pharmaceutically acceptable carriers (see, e.g., U.S. Pat. No. 4,522,811). The composition preparation can be formulated and enclosed in ampoules, disposable syringes, or multiple-dose vials. Where necessary (e.g., in injectable formulations), proper fluidity can be maintained, for example, by the use of a coating such as lecithin or a surfactant. Absorption of the antibody or antigen-binding fragment thereof can be prolonged by including an agent that delays absorption (e.g., aluminum monostearate and gelatin). Alternatively, sustained release can be achieved by implants and microencapsulated delivery systems, which can include biodegradable, biocompatible polymers (e.g., ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid; Alza Corporation and Nova Pharmaceutical, Inc.).

[0341] Compositions containing one or more of any of the antibodies or antigen-binding fragments described herein can be formulated for parenteral (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal) administration in unit dosage form (i.e., physically discrete units containing a predetermined amount of active compound(s) for ease of administration and uniformity of dosage).

[0342] Pharmaceutical compositions for parenteral administration are preferably sterile, substantially isotonic, and manufactured under Good Manufacturing Practice (GMP) conditions. Pharmaceutical compositions may be provided in unit dosage form (i.e., a dose for a single administration). Pharmaceutical compositions may be formulated using one or more physiologically acceptable carriers, diluents, excipients, or adjuvants. The formulation depends on the chosen route of administration. For injection, the antibody may be formulated in an aqueous solution, preferably in a physiologically compatible buffer, to reduce discomfort at the injection site. The solution may contain formulating agents such as suspending agents, stabilizing agents, and / or dispersing agents. Alternatively, the antibody may be in lyophilized form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use.

[0343] Compositions comprising recombinant cells for administration are provided, including pharmaceutical compositions and formulations, such as unit dose compositions containing the number of cells to administer in a given dose or fraction thereof. Pharmaceutical compositions and formulations can include one or more optional pharmaceutically acceptable carriers or excipients.

[0344] The toxicity and therapeutic efficacy of a composition can be determined by standard pharmaceutical procedures in cell cultures or experimental animals (e.g., monkeys). For example, the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population) can be determined, and the therapeutic index is the ratio of LD50:ED50. Drugs that exhibit a high therapeutic index are preferred. If a drug exhibits undesirable side effects, care should be taken to minimize the potential for harm (i.e., reduce the undesirable side effects). Toxicity and therapeutic efficacy can be determined by other standard pharmaceutical procedures.

[0345] Data obtained from cell culture assays and animal studies can be used in formulating an appropriate dose of any given agent for use in a subject (e.g., a human). A therapeutically effective amount of one or more (e.g., one, two, three, or four) antibodies or antigen-binding fragments thereof (e.g., any of the antibodies or antibody fragments described herein) is an amount that treats the disease in a subject (e.g., a human subject identified as having cancer) or a subject identified as at risk for developing the disease (e.g., a subject who previously developed cancer but has now been cured), (e.g., an amount that reduces the severity, frequency, and / or duration of one or more symptoms of the disease in the subject (e.g., a human). The efficacy and administration of any of the antibodies or antigen-binding fragments described herein can be determined by a health care professional or veterinary professional using methods well known in the art, as well as by observing one or more symptoms of the disease in the subject (e.g., a human). Certain factors can affect the dosage and timing required to effectively treat a subject (e.g., the severity of the disease or disorder, previous treatments, the overall health and / or age of the subject, and the presence of other diseases).

[0346] Exemplary doses include amounts (milligrams or micrograms) of any of the antibodies or antigen-binding fragments described herein per kilogram of subject body weight (e.g., about 1 μg / kg to about 500 mg / kg; about 100 μg / kg to about 500 mg / kg; about 100 μg / kg to about 50 mg / kg; about 10 μg / kg to about 5 mg / kg; about 10 μg / kg to about 0.5 mg / kg; or about 1 μg / kg to about 50 μg / kg). While these doses cover a wide range, those of skill in the art will appreciate that the efficacy and effective amounts of therapeutic agents comprising antibodies and antigen-binding fragments thereof can be determined by methods well known in the art. Typically, a relatively low dose is administered initially, and the dose can be subsequently and gradually increased by the attending health care professional or veterinary professional (for therapeutic uses) or by a researcher (if still working in the development phase) until an appropriate response is obtained. In addition, it is understood that the specific dose level for any particular subject will depend on a variety of factors, including the activity of the specific compound used, the subject's age, weight, general health, sex, and diet, time of administration, route of administration, rate of excretion, and half-life of the antibody or antibody fragment in vivo.

[0347] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.The present disclosure also provides methods for producing antibodies or antigen-binding fragments thereof for the various uses described herein. [Example]

[0348] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0349] Example 1. Generation of anti-WT1 / HLA antibodies MHC-I VH / VL mice (a detailed description of MHC-I VH / VL mice can be found in PCT / CN2022 / 081924, which is incorporated herein by reference in its entirety) were transfected with WT1 (126-134) peptide (WT1), which is specifically presented by human HLA-A2.RMF , RMFPNAPYL, SEQ ID NO: 100). MHC-I VH / VL mice are mice with a humanized light chain immunoglobulin locus, a humanized heavy chain immunoglobulin locus, and a humanized major histocompatibility complex (MHC) protein complex.

[0350] Antigen-positive immune cells were isolated from the immunized mice and fused with mouse myeloma cells to form hybridoma cells. Hybridoma cells secreting antigen-specific monoclonal antibodies were screened to obtain antigen-specific antibodies. The heavy and light chain variable region sequences of antigen-specific antibodies can also be obtained directly from the immunized mice by isolating antigen-positive B cells. For example, single-cell technology (e.g., Beacon® Optofluidic System, Berkeley Lights Inc.) can be used to screen and isolate plasma cells secreting antigen-specific monoclonal antibodies, followed by reverse transcription and PCR sequencing to obtain the antibody variable region sequences. The sequences can then be used to express antibodies. WT1 RMF The specificity of the expressed antibodies to bind to the HLA-A2 / HLA-A2 complex can be verified by FACS (fluorescence activated cell sorting). Exemplary antibodies obtained by this method included 1E9, 1G7, 3A6, 3E6, 4F10, and 7B9.

[0351] In another experiment, phage display was performed to identify WT1 RMF Monoclonal antibodies specific for the HLA-A1 / HLA-A2 complex were screened and discovered. Exemplary antibodies obtained by this method include P01199, P01218, and P01264.

[0352] The heavy and light chain variable regions of 1E9, 1G7, 3A6, 3E6, 4F10, 7B9, P01199, P01218, and P01264 are shown in Figure 9. Figures 2 and 3 show the heavy and light chain CDR sequences of 1E9, 1G7, 3A6, 3E6, 4F10, 7B9, P01199, P01218, and P01264, respectively, under the Kabat and Chothia definitions.

[0353] Various IgG1, IgG2, and IgG4 antibodies were produced. When antibody VH / VL are linked to various isotypes, the isotype is added to the name. For example, when the VH and VL of 1G7 are linked to the IgG1 constant region, the antibody is named 1G7-IgG1. Examples of other isotypes include 1G7-IgG2 and 1G7-IgG4. The constant region can also contain some mutations. For example, SI mutations (EU numbering: S239D and I332E mutations) are introduced into the Fc region of 1G7-IgG1, and the resulting antibody is named 1G7-IgG1-SI.

[0354] Example 2. Binding affinity of anti-WT1 / HLA antibodies Anti-WT1 / HLA antibody, WT1 RMF The affinity for the / HLA-A2 complex was measured by surface plasmon resonance (SPR) using an 8K biosensor equipped with a pre-immobilized protein A sensor chip on a Biacore (Biacore, INC, Piscataway NJ).

[0355] The purified anti-WT1 / HLA antibody was diluted to 1 μg / mL and then injected onto a Biacore 8K biosensor at 10 μL / min for approximately 50 seconds to achieve the desired protein density (e.g., approximately 50 response units (RU)). His-tagged WT1 was then injected at concentrations of 200, 100, 50, 25, 6.25, or 1.56 nM. RMF The WT1 / HLA-A2 complex (WT1(126-134) peptide, specifically presented by human HLA-A2) was injected at 30 μL / min for 120 s. Dissociation was monitored for 600 s. After the last injection of each titration, the chip was regenerated with glycine (pH 2.0, 30 μL / min for 30 s).

[0356] Kinetic association rates (k) and dissociation rates (k) were obtained simultaneously by fitting the entire data set to a 1:1 Langmuir binding model (Karlsson, R. Roos, H. Fagerstam, L. Petersson, B., 1994. Methods Enzymology 6.99-110) using Biacore 8K Evaluation software 3.0. The affinity was estimated from the quotient of the kinetic rate constants (K = k / k).

[0357] As one of skill in the art would understand, the same method was performed for each antibody tested, adjusting parameters (e.g., antibody concentration) appropriately. The results for the antibodies tested are summarized in the table below.

[0358] [Table 1]

[0359] All nine antibodies were RMF The results showed that the antibody showed good binding affinity to the IgG / HLA-A2 complex.

[0360] Example 3. WT1 RMF Verification of the binding activity of anti-WT1 / HLA antibodies to T2 cells pulsed with WT1 WT1 RMF The binding activity of the anti-WT1 / HLA antibody to T2 cells (ATCC, catalog number: CRL-1992) pulsed with WT1 was examined by flow cytometry.

[0361] T2 cells, WT1 RMFThe cells were incubated with WT1 / HLA antibody (50 μM) for 16 hours at 37°C in 5% CO. After washing, the cells were suspended in cold PBS containing serially diluted anti-WT1 / HLA antibodies (45 nM, 15 nM, 3 nM, 0.6 nM, 0.12 nM, 0.024 nM, 0.0048 nM, and 0.00096 nM) for 30 minutes at 4°C, followed by incubation with Alexa Fluor® 647 anti-human IgG Fcγ (Jackson Immuno Research Laboratories, Inc., catalog number 109-606-170) for 30 minutes.

[0362] Cells were harvested and mean fluorescence intensity (MFI) was measured. A curve fit was generated using antibody concentration (nM) as the X-axis and MFI as the Y-axis to obtain binding EC50 values. The results are shown in the table below.

[0363] [Table 2]

[0364] ESK1 is WT1 RMF ESK1 is a fully human TCR-like monoclonal IgG1 antibody that targets the HLA-A1 / HLA-A2 complex. The VH and VL sequences of ESK1 are shown as SEQ ID NO: 111 and SEQ ID NO: 112, respectively.

[0365] Anti-WT1 / HLA antibodies 1E9-IgG1-SI, 1G7-IgG1-SI, 3A6-IgG1-SI, 3E6-IgG1-SI, 4F10-IgG1-SI, 7B9-IgG1-SI, P01218-IgG1-SI, and P01264-IgG1-SI are WT1 RMF The results showed that the antibody showed good binding activity to T2 cells pulsed with IgG.

[0366] Example 4. Determination of binding sites of anti-WT1 / HLA antibodies by alanine scanning assay WT1 RMF To investigate the binding site of anti-WT1 / HLA antibodies to WT1, an alanine scanning assay was used to determine the epitopes recognized by anti-WT1 / HLA antibodies.RMF The amino acids at positions 1, 3, 4, 5, 6, 7, and 8 of the formula (I) were individually replaced with alanine to obtain a series of peptides (as shown in the table below). Flow cytometry was used to measure the binding of T2 cells pulsed with these peptides (peptide concentration: 25 μM) and anti-WT1 / HLA antibodies (antibody concentration: 10 μg / mL). The secondary antibody was Alexa Fluor® 647 anti-human IgG Fcγ (Jackson Immuno Research Laboratories, Inc., catalog number: 109-606-170). The mean fluorescence intensity (MFI) results are shown in Figures 4A to 4J.

[0367] [Table 3]

[0368] Example 5. Binding of anti-WT1 / HLA antibodies to potential off-target peptides Anti-WT1 / HLA antibody, WT1 RMF Binding to the IgG1 and potential off-target peptides (sequence-similar peptides derived from the proteins MED13L and PIGQ) was verified by flow cytometry.

[0369] By pulsing HLA-A2 cells with various peptides, binding of anti-WT1 / HLA antibodies to peptides was measured in a TAP1 / 2-deficient T2 lymphoblastoid cell line.

[0370] T2 cells were treated with 25 μM WT1 RMFThe cells were incubated with either the MED13L peptide (RMFPTPPSL, SEQ ID NO: 107), or the PIGQ peptide (RMFPGEVAL, SEQ ID NO: 108) at 37°C and 5% CO for 16 hours. After washing, the cells were suspended in cold PBS and incubated with 10 μg / mL of anti-WT1 / HLA antibody for 30 minutes at 4°C, followed by incubation with Alexa Fluor® 647 anti-human IgG Fcγ (Jackson Immuno Research Laboratories, Inc., catalog number: 109-606-170). The results are shown in the table below.

[0371] [Table 4]

[0372] The PIGQ peptide (SEQ ID NO: 107) and the MED13L peptide (SEQ ID NO: 108) were synthesized by the WT1 RMF The ESK1 analog binds to both the PIGQ peptide and the MED13L peptide, and has a similar sequence to the peptide.

[0373] The PIGQ peptide occurs ubiquitously in healthy human tissues. Binding to the PIGQ / HLA complex is therefore highly undesirable. Five of the nine amino acids of the PIGQ peptide are identical to those of the WT1 peptide.

[0374] Anti-WT1 / HLA antibodies 3A6-IgG1-SI, 3E6-IgG1-SI, 7B9-IgG1-SI, P01199-IgG1-SI, P01218-IgG1-SI, P01264-IgG1-SI, 1E9-IgG1-SI, 1G7-IgG1-SI, and 4F10-IgG1-SI are WT1 RMF The results showed that the positive control ESK1 analog showed good binding affinity to the WT1 / HLA complex, but not to the MED13L / HLA complex and the PIGQ / HLA complex. RMFThese nine antibodies bound not only to the WT1 / HLA complex but also to MED13L-pulsed and PIGQ-pulsed T2 cells at high rates (>50%). Therefore, these nine antibodies bound not to the potential off-target peptides, PIGQ / HLA complex, and MED13L / HLA complex, but to the WT1 / HLA complex. RMF It showed specificity for the / HLA complex.

[0375] In another similar experiment, 50 μM WT1 RMF The binding activity of 1G7-IgG1-SI or RG6007-WT1 to T2 cells pulsed with RG6007-WT1, MED13L peptide, or PIGQ peptide was examined by flow cytometry. The results are shown in Figures 15A and 15B, and show that 1G7-IgG1-SI binds to WT1 more effectively than RG6007-WT1. RMF This indicates that the antibody showed good binding specificity to the antibody.

[0376] RG6007 is a T cell bispecific (TCB) trivalent antibody construct targeting the HLA-A2-restricted Wilms tumor gene (WT1) and human CD3e, and is currently in Phase I clinical trials at Roche as an intravenous infusion for the treatment of adult patients with hematologically and molecularly relapsed / refractory acute myeloid leukemia. The VH (WT1 and CD3 portions) and VL (WT1 and CD3 portions) sequences of RG6007 are shown as SEQ ID NOs: 117, 118, 119, and 120, respectively. RG6007-WT1 was constructed by linking the VH (WT1 portion) and VL (WT1 portion) of RG6007 to an IgG1 Fc with an SI mutation.

[0377] Example 6. Preparation of anti-WT1 / CD3 bispecific antibody Various bispecific antibodies can be produced using VH and VL sequences derived from anti-WT1 / HLA antibodies (e.g., 1E9, 1G7, 3A6, 3E6, 4F10, 7B9, P01199, P01218, and P01264) and the anti-CD3 antibodies described herein. Some exemplary antibody structures of bispecific antibodies are shown in Figures 1A to 1C.

[0378] Structural form I WT1-scFv (1E9-scFv, 1G7-scFv, 3A6-scFv, 3E6-scFv, 4F10-scFv, 7B9-scFv, P01199-scFv, P01218-scFv, or P01264-scFv) and CD3-scFv (VH SEQ ID NO: 109, VL SEQ ID NO: 110) can be paired to form a bispecific antibody.

[0379] The structural form of the bispecific antibody is a BITE (bispecific T cell engager) molecule linked to the Fc region of an IgG1 (containing the N297G mutation). The BITE molecule consists of two single-chain variable fragments (scFvs) connected in tandem by a flexible fusion linker; one scFv binds to the cell surface protein CD3, and the other scFv binds to the tumor cell surface antigen WT1. An exemplary structure is shown in Figure 1B. Exemplary bispecific antibodies obtained included 1E9-CD3, 1G7-CD3, 3A6-CD3, 3E6-CD3, 4F10-CD3, 7B9-CD3, P01199-CD3, P01218-CD3, and P01264-CD3.

[0380] Structural form II The bispecific antibody has an anti-WT1 arm comprising a heavy chain and a light chain, and an anti-CD3 arm comprising the variable domain of the heavy chain of a heavy chain antibody (VHH) (e.g., CD3 VHH, SEQ ID NO: 113) connected to the CH2 and CH3 domains of human IgG1. In the Fc region, knob-into-hole mutations were introduced into the anti-WT1 arm heavy chain and the anti-CD3 arm heavy chain. Exemplary antibody structures are shown in Figure 1C. The resulting exemplary bispecific antibodies included 1E9-CD3 (VHH), 1G7-CD3 (VHH), 3A6-CD3 (VHH), 3E6-CD3 (VHH), 4F10-CD3 (VHH), 7B9-CD3 (VHH), P01199-CD3 (VHH), P01218-CD3 (VHH), and P01264-CD3 (VHH). For example, 1E9-CD3 (VHH) is a bispecific antibody having an IgG1 heavy chain constant region, where the heavy chain constant region of CD3 contains a knob mutation and the heavy chain constant region of 1E9 contains a hole mutation. The sequences of the light chain constant region, the heavy chain constant region with the knob mutation, and the heavy chain constant region with the hole mutation are shown in SEQ ID NO: 114, SEQ ID NO: 115, and SEQ ID NO: 116, respectively.

[0381] Chinese hamster ovary (CHO) cells were transfected with an anti-WT1 / HLA x anti-CD3 bispecific antibody expression vector to express the bispecific antibody. The CHO cell supernatant containing the anti-WT1 / HLA x anti-CD3 bispecific antibody was collected and purified to obtain the anti-WT1 / HLA x anti-CD3 bispecific antibody. The cell supernatant was collected and purified by protein A affinity chromatography.

[0382] Example 7. Binding affinity of anti-WT1 / CD3 bispecific antibodies The effects of anti-WT1 / HLA antibodies and anti-WT1 / CD3 bispecific antibodies (1E9-CD3, 1G7-CD3, 3A6-CD3, 3E6-CD3, 4F10-CD3, 7B9-CD3, P01199-CD3, P01218-CD3, and P01264-CD3) on HCT116 cells, HCT116-WT1 cells (WT1 RMFBinding to HCT116 cells expressing the HLA-A2 complex, Jurkat cells, and / or CD3+ T cells was assessed by flow cytometry. Among these cells tested, CD3 expression was not detected in HCT116 or HCT116-WT1 cells.

[0383] Specifically, HCT116 cells, HCT116-WT1 cells, Jurkat cells, and T cells were incubated with anti-WT1 / HLA antibody or anti-WT1 / CD3 bispecific antibody for 30 min at 4°C, respectively, and then incubated with His-tagged FITC-conjugated antibody for an additional 30 min, and the mean fluorescence intensity (MFI) was measured.

[0384] The results showed that none of the tested anti-WT1 / HLA antibodies or anti-WT1 / CD3 bispecific antibodies showed binding to HCT116 cells. In comparison, a positive signal was detected in HCT116-WT1 cells, indicating binding. Both CD3+ T cells and Jurkat cells showed binding to the nine anti-WT1 / CD3 bispecific antibodies, but none showed binding to the anti-WT1 / HLA antibodies. Taken together, the anti-WT1 / CD3 bispecific antibodies can selectively recognize tumor cells (expressing WT1 and HLA-A2) and CD3+ T cells.

[0385] Example 8. Cytotoxicity and T cell activation in recombinant WT1 and HLA-A2-positive tumor cells (HCT116-WT1 cells) Anti-WT1 / CD3 bispecific antibodies (1G7-CD3, 7B9-CD3, P01199-CD3, P01218-CD3, and P01264-CD3) at various concentrations (0.00001 μg / mL, 0.001 μg / mL, 0.1 μg / mL, and 10 μg / mL) were co-incubated with purified effector cells (CD3+ T cells) and target cells (HCT116-WT1 cells) at a 10:1 ratio (E:T) for 24 h. In the control group, target cells were replaced with HCT116 cells or T cells to test the specificity of cell killing. After 24 h of co-incubation, the cells were centrifuged at 500 g for 5 min at 4°C. The supernatant was collected and the amount of the cytokine IFN-γ was detected. The cell pellet was stained with dead and live dyes for flow cytometry. The results are shown in Table 5 and Figures 5A to 5D.

[0386] [Table 5]

[0387] The results showed that the five anti-WT1 / CD3 bispecific antibodies induced cytotoxicity against HCT116-WT1 cells in a dose-dependent manner.

[0388] Human IFN-γ cytokine release in the supernatants was measured by ELISA at 24 h, and the results are shown in the table below and in FIG.

[0389] [Table 6]

[0390] When HCT116-WT1 cells were used as target cells, significant induction of human IFN-γ secretion was detected within 24 hours, and dose-dependent induction of IFN-γ release was observed at certain concentrations of five anti-WT1 / CD3 bispecific antibodies. In contrast, when HCT116 cells or T cells were used as target cells, IFN-γ release was not induced.

[0391] Example 9. In vivo results of bispecific antibodies The effect of anti-WT1 / CD3 bispecific antibodies (P01264-CD3 and P01218-CD3) on tumor growth in vivo was examined in a colon carcinoma model. 6 HCT116-WT1 cells were subcutaneously injected into B-NDG mice (Biocytogen, Catalog No. B-CM-001). On the day of tumor cell injection, each mouse was also injected with PBMCs (5E6) (via iv injection). Tumors in the mice grew to a size of 60-120 mm. 3 Once the tumor volume reached 100 μg / ml, the mice were randomly assigned to different groups based on tumor volume and the amount of hCD45. Treatment groups were randomly selected for treatment with anti-WT1 / CD3 bispecific antibodies (P01264-CD3 or P01218-CD3) by intraperitoneal (ip) administration. Control group mice were injected with an equal volume of phosphate-buffered saline (PBS). The administration frequency was twice a week (5 administrations in total). Details are shown in the table below.

[0392] [Table 7]

[0393] The lengths of the long and short axes of the tumor were measured, and the tumor volume was calculated as 0.5 × (long axis) × (short axis). 2 It was calculated as:

[0394] Tumor growth inhibition percentage (TGI%) is calculated using the following formula: (TGI%)=[1-(Ti-T0) / (Vi-V0)]×100%. Ti is the mean tumor volume in the treatment group on day i. T0 is the mean tumor volume in the treatment group on day 0. Vi is the mean tumor volume in the control group on day i. V0 is the mean tumor volume in the control group on day 0.

[0395] Values ​​are expressed as mean ± SEM (standard error of the mean). T-test was performed for statistical analysis. A TGI% of more than 60% indicates a clear inhibition of tumor growth. P<0.05 was the threshold for indicating a significant difference.

[0396] The weight of the mice was monitored throughout the treatment period. The weights of the mice in different groups all decreased to different degrees, but the weights of the mice in different groups were not significantly different from each other.

[0397] The tumor sizes in the anti-WT1 / CD3 bispecific antibody-treated groups are shown in Figure 7. The table below lists the results of this experiment, including tumor volumes on the day of grouping (day 0), 14 days after grouping (day 14), and at the end of the experiment (day 21); tumor growth inhibition scores (TGI); and statistical differences (P values) in tumor volume and body weight between the treatment and control groups.

[0398] [Table 8]

[0399] Results: Tumor volumes in the treatment group were smaller than those in the PBS group. The anti-WT1 / -CD3 bispecific antibodies P01218-CD3 and P01264-CD3 demonstrated sustained and potent tumor-suppressing effects. P01218-CD3 at a dose of 0.5 mg / kg even exhibited a better tumor-suppressing effect than P01264-CD3 at a dose of 3 mg / kg.

[0400] Example 10. Antitumor activity of CAR-T cells Single-chain variable fragments (scFv) of anti-WT1 / HLA antibodies (1G7-scFv, 3E6-scFv, P01264-scFv, and ESK1-scFv) were used to generate WT1-targeting CARs. In some embodiments, the CARs have the structure shown in Figure ID. The CARs contain the following elements linked in series: (1) a CD8α signal peptide, (2) an anti-WT1 / HLA antigen-binding portion (e.g., 1G7-scFv, 3E6-scFv, P01264-scFv, or ESK1-scFv), (3) a CD8α hinge region, (4) a CD8TM transmembrane domain, (5) a 4-1BB intracellular domain, and (6) a CD3ζ intracellular domain. The resulting CAR-T cells were given different names based on the order of VH and VL in the scFv. "VH-VL" means that the VH is coupled to the CD8α signal peptide and the VL is coupled to the CD8α hinge region. In contrast, "VL-VH" means that the VL is coupled to the CD8α signal peptide and the VH is coupled to the CD8α hinge region. Thus, the resulting CAR-T cells included 1G7-CAR-T(VH-VL), 3E6-CAR-T(VH-VL), P01264-CAR-T(VH-VL), ESK1-CAR-T(VH-VL), 1G7-CAR-T(VL-VH), 3E6-CAR-T(VL-VH), P01264-CAR-T(VL-VH), 1E9-CAR-T(VL-VH), 3A6-CAR-T(VL-VH), 4F10-CAR-T(VL-VH), 7B9-CAR-T(VL-VH), P01199-CAR-T(VL-VH), and P01218-CAR-T(VL-VH).

[0401] Antitumor activity of CAR-T cells in a colon cancer model The effect of CAR-T cells (1G7-CAR-T(VH-VL), 3E6-CAR-T(VH-VL), P01264-CAR-T(VH-VL), ESK1-CAR-T(VH-VL), 1G7-CAR-T(VL-VH), 3E6-CAR-T(VL-VH), P01264-CAR-T(VL-VH)) on in vivo tumor growth in a colorectal cancer model was tested. Approximately 5 × 10 6HCT116-WT1 cells were subcutaneously injected into B-NDG mice. The tumor volume in the mice was approximately 50-100 mm. 3 Once the tumor volume reached 3000 mm, the mice were randomly divided into different groups based on tumor volume. Then, on the same day after grouping, the mice were intravenously (iv) injected with an equal volume of saline, mock T cells, or CAR-T cells. The administration frequency was one administration in total. The tumor volume was measured twice a week, and the body weight of the mice was also measured. When the tumor volume of the mice reached 3000 mm, the mice were randomly divided into different groups based on tumor volume. Then, on the same day after grouping, the mice were intravenously (iv) injected with an equal volume of saline, mock T cells, or CAR-T cells. The administration frequency was one administration in total. The tumor volume was measured twice a week, and the body weight of the mice was also measured. 3 Animals were ethically sacrificed when the age exceeded 100. Details are shown in the table below.

[0402] [Table 9]

[0403] The weights of all mice in the different groups increased. On the day of group assignment (day 0), the mean weight of each group ranged from 18.9 g to 19.4 g. At the end of the experiment (day 30), the mean weight of each group ranged from 19.1 g to 22.4 g, and the mean weight change of each group ranged from 99.9% to 117.6%. The results showed that the tested CARs were well tolerated and had no obvious toxicity to mice.

[0404] Tumor sizes in the CAR-T cell-treated groups are shown in Figure 8. The table below lists the results for this experiment, including tumor volumes on the day of grouping (day 0), 13 days after grouping (day 13), 20 days after grouping (day 20), and 30 days after grouping (day 30); mouse survival rates; tumor growth inhibition values ​​(TGI); and statistical differences (P values) in tumor volume and body weight between the treatment and control groups.

[0405] [Table 10]

[0406] The tumor volumes in the treatment groups were smaller than those in the control groups (saline and mock T). CAR-T cells demonstrated a sustained and potent tumor-suppressing effect. Complete tumor suppression was achieved through a series of CAR-T cell treatments. In addition, the experiment was continued until 56 days after group allocation (day 56), and all treatment groups still demonstrated complete tumor suppression.

[0407] In another similar experiment, we tested the effects of saline (G1), mock T cells (G2), and CART cells 1E9-CAR-T(VL-VH) (G3), 3A6-CAR-T(VL-VH) (G4), 4F10-CAR-T(VL-VH) (G5), P01199-CAR-T(VL-VH) (G6), and P01218-CAR-T(VL-VH) (G7) on in vivo tumor growth in the HCT116-WT1 colon cancer model.

[0408] All mice in groups G3 to G8 gained weight, indicating that the tested CAR-T cells were well tolerated and had no obvious toxicity to the mice.

[0409] Tumor sizes in the CAR-T cell-treated groups are shown in Figure 10. The table below lists the results for this experiment, including tumor volumes on the day of grouping (day 0), 14 days after grouping (day 14), and 28 days after grouping (day 28); survival and tumor-free mice 56 days after grouping (day 56); tumor growth inhibition values ​​(TGI); and statistical differences (P values) in tumor volumes between the treatment and control groups.

[0410] [Table 11]

[0411] At the end of the experiment, all mice in treatment groups G3 to G7 survived and their tumors regressed, while all mice in control groups G1 and G2 died, indicating that all five anti-WT1 CAR-T cells had a sustained and potent tumor-suppressing effect.

[0412] Antitumor effects of CAR-T cells in leukemia models Approximately 1×10 6 THP-1-luc cells (Cobioer, Cat. No. CBP30129L) were injected into B-NDG mice via the tail vein. Tumor growth in the mice was observed using an in vivo imaging system. The luminescence signature value was 1 × 10 6 When the mice reached 100% IR, they were randomly assigned to different groups. Then, on the same day of grouping, the mice were injected intravenously (iv) with an equal volume of saline, mock T cells, or CAR-T cells. Plots of mouse radiance (p / sec / cm) are shown. 2 The blood glucose (g / sr) was measured twice a week, and the weight of the mice was also measured. The details are shown in the table below.

[0413] [Table 12]

[0414] Tumor growth inhibition percentage (TGI%) is calculated using the following formula: (TGI%)=[1-(Ti-T1) / (Ri-R1)]×100%. Ti is the mean plot of radiance for the treatment group on day i. T1 is the mean plot of radiance for the treatment group on day 1. Ri is the mean plot of radiance for the control group on day i. R1 is the mean plot of radiance for the control group on day 1.

[0415] Values ​​are expressed as mean ± SEM (standard error of the mean). For statistical analysis, a T-test was performed. P<0.05 was the threshold for indicating a significant difference.

[0416] The weight of the mice was monitored throughout the treatment period, and the weights of the mice in the different groups were not significantly different from each other.

[0417] The results showed that the CAR-T cells in the treatment groups (G3 to G8) all exhibited different tumor-suppressing effects, with P01218-CAR-T (VL-VH) exhibiting the highest tumor-suppressing effect with a TGI of 96.6% on day 26.

[0418] In addition, the experiment was continued until 57 days after group allocation (day 57), and the survival rates of all groups are shown in Figure 11. Mice in all six anti-WT1 CAR-T treatment groups showed longer survival times than mice in the control group. In particular, the P01218-CAR-T (VL-VH) treatment group showed the longest survival time and highest survival rate among all treatment groups.

[0419] Example 11. Cytotoxic effect of anti-WT1 / CD3 bispecific antibody Anti-WT1 / CD3 bispecific antibodies (1E9-CD3, 1G7-CD3, 3E6-CD3, 7B9-CD3, P01199-CD3, and P01218-CD3) were serially diluted (5-fold, 9 gradients) at a maximum concentration of 100 nM and then incubated with purified effector cells (CD3+ T cells) and target cells THP-1 cells (HLA-A2) for 24 or 48 hours, respectively. + , WT1 RMF + ), or OVCAR3 cells (ATCC, Catalog No.: HTB-161, HLA-A2 + WT1 RMF + ) at a 10:1 ratio (E:T) to test the cell-killing activity. In the control group, the target cells were replaced with HCT116 cells. After co-incubation, the cells were centrifuged at 500g for 5 min at 4°C. The cell pellets were stained with dead and live dyes for flow cytometry. The supernatants were collected to detect the expression level of the cytokine IFN-γ.

[0420] The results are shown in Figures 12 and 13, which show that 1E9-CD3, 1G7-CD3, 3E6-CD3, 7B9-CD3, P01199-CD3, and P01218-CD3 can kill THP-1 and OVCAR3 cells, but not WT1. RMFThe negative control HCT116 cells, which do not express RG6007, did not kill them. However, the positive control RG6007 analogs exhibited cytotoxicity against HCT116 cells at 48 hours, indicating that 1E9-CD3, 1G7-CD3, 3E6-CD3, 7B9-CD3, P01199-CD3, and P01218-CD3 exhibited better specific targeting and killing performance than the RG6007 analogs in OVCAR3 cells.

[0421] In another similar experiment, the cytotoxicity of the anti-WT1 / CD3 bispecific antibodies 1E9-CD3(VHH), 1G7-CD3(VHH), 3A6-CD3(VHH), 3E6-CD3(VHH), 4F10-CD3(VHH), 7B9-CD3(VHH), P01199-CD3(VHH), P01218-CD3(VHH), and P01264-CD3(VHH) in natural tumor cells was tested. The results are shown in Figure 14, which indicates that all nine bispecific antibodies were able to kill THP-1 and / or OVCAR3 cells, but not WT1. RMF It was shown that HCT116 cells that do not express β-glucan were not killed.

[0422] Other embodiments While the present invention has been described in conjunction with its detailed description, it should be understood that the above description is intended to be illustrative and not limiting of the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. An antibody or antigen-binding fragment thereof that binds to a complex comprising a WT1 peptide and an MHC molecule, a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH CDR1 region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of a selected VH CDR1, the VH CDR2 region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of a selected VH CDR2, and the VH CDR3 region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of a selected VH CDR3; a light chain variable region (VL) comprising CDR1, 2, and 3, wherein the VL CDR1 region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of a selected VL CDR1, the VL CDR2 region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of a selected VL CDR2, and the VL CDR3 region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of a selected VL CDR3; The amino acid sequences of the selected VH CDR1, 2, and 3 and the amino acid sequences of the selected VL CDR1, 2, and 3 are one of the following: (1) The amino acid sequences of the selected VH CDR1, 2, and 3 are set forth in SEQ ID NOs: 1 to 3, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are set forth in SEQ ID NOs: 55 to 57, respectively; (2) The amino acid sequences of the selected VH CDR1, 2, and 3 are set forth in SEQ ID NOs: 4 to 6, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are set forth in SEQ ID NOs: 58 to 60, respectively; (3) The amino acid sequences of the selected VH CDR1, 2, and 3 are set forth in SEQ ID NOs: 7 to 9, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are set forth in SEQ ID NOs: 61 to 63, respectively; (4) The amino acid sequences of the selected VH CDR1, 2, and 3 are set forth in SEQ ID NOs: 10 to 12, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are set forth in SEQ ID NOs: 64 to 66, respectively; (5) The amino acid sequences of the selected VH CDR1, 2, and 3 are set forth in SEQ ID NOs: 13 to 15, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are set forth in SEQ ID NOs: 67 to 69, respectively; (6) The amino acid sequences of the selected VH CDR1, 2, and 3 are set forth in SEQ ID NOs: 16 to 18, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are set forth in SEQ ID NOs: 70 to 72, respectively; (7) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 19 to 21, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 73 to 75, respectively; (8) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 22 to 24, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 76 to 78, respectively; (9) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 25 to 27, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 79 to 81, respectively; (10) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 28 to 30, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 55 to 57, respectively; (11) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 31 to 33, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 58 to 60, respectively; (12) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 34 to 36, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 61 to 63, respectively; (13) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 37 to 39, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 64 to 66, respectively; (14) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 40 to 42, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 67 to 69, respectively; (15) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 43 to 45, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 70 to 72, respectively; (16) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 46 to 48, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 73 to 75, respectively; (17) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 49 to 51, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 76 to 78, respectively; (18) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 52 to 54, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 79 to 81, respectively.

2. 2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the VH comprises CDR1, CDR2, and CDR3 having the amino acid sequences set forth in SEQ ID NOs: 1 to 3, respectively, and the VL comprises CDR1, CDR2, and CDR3 having the amino acid sequences set forth in SEQ ID NOs: 55 to 57, respectively, according to the Kabat definition.

3. 2. The antibody or antigen-binding fragment thereof according to claim 1, wherein, according to the Kabat definition, the VH comprises CDR1, 2, and 3 having the amino acid sequences set forth in SEQ ID NOs: 4 to 6, respectively, and the VL comprises CDR1, 2, and 3 having the amino acid sequences set forth in SEQ ID NOs: 58 to 60, respectively.

4. 2. The antibody or antigen-binding fragment thereof according to claim 1, wherein, according to the Kabat definition, the VH comprises CDR1, CDR2, and CDR3 having the amino acid sequences set forth in SEQ ID NOs: 7 to 9, respectively, and the VL comprises CDR1, CDR2, and CDR3 having the amino acid sequences set forth in SEQ ID NOs: 61 to 63, respectively.

5. 2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the VH comprises CDR1, CDR2, and CDR3 having the amino acid sequences set forth in SEQ ID NOs: 10 to 12, respectively, and the VL comprises CDR1, CDR2, and CDR3 having the amino acid sequences set forth in SEQ ID NOs: 64 to 66, respectively, according to the Kabat definition.

6. 2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the VH comprises CDR1, CDR2, and CDR3 having the amino acid sequences set forth in SEQ ID NOs: 13 to 15, respectively, and the VL comprises CDR1, CDR2, and CDR3 having the amino acid sequences set forth in SEQ ID NOs: 67 to 69, respectively, according to the Kabat definition.

7. 2. The antibody or antigen-binding fragment thereof according to claim 1, wherein, according to the Kabat definition, the VH comprises CDR1, CDR2, and CDR3 having the amino acid sequences set forth in SEQ ID NOs: 16 to 18, respectively, and the VL comprises CDR1, CDR2, and CDR3 having the amino acid sequences set forth in SEQ ID NOs: 70 to 72, respectively.

8. 2. The antibody or antigen-binding fragment thereof according to claim 1, wherein, according to the Kabat definition, the VH comprises CDR1, CDR2, and CDR3 having the amino acid sequences set forth in SEQ ID NOs: 19 to 21, respectively, and the VL comprises CDR1, CDR2, and CDR3 having the amino acid sequences set forth in SEQ ID NOs: 73 to 75, respectively.

9. 2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the VH comprises CDR1, CDR2, and CDR3 having the amino acid sequences set forth in SEQ ID NOs: 22 to 24, respectively, and the VL comprises CDR1, CDR2, and CDR3 having the amino acid sequences set forth in SEQ ID NOs: 76 to 78, respectively, according to the Kabat definition.

10. 2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the VH comprises CDR1, CDR2, and CDR3 having the amino acid sequences set forth in SEQ ID NOs: 25 to 27, respectively, and the VL comprises CDR1, CDR2, and CDR3 having the amino acid sequences set forth in SEQ ID NOs: 79 to 81, respectively, according to the Kabat definition.

11. 2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the VH comprises CDR1, CDR2, and CDR3 having the amino acid sequences set forth in SEQ ID NOs: 28 to 30, respectively, and the VL comprises CDR1, CDR2, and CDR3 having the amino acid sequences set forth in SEQ ID NOs: 55 to 57, respectively, according to the Chothia definition.

12. 2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the VH comprises CDR1, CDR2, and CDR3 having the amino acid sequences set forth in SEQ ID NOs: 31 to 33, respectively, and the VL comprises CDR1, CDR2, and CDR3 having the amino acid sequences set forth in SEQ ID NOs: 58 to 60, respectively, according to the Chothia definition.

13. 2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the VH comprises CDR1, CDR2, and CDR3 having the amino acid sequences set forth in SEQ ID NOs: 34 to 36, respectively, and the VL comprises CDR1, CDR2, and CDR3 having the amino acid sequences set forth in SEQ ID NOs: 61 to 63, respectively, according to the Chothia definition.

14. 2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the VH comprises CDR1, CDR2, and CDR3 having the amino acid sequences set forth in SEQ ID NOs: 37 to 39, respectively, and the VL comprises CDR1, CDR2, and CDR3 having the amino acid sequences set forth in SEQ ID NOs: 64 to 66, respectively, according to the Chothia definition.

15. 2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the VH comprises CDR1, CDR2, and CDR3 having the amino acid sequences set forth in SEQ ID NOs: 40 to 42, respectively, and the VL comprises CDR1, CDR2, and CDR3 having the amino acid sequences set forth in SEQ ID NOs: 67 to 69, respectively, according to the Chothia definition.

16. 2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the VH comprises CDR1, 2, and 3 having the amino acid sequences set forth in SEQ ID NOs: 43 to 45, respectively, and the VL comprises CDR1, 2, and 3 having the amino acid sequences set forth in SEQ ID NOs: 70 to 72, respectively, according to the Chothia definition.

17. 2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the VH comprises CDR1, CDR2, and CDR3 having the amino acid sequences set forth in SEQ ID NOs: 46 to 48, respectively, and the VL comprises CDR1, CDR2, and CDR3 having the amino acid sequences set forth in SEQ ID NOs: 73 to 75, respectively, according to the Chothia definition.

18. 2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the VH comprises CDR1, CDR2, and CDR3 having the amino acid sequences set forth in SEQ ID NOs: 49 to 51, respectively, and the VL comprises CDR1, CDR2, and CDR3 having the amino acid sequences set forth in SEQ ID NOs: 76 to 78, respectively, according to the Chothia definition.

19. 2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the VH comprises CDR1, CDR2, and CDR3 having the amino acid sequences set forth in SEQ ID NOs: 52 to 54, respectively, and the VL comprises CDR1, CDR2, and CDR3 having the amino acid sequences set forth in SEQ ID NOs: 79 to 81, respectively, according to the Chothia definition.

20. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 19, which specifically binds to a complex comprising a human WT1 peptide and an MHC molecule.

21. The antibody or antigen-binding fragment thereof of any one of claims 1 to 20, wherein the WT1 peptide comprises an sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence of SEQ ID NO:

100.

22. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 21, wherein the MHC is HLA (e.g., HLA-A2).

23. 23. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 22, wherein the antibody or antigen-binding fragment thereof is a human or humanized antibody or antigen-binding fragment thereof, a single-chain variable fragment (scFv), a single-arm antibody, and / or a multispecific antibody (e.g., a bispecific antibody).

24. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 23, wherein the antibody or antigen-binding fragment thereof is a human IgG1 antibody or antigen-binding fragment thereof, a human IgG2 antibody or antigen-binding fragment thereof, or a human IgG4 antibody or antigen-binding fragment thereof.

25. A nucleic acid comprising a polynucleotide encoding a polypeptide, (1) An immunoglobulin heavy chain or a fragment thereof, comprising a heavy chain variable region (VH) including complementarity-determining regions (CDRs) 1, 2, and 3 containing the amino acid sequences set forth in SEQ ID NOs: 1 to 3, respectively, wherein the VH binds to a complex containing a WT1 peptide and an MHC molecule when paired with a light chain variable region (VL) containing the amino acid sequence set forth in SEQ ID NO: 91; (2) An immunoglobulin light chain or a fragment thereof comprising a VL comprising complementarity-determining regions (CDRs) 1, 2, and 3 each comprising the amino acid sequences set forth in SEQ ID NOs: 55 to 57, wherein the VL binds to a complex comprising a WT1 peptide and an MHC molecule when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 82; (3) An immunoglobulin heavy chain or a fragment thereof, comprising a heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3 each comprising the amino acid sequence set forth in SEQ ID NO: 4 to 6, respectively, wherein the VH binds to a complex comprising a WT1 peptide and an MHC molecule when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 92; (4) An immunoglobulin light chain or a fragment thereof comprising a VL comprising complementarity-determining regions (CDRs) 1, 2, and 3 each comprising the amino acid sequences set forth in SEQ ID NOs: 58 to 60, wherein the VL binds to a complex comprising a WT1 peptide and an MHC molecule when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 83; (5) An immunoglobulin heavy chain or a fragment thereof, comprising a heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3 each comprising the amino acid sequence set forth in SEQ ID NO: 7 to 9, wherein the VH binds to a complex comprising a WT1 peptide and an MHC molecule when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 93; (6) An immunoglobulin light chain or a fragment thereof comprising a VL comprising complementarity-determining regions (CDRs) 1, 2, and 3 each comprising the amino acid sequences set forth in SEQ ID NOs: 61 to 63, wherein the VL binds to a complex comprising a WT1 peptide and an MHC molecule when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 84; (7) An immunoglobulin heavy chain or a fragment thereof comprising a VH comprising complementarity-determining regions (CDRs) 1, 2, and 3 each comprising the amino acid sequence set forth in SEQ ID NO: 10 to 12, wherein the VH binds to a complex comprising a WT1 peptide and an MHC molecule when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 94; (8) An immunoglobulin light chain or a fragment thereof comprising a VL comprising CDR1, 2, and 3 each comprising the amino acid sequence set forth in SEQ ID NO: 64 to 66, wherein the VL binds to a complex comprising a WT1 peptide and an MHC molecule when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 85; (9) An immunoglobulin heavy chain or a fragment thereof, comprising a VH comprising complementarity-determining regions (CDRs) 1, 2, and 3 each comprising the amino acid sequence set forth in SEQ ID NO: 13 to 15, wherein the VH binds to a complex comprising a WT1 peptide and an MHC molecule when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 95; (10) An immunoglobulin light chain or a fragment thereof comprising a VL comprising CDR1, 2, and 3 each comprising the amino acid sequence set forth in SEQ ID NO: 67 to 69, wherein the VL binds to a complex comprising a WT1 peptide and an MHC molecule when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 86; (11) An immunoglobulin heavy chain or a fragment thereof comprising a VH comprising complementarity-determining regions (CDRs) 1, 2, and 3 each comprising the amino acid sequence set forth in SEQ ID NO: 16 to 18, wherein the VH binds to a complex comprising a WT1 peptide and an MHC molecule when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 96; (12) An immunoglobulin light chain or a fragment thereof comprising a VL comprising CDR1, 2, and 3 each comprising the amino acid sequence set forth in SEQ ID NO: 70 to 72, wherein the VL binds to a complex comprising a WT1 peptide and an MHC molecule when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 87; (13) An immunoglobulin heavy chain or a fragment thereof comprising a VH comprising complementarity-determining regions (CDRs) 1, 2, and 3 each comprising the amino acid sequence set forth in SEQ ID NO: 19 to 21, wherein the VH binds to a complex comprising a WT1 peptide and an MHC molecule when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 97; (14) An immunoglobulin light chain or a fragment thereof comprising a VL comprising CDR1, 2, and 3 each comprising the amino acid sequence set forth in SEQ ID NO: 73 to 75, wherein the VL binds to a complex comprising a WT1 peptide and an MHC molecule when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 88; (15) An immunoglobulin heavy chain or a fragment thereof comprising a VH comprising complementarity-determining regions (CDRs) 1, 2, and 3 each comprising the amino acid sequence set forth in SEQ ID NOs: 22 to 24, wherein the VH binds to a complex comprising a WT1 peptide and an MHC molecule when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 98; (16) An immunoglobulin light chain or a fragment thereof comprising a VL comprising CDR1, 2, and 3 each comprising the amino acid sequence set forth in SEQ ID NO: 76 to 78, wherein the VL binds to a complex comprising a WT1 peptide and an MHC molecule when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 89; (17) An immunoglobulin heavy chain or a fragment thereof, comprising a VH comprising complementarity-determining regions (CDRs) 1, 2, and 3 each comprising the amino acid sequence set forth in SEQ ID NOs: 25 to 27, wherein the VH binds to a complex comprising a WT1 peptide and an MHC molecule when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 99; (18) An immunoglobulin light chain or a fragment thereof comprising a VL comprising CDR1, 2, and 3 each comprising the amino acid sequence set forth in SEQ ID NO: 79 to 81, wherein the VL binds to a complex comprising a WT1 peptide and an MHC molecule when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 90; (19) An immunoglobulin heavy chain or a fragment thereof, comprising a heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3 each comprising the amino acid sequence set forth in SEQ ID NOs: 28 to 30, wherein the VH binds to a complex comprising a WT1 peptide and an MHC molecule when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 91; (20) An immunoglobulin heavy chain or a fragment thereof, comprising a heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3 each comprising the amino acid sequence set forth in SEQ ID NOs: 31 to 33, respectively, wherein the VH binds to a complex comprising a WT1 peptide and an MHC molecule when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 92; (21) An immunoglobulin heavy chain or a fragment thereof, comprising a heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3 each comprising the amino acid sequences set forth in SEQ ID NOs: 34 to 36, respectively, wherein the VH binds to a complex comprising a WT1 peptide and an MHC molecule when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 93; (22) An immunoglobulin heavy chain or a fragment thereof comprising a VH comprising complementarity-determining regions (CDRs) 1, 2, and 3 each comprising the amino acid sequence set forth in SEQ ID NO: 37 to 39, wherein the VH binds to a complex comprising a WT1 peptide and an MHC molecule when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 94; (23) An immunoglobulin heavy chain or a fragment thereof comprising a VH comprising complementarity-determining regions (CDRs) 1, 2, and 3 each comprising the amino acid sequence set forth in SEQ ID NOs: 40 to 42, wherein the VH binds to a complex comprising a WT1 peptide and an MHC molecule when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 95; (24) An immunoglobulin heavy chain or a fragment thereof comprising a VH comprising complementarity-determining regions (CDRs) 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 43 to 45, respectively, wherein the VH binds to a complex comprising a WT1 peptide and an MHC molecule when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 96; (25) An immunoglobulin heavy chain or a fragment thereof comprising a VH comprising complementarity-determining regions (CDRs) 1, 2, and 3 each comprising the amino acid sequence set forth in SEQ ID NOs: 46 to 48, wherein the VH binds to a complex comprising a WT1 peptide and an MHC molecule when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 97; (26) An immunoglobulin heavy chain or a fragment thereof comprising a VH comprising complementarity-determining regions (CDRs) 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 49 to 51, respectively, wherein the VH binds to a complex comprising a WT1 peptide and an MHC molecule when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 98; or (27) An immunoglobulin heavy chain or a fragment thereof comprising a VH comprising complementarity-determining regions (CDRs) 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 52 to 54, respectively, wherein the VH binds to a complex comprising a WT1 peptide and an MHC molecule when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO:

99. A nucleic acid comprising:

26. The nucleic acid of claim 25, wherein the VH, when paired with the VL, specifically binds to a complex comprising a WT1 peptide and an MHC molecule, or the VL, when paired with the VH, specifically binds to a complex comprising a human WT1 peptide and an MHC molecule.

27. 27. The nucleic acid of claim 25 or 26, wherein the immunoglobulin heavy chain or fragment thereof is a human immunoglobulin heavy chain or fragment thereof (e.g., a human IgG1 heavy chain or fragment thereof, a human IgG2 heavy chain or fragment thereof, or a human IgG4 heavy chain or fragment thereof), and the immunoglobulin light chain or fragment thereof is a human immunoglobulin light chain or fragment thereof.

28. 28. The nucleic acid of any one of claims 25 to 27, wherein the nucleic acid encodes a single-chain variable fragment (scFv), a single-armed antibody, a multispecific antibody (e.g., a bispecific antibody), or a chimeric antigen receptor (CAR).

29. The nucleic acid according to any one of claims 25 to 28, wherein the nucleic acid is a cDNA.

30. A vector comprising one or more of the nucleic acids according to any one of claims 25 to 29.

31. A vector comprising two of the nucleic acids according to any one of claims 25 to 29, wherein the vector encodes the VL region and the VH region, each of which binds to a complex comprising a WT1 peptide and an MHC molecule.

32. A pair of vectors, each vector comprising one of the nucleic acids of any one of claims 25 to 29, and the pair of vectors collectively encoding the VL region and the VH region, respectively, that bind to a complex comprising a WT1 peptide and an MHC molecule.

33. A cell comprising a vector according to claim 30 or 31, or a pair of vectors according to claim 32.

34. 34. The cell of claim 33, wherein the cell is a CHO cell.

35. A cell comprising one or more nucleic acids according to any one of claims 25 to 29.

36. A cell comprising two of the nucleic acids according to any one of claims 25 to 29.

37. The cell of claim 36, wherein the two nucleic acids collectively encode the VL region and the VH region, respectively, that bind to a complex containing a WT1 peptide and an MHC molecule.

38. 1. A method for producing an antibody or antigen-binding fragment thereof, comprising: (a) culturing the cell of any one of claims 33 to 37 under conditions sufficient for the cell to produce the antibody or antigen-binding fragment; (b) recovering the antibody or antigen-binding fragment produced by the cell; and A method comprising:

39. An antibody or antigen-binding fragment thereof that binds to a complex comprising a WT1 peptide and an MHC molecule, 1. An antibody or antigen-binding fragment thereof comprising: a heavy chain variable region (VH) comprising an amino acid sequence that is at least 90% identical to a selected VH sequence; and a light chain variable region (VL) comprising an amino acid sequence that is at least 90% identical to a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following: (1) the selected VH sequence is SEQ ID NO: 82 and the selected VL sequence is SEQ ID NO: 91; (2) the selected VH sequence is SEQ ID NO: 83 and the selected VL sequence is SEQ ID NO: 92; (3) the selected VH sequence is SEQ ID NO: 84 and the selected VL sequence is SEQ ID NO: 93; (4) the selected VH sequence is SEQ ID NO: 85 and the selected VL sequence is SEQ ID NO: 94; (5) the selected VH sequence is SEQ ID NO: 86 and the selected VL sequence is SEQ ID NO: 95; (6) the selected VH sequence is SEQ ID NO: 87 and the selected VL sequence is SEQ ID NO: 96; (7) The selected VH sequence is SEQ ID NO: 88 and the selected VL sequence is SEQ ID NO:

97. (8) the selected VH sequence is SEQ ID NO: 89 and the selected VL sequence is SEQ ID NO: 98; and (9) The selected VH sequence is SEQ ID NO: 90 and the selected VL sequence is SEQ ID NO:

99.

40. 40. The antibody or antigen-binding fragment thereof of claim 39, wherein the VH comprises the sequence of SEQ ID NO: 82 and the VL comprises the sequence of SEQ ID NO:

91.

41. 40. The antibody or antigen-binding fragment thereof of claim 39, wherein the VH comprises the sequence of SEQ ID NO: 83 and the VL comprises the sequence of SEQ ID NO:

92.

42. 40. The antibody or antigen-binding fragment thereof of claim 39, wherein the VH comprises the sequence of SEQ ID NO: 84 and the VL comprises the sequence of SEQ ID NO:

93.

43. 40. The antibody or antigen-binding fragment thereof of claim 39, wherein the VH comprises the sequence of SEQ ID NO: 85 and the VL comprises the sequence of SEQ ID NO:

94.

44. 40. The antibody or antigen-binding fragment thereof of claim 39, wherein the VH comprises the sequence of SEQ ID NO: 86 and the VL comprises the sequence of SEQ ID NO:

95.

45. 40. The antibody or antigen-binding fragment thereof of claim 39, wherein the VH comprises the sequence of SEQ ID NO: 87 and the VL comprises the sequence of SEQ ID NO:

96.

46. 40. The antibody or antigen-binding fragment thereof of claim 39, wherein the VH comprises the sequence of SEQ ID NO: 88 and the VL comprises the sequence of SEQ ID NO:

97.

47. 40. The antibody or antigen-binding fragment thereof of claim 39, wherein the VH comprises the sequence of SEQ ID NO: 89 and the VL comprises the sequence of SEQ ID NO:

98.

48. 40. The antibody or antigen-binding fragment thereof of claim 39, wherein the VH comprises the sequence of SEQ ID NO: 90 and the VL comprises the sequence of SEQ ID NO:

99.

49. An antibody or antigen-binding fragment thereof that binds to a complex comprising a WT1 peptide and an MHC molecule, an antibody or antigen-binding fragment thereof comprising: a heavy chain variable region (VH) comprising VH CDR1, VH CDR2, and VH CDR3 identical to the VH CDR1, VH CDR2, and VH CDR3 of a selected VH sequence; and a light chain variable region (VL) comprising VL CDR1, VL CDR2, and VL CDR3 identical to the VL CDR1, VL CDR2, and VL CDR3 of a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following: (1) the selected VH sequence is SEQ ID NO: 82 and the selected VL sequence is SEQ ID NO: 91; (2) the selected VH sequence is SEQ ID NO: 83 and the selected VL sequence is SEQ ID NO: 92; (3) the selected VH sequence is SEQ ID NO: 84 and the selected VL sequence is SEQ ID NO: 93; (4) the selected VH sequence is SEQ ID NO: 85 and the selected VL sequence is SEQ ID NO: 94; (5) the selected VH sequence is SEQ ID NO: 86 and the selected VL sequence is SEQ ID NO: 95; (6) the selected VH sequence is SEQ ID NO: 87 and the selected VL sequence is SEQ ID NO: 96; (7) The selected VH sequence is SEQ ID NO: 88 and the selected VL sequence is SEQ ID NO:

97. (8) the selected VH sequence is SEQ ID NO: 89 and the selected VL sequence is SEQ ID NO: 98; and (9) The selected VH sequence is SEQ ID NO: 90 and the selected VL sequence is SEQ ID NO:

99.

50. The antibody or antigen-binding fragment thereof according to any one of claims 39 to 49, which specifically binds to a complex comprising a human WT1 peptide and an MHC molecule.

51. The antibody or antigen-binding fragment thereof of any one of claims 39 to 50, wherein the WT1 peptide comprises an sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence of SEQ ID NO:

100.

52. The antibody or antigen-binding fragment thereof of any one of claims 39 to 51, wherein the MHC is HLA (e.g., HLA-A2).

53. 53. The antibody or antigen-binding fragment thereof of any one of claims 39 to 52, wherein the antibody or antigen-binding fragment thereof is a human or humanized antibody or antigen-binding fragment thereof, a single-chain variable fragment (scFv), a mono-armed antibody, and / or a multispecific antibody (e.g., a bispecific antibody).

54. The antibody or antigen-binding fragment thereof according to any one of claims 39 to 53, wherein the antibody or antigen-binding fragment thereof is a human IgG1 antibody or antigen-binding fragment thereof, a human IgG2 antibody or antigen-binding fragment thereof, or a human IgG4 antibody or antigen-binding fragment thereof.

55. An antibody or an antigen-binding fragment thereof that cross-competes with the antibody or antigen-binding fragment thereof according to any one of claims 1 to 24 and 39 to 54.

56. The antibody or antigen-binding fragment thereof of any one of claims 1 to 24 and 39 to 55, wherein the antibody or antigen-binding fragment thereof comprises a fragment crystallizable region (Fc region).

57. (1) a first functional portion comprising an antigen-binding fragment thereof according to any one of claims 1 to 24 and 39 to 55; and (2) a second functional moiety comprising a T cell binding molecule; A protein construct that binds to a complex containing a WT1 peptide and an MHC molecule, comprising:

58. 58. The protein construct of claim 57, wherein the T cell binding molecule (e.g., scFv or VHH) targets human CD3.

59. 59. The protein construct of claim 57 or 58, wherein the WT1 peptide comprises a sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence of SEQ ID NO:

100.

60. A protein construct according to any one of claims 57 to 59, wherein the MHC molecule is HLA (e.g., HLA-A2).

61. 61. The protein construct of any one of claims 57 to 60, wherein the first functional portion and the second functional portion are connected via a linker.

62. (1) a first functional portion comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 24 and 39 to 55; and (2) a second functional moiety comprising a T cell binding molecule, and (3) a third functional portion comprising a single-chain human crystallizable fragment; A protein construct comprising:

63. The protein construct of claim 62, wherein the T cell binding molecule is an scFv or VHH targeting human CD3.

64. 64. The protein construct of claim 62 or 63, wherein the first functional portion, the second functional portion, and the third functional portion are connected via one or more linkers.

65. 65. The protein construct of any one of claims 62 to 64, wherein the WT1 peptide comprises a sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence of SEQ ID NO:

100.

66. A protein construct according to any one of claims 62 to 65, wherein the MHC molecule is HLA (e.g., HLA-A2).

67. 67. An antibody drug conjugate comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 24 and 39 to 55, or the protein construct of any one of claims 57 to 66, covalently linked to a therapeutic agent.

68. 68. The antibody drug conjugate of claim 67, wherein the therapeutic agent is a cytotoxic or cytostatic agent.

69. A recombinant receptor comprising an antigen-binding fragment according to any one of claims 1 to 24 and 39 to 55.

70. 70. The recombinant receptor of claim 69, wherein the recombinant receptor further comprises a transmembrane region and an intracellular signaling domain.

71. 71. The recombinant receptor of claim 69 or 70, wherein the recombinant receptor is a chimeric antigen receptor ("CAR").

72. The recombinant receptor of any one of claims 69 to 71, further comprising a hinge region.

73. 73. The recombinant receptor of any one of claims 70 to 72, wherein the transmembrane domain comprises the transmembrane domain of CD4, CD8, and / or CD28, or a portion thereof.

74. 74. The recombinant receptor of any one of claims 70 to 73, wherein the intracellular signaling domain comprises a primary intracellular signaling sequence of an immune effector cell.

75. 75. The recombinant receptor of claim 74, wherein the intracellular signaling domain is or comprises a functional signaling domain of CD3 zeta.

76. 76. The recombinant receptor of any one of claims 70 to 75, wherein the intracellular signaling domain further comprises a costimulatory signal domain.

77. The costimulatory signal domains include those of MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), activating NK cell receptors, BTLA, Toll ligand receptors, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1, CD11a / CD18, 4-1BB (CD137), B7-H3, C DS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD8 beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, C D11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG 2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 77. The recombinant receptor of claim 76, comprising a functional signaling domain derived from a protein selected from the group consisting of (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, and CD83 ligand.

78. 77. The recombinant receptor of claim 76, wherein the costimulatory signal domain comprises the intracellular signaling domain of 4-1BB and / or CD28.

79. 79. The recombinant receptor of any one of claims 69 to 78, wherein the recombinant receptor comprises a signal peptide.

80. 70. The recombinant receptor of claim 69, wherein the recombinant receptor is a chimeric T cell receptor ("cTCR").

81. A polynucleotide encoding the recombinant receptor of any one of claims 69 to 80.

82. A vector comprising the polynucleotide of claim 81.

83. 83. The vector of claim 82, wherein the vector is a viral vector.

84. A recombinant cell expressing the recombinant receptor of any one of claims 69 to 80.

85. 85. The recombinant cell of claim 84, wherein the recombinant cell is an immune cell.

86. 86. The recombinant cell of claim 85, wherein the immune cell is a NK cell or a T cell.

87. 87. The recombinant cell of claim 86, wherein the recombinant cell is a T cell.

88. 88. The recombinant cell of claim 87, wherein the T cell is selected from the group consisting of a cytotoxic T cell, a helper T cell, a natural killer T (NK-T) cell, and a γδ T cell.

89. 84. A method for producing a recombinant cell, comprising introducing the vector of claim 82 or 83 into a cell in vitro or ex vivo.

90. 90. The method of claim 89, wherein the vector is a viral vector and the introduction is by transduction.

91. 10. A method of treating a subject having cancer, comprising administering to the subject a therapeutically effective amount of a composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 24 and 39 to 56, the protein construct of any one of claims 57 to 66, the recombinant cell of any one of claims 84 to 88, or the antibody drug conjugate of claim 67 or 68.

92. 92. The method of claim 91, wherein the subject has a solid tumor.

93. 92. The method of claim 91, wherein the cancer is leukemia, breast cancer, ovarian cancer, glioblastoma, colorectal cancer, hematological malignancies, or soft tissue sarcoma.

94. 92. The method of claim 91, wherein the subject is further treated with an effective amount of an anti-4-1BB antibody, an anti-OX40 antibody, an anti-CD40 antibody, an anti-PD-1 antibody, an anti-CTLA4 antibody, or an anti-PD-L1 antibody.

95. 1. A method for reducing tumor growth rate, comprising: contacting tumor cells with an effective amount of a composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 24 and 39 to 56, a protein construct according to any one of claims 57 to 66, a recombinant cell according to any one of claims 84 to 88, or an antibody drug conjugate according to claim 67 or 68; A method comprising:

96. 1. A method for killing tumor cells, comprising: contacting tumor cells with an effective amount of a composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 24 and 39 to 56, a protein construct according to any one of claims 57 to 66, a recombinant cell according to any one of claims 84 to 88, or an antibody drug conjugate according to claim 67 or 68; A method comprising:

97. 1. A method of increasing an immune response in a subject, comprising: administering to the subject an effective amount of a composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 24 and 39 to 56, the protein construct of any one of claims 57 to 66, the recombinant cell of any one of claims 84 to 88, or the antibody drug conjugate of claim 67 or 68; A method comprising:

98. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 24 and 39 to 56, a protein construct according to any one of claims 57 to 66, a recombinant cell according to any one of claims 84 to 88, or an antibody drug conjugate according to claim 67 or 68, and a pharmaceutically acceptable carrier.

99. 68. The antibody drug conjugate of claim 67, wherein the drug-to-antibody ratio (DAR) of the drug to the antibody is about 4.

100. An antibody or antigen-binding fragment thereof that binds to a complex comprising a WT1 peptide and an MHC molecule, wherein the antibody or antigen-binding fragment thereof specifically binds to an epitope within the WT1 peptide, and the epitope is one or more of the following: (1) an amino acid residue corresponding to R1 in SEQ ID NO: 100; (2) amino acid residues corresponding to F3 of SEQ ID NO: 100; (3) an amino acid residue corresponding to P4 of SEQ ID NO: 100; (4) an amino acid residue corresponding to N5 of SEQ ID NO: 100; and (5) An amino acid residue corresponding to Y8 in SEQ ID NO: 100.

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