VH4-34 antigen-binding molecule

Abstract

VH4-34 antigen-binding molecules (e.g., VH4-34 antibodies), compositions containing the same, and methods of use are provided herein.

Description

[Technical Field]

[0001] cross reference

[0001] This application claims the interests and priority of U.S. Provisional Patent Application No. 63 / 451,053, filed on 9 March 2023, which is incorporated in its entirety by reference herein.

[0002]

[0002] This disclosure relates to the field of molecular biology, more specifically to antibody technology. This disclosure also relates to methods of medical treatment and prevention. [Background technology]

[0003]

[0003] The generation of B cells that express BCRs specific to self-antigens is inherent in the process of B cell development. In healthy individuals, approximately 75% of newly formed B cells are self-reactive. However, through negative selection, self-reactive B cells are either eliminated or made anerogenic, thus preventing them from maturing into memory or plasma B cells. Failure of the B cell tolerance mechanism can lead to the presence of mature B cells expressing self-reactive antibodies in the periphery.

[0004]

[0004] VH4-34 is an endogenous autoreactive antibody heavy chain variable region (VH) subregion containing a hydrophobic patch in the first framework region (FR1) that binds to carbohydrate moieties expressed on red blood cells and some glycoproteins (Young et al. PNAS USA (2015) 112(44): pp. 13447-54). Antibodies containing VH4-34 are not normally present in the serum of healthy individuals. However, in diseases such as systemic lupus erythematosus (SLE), autoreactive B cells expressing VH4-34 are overpresented in the memory B cell and plasma B cell compartments and are involved in disease pathogenesis. The monoclonal rat anti-idiotype antibody 9G4 binds to antibodies containing VH4-34 (Stevenson et al. Blood (1986) 68: p. 430, Richardson et al. J Immunol (2013) 191(10): pp. 4926-4939).

Summary of the Invention

Means for Solving the Problems

[0005]

[0005] In a particular embodiment, (a) (i) An HC-CDR1 having the amino acid sequence of SEQ ID NO: 172, An HC-CDR2 having the amino acid sequence of SEQ ID NO: 55, An HC-CDR3 having the amino acid sequence of SEQ ID NO: 56 A heavy chain variable (VH) region comprising, and (ii) An LC-CDR1 having the amino acid sequence of SEQ ID NO: 144, An LC-CDR2 having the amino acid sequence YAS, An LC-CDR3 having the amino acid sequence of SEQ ID NO: 64 A light chain variable (VL) region comprising, or (b) (i) An HC-CDR1 having the amino acid sequence of SEQ ID NO: 142, An HC-CDR2 having the amino acid sequence of SEQ ID NO: 55, An HC-CDR3 having the amino acid sequence of SEQ ID NO: 56 A heavy chain variable (VH) region comprising, and (ii) An LC-CDR1 having the amino acid sequence of SEQ ID NO: 144, An LC-CDR2 having the amino acid sequence YAS, An LC-CDR3 having the amino acid sequence of SEQ ID NO: 64 A light chain variable (VL) region comprising, or (c) (i) An HC-CDR1 having the amino acid sequence of SEQ ID NO: 38, An HC-CDR2 having the amino acid sequence of SEQ ID NO: 39, An HC-CDR3 having the amino acid sequence of SEQ ID NO: 40 A heavy chain variable (VH) region comprising, and (ii) An LC-CDR1 having the amino acid sequence of SEQ ID NO: 46, An LC-CDR2 having the amino acid sequence AAS, LC-CDR3 having the amino acid sequence of SEQ ID NO: 48 Light chain variable (VL) region including, or (d) (i) HC-CDR1 having the amino acid sequence of SEQ ID NO: 54, HC-CDR2 having the amino acid sequence of SEQ ID NO: 55, HC-CDR3 having the amino acid sequence of SEQ ID NO: 56 Heavy chain variable (VH) region including, (ii) LC-CDR1 having the amino acid sequence of SEQ ID NO: 62, LC-CDR2 having the amino acid sequence YAS, LC-CDR3 having the amino acid sequence of SEQ ID NO: 64 Light chain variable (VL) region including, or (e) (i) HC-CDR1 having the amino acid sequence of SEQ ID NO: 6, HC-CDR2 having the amino acid sequence of SEQ ID NO: 7, HC-CDR3 having the amino acid sequence of SEQ ID NO: 8 Heavy chain variable (VH) region including, (ii) LC-CDR1 having the amino acid sequence of SEQ ID NO: 131, LC-CDR2 having the amino acid sequence LVS, LC-CDR3 having the amino acid sequence of SEQ ID NO: 16 Light chain variable (VL) region including, or (f) (i) HC-CDR1 having the amino acid sequence of SEQ ID NO: 22, HC-CDR2 having the amino acid sequence of SEQ ID NO: 23, HC-CDR3 having the amino acid sequence of SEQ ID NO: 24 Heavy chain variable (VH) region including, (ii) LC-CDR1 having the amino acid sequence of SEQ ID NO: 30, LC-CDR2 having the amino acid sequence YTS, LC-CDR3 having the amino acid sequence of SEQ ID NO: 32 Light chain variable (VL) region including, or (g) (i) HC-CDR1 having the amino acid sequence of SEQ ID NO: 6, HC-CDR2 having the amino acid sequence of SEQ ID NO: 7, HC-CDR3 having the amino acid sequence of SEQ ID NO: 8 Heavy chain variable (VH) region including, (ii) LC-CDR1 having the amino acid sequence of SEQ ID NO: 162, LC-CDR2 having the amino acid sequence LVS, LC-CDR3 having the amino acid sequence of SEQ ID NO: 16 Light chain variable (VL) region including, or (h) (i) HC-CDR1 having the amino acid sequence of SEQ ID NO: 142, HC-CDR2 having the amino acid sequence of SEQ ID NO: 55, HC-CDR3 having the amino acid sequence of SEQ ID NO: 56 Heavy chain variable (VH) region including, (ii) LC-CDR1 having the amino acid sequence of SEQ ID NO: 144, LC-CDR2 having the amino acid sequence YAS, LC-CDR3 having the amino acid sequence of SEQ ID NO: 64 Light chain variable (VL) region including, or (i) (i) HC-CDR1 having the amino acid sequence of SEQ ID NO: 185, HC-CDR2 having the amino acid sequence of SEQ ID NO: 55, HC-CDR3 having the amino acid sequence of SEQ ID NO: 56 Heavy chain variable (VH) region including, (ii) LC-CDR1 having the amino acid sequence of SEQ ID NO: 144, LC-CDR2 having the amino acid sequence YAS, LC-CDR3 having the amino acid sequence of SEQ ID NO: 64 Light chain variable (VL) region including, or (j) (i) HC-CDR1 having the amino acid sequence of SEQ ID NO: 6, HC-CDR2 having the amino acid sequence of SEQ ID NO: 7, HC-CDR3 having the amino acid sequence of SEQ ID NO: 8 Heavy chain variable (VH) region including, (ii) LC-CDR1 having the amino acid sequence of SEQ ID NO: 14, LC-CDR2 having the amino acid sequence LVS, LC-CDR3 having the amino acid sequence of SEQ ID NO: 16 Light chain variable (VL) region including VH4-34 antigen-binding molecules containing the above are described herein.

[0006]

[0006] In some embodiments, the VH4-34 antigen-binding molecule includes a VH region containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NOs. 5, 21, 37, 53, 125, 141, 151, 156, 167, 171, 176, 181, 182, or 184, and a VL region containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NOs. 13, 29, 45, 61, 130, 143, 147, 153, 161, 189, 193, 198, 201, 205, or 209.

[0007]

[0007] In some embodiments, the VH4-34 antigen-binding molecule comprises (i) a VH region containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 182 and a VL region containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 198, or (ii) a VH region containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 182 and a VL region containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 193, or (iii) a VH region containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 167 and a VL region containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 189, or (iv) a VH region containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 21 and a VL region containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 29, or (v) the amino acid sequence of SEQ ID NO: 37 (vi) A VH region containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence, and a VL region containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 45, or (vi) A VH region containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 53, and a VL region containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 61, or (vii) A VH region containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 125, and a VL region containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 130, or (viii) A VH region containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 141, and a VL region containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 143, or (ix) A VH region containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 141,(x) A VL region containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 147, or (x) A VH region containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 156, and a VL region containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 161, or (xi) A VH region containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 151, and at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 153 VL region containing an amino acid sequence having sequence identity, or (xii) VH region containing an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 184, and VL region containing an amino acid sequence having at least 80% sequence identity to the amino acid sequences of SEQ ID NO: 189, 193, 198, 201, 205 or 209, or (xiii) VH region containing an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 167, and A A VL region containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence, or a VH region containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence of (xiv) SEQ ID NO: 171, and a VL region containing an amino acid sequence having at least 80% sequence identity with the amino acid sequences of SEQ ID NOs: 189, 193, 198, 201, 205 or 209, or a VH region containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence of (xv) SEQ ID NO: 176, and SEQ ID NOs: 189, 193, 198 , a VL region containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence of 201, 205 or 209, or a VH region containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence of (xvi) SEQ ID NO: 181, and a VL region containing an amino acid sequence having at least 80% sequence identity with the amino acid sequences of 189, 193, 198, 201, 205 or 209, or a VH region containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence of (xvii) SEQ ID NO: 182,and a VL region containing an amino acid sequence having at least 80% sequence identity with the amino acid sequences of SEQ ID NOs. 189, 193, 198, 201, 205 or 209, or a VH region containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO. 5, and a VL region containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO. 13, Includes.

[0008]

[0008] In some embodiments, the VH4-34 antigen-binding molecule inhibits the interaction between VH4-34 and a molecule containing the N-acetyllactosamine moiety, and optionally, the molecule containing the N-acetyllactosamine moiety is selected from I / I carbohydrates and CD45 isoform B220. In some embodiments, the VH4-34 antigen-binding molecule further includes an Fc region. In some embodiments, the VH4-34 antigen-binding molecule is a multispecific antigen-binding molecule, and the VH4-34 antigen-binding molecule further includes an antigen-binding domain that binds to antigens other than VH4-34.

[0009]

[0009] In certain embodiments, a chimeric antigen receptor (CAR) comprising a VH4-34 antigen-binding molecule described herein is described herein.

[0010] In certain embodiments, optionally isolated nucleic acids or a group of nucleic acids encoding a VH4-34 antigen-binding molecule or a CAR described herein are described herein.

[0010]

[0011] In certain embodiments, an expression vector or a plurality of expression vectors comprising the nucleic acid or plurality of nucleic acids described herein is described herein.

[0012] In certain embodiments, cells comprising a VH4-34 antigen-binding molecule described herein, a CAR described herein, a nucleic acid or multiple nucleic acids described herein, or an expression vector or multiple expression vectors described herein are described herein.

[0011]

[0013] In certain embodiments, a method described herein includes the step of culturing the cells described herein under conditions suitable for the expression of antigen-binding molecules or CARs by the cells.

[0012]

[0014] In certain embodiments, compositions comprising a VH4-34 antigen-binding molecule described herein, a CAR described herein, a nucleic acid or multiple nucleic acids described herein, an expression vector or multiple expression vectors or cells described herein, and a pharmaceutically acceptable carrier, diluent, excipient or adjuvant are described herein.

[0013]

[0015] In certain embodiments, a VH4-34 antigen-binding molecule, a CAR, a nucleic acid or multiple nucleic acids, an expression vector or multiple expression vectors, or cells described herein are described herein for use in a method of treating or preventing a disease or condition characterized by the expression of VH4-34.

[0014]

[0016] In certain embodiments, a VH4-34 antigen-binding molecule, a CAR, a nucleic acid or multiple nucleic acids, an expression vector or multiple expression vectors, or cells described herein are described herein for use in a medical treatment or preventive method.

[0015]

[0017] In certain embodiments, VH4-34 antigen-binding molecules, CARs, nucleic acids or multiple nucleic acids, expression vectors or multiple expression vectors, or cells described herein are described herein for use in methods of treating or preventing diseases or conditions characterized by VH4-34 expression. In certain embodiments, VH4-34 antigen-binding molecules, CARs, nucleic acids or multiple nucleic acids, expression vectors or multiple expression vectors, or cells described herein are further described herein for use in the manufacture of pharmaceuticals for treating or preventing diseases or conditions characterized by VH4-34 expression. In some embodiments, autoimmune diseases are examples of diseases or conditions characterized by VH4-34 expression. In some embodiments, the autoimmune disease is selected from the group consisting of autoimmune hemolytic anemia (AIHA), unclassified immunodeficiency (CVID), rheumatoid arthritis, systemic lupus erythematosus (SLE), lupus nephritis, central nervous system (CNS) lupus, cold agglutinin disease (CAD), and Wiscott-Aldrich syndrome. In some embodiments, the autoimmune disease is cold agglutinin disease (CAD). In some embodiments, the autoimmune disease is systemic lupus erythematosus (SLE). In some embodiments, the disease or condition characterized by VH4-34 expression is cancer. In some embodiments, cancer is a B-cell malignancy. In some embodiments, B-cell malignancies are selected from the group consisting of: B-cell leukemia, Hairy cell leukemia (HCL), Hairy cell leukemia subtype (HCL-V), chronic lymphocytic leukemia (CLL), mutagenic chronic lymphocytic leukemia (M-CLL), acute lymphoblastic leukemia (ALL), B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), activated B-cell diffuse large B-cell lymphoma (ABC-DLBCL), germinal center B-cell diffuse large B-cell lymphoma (GBC-DLBCL), primary central nervous system (CNS) lymphoma, and mantle cell lymphoma. In some embodiments, the B-cell malignancy is chronic lymphocytic leukemia (CLL).In some embodiments, the disease or condition characterized by the expression of VH4-34 is selected from: B-cell leukemia, Hairy cell leukemia (HCL), Hairy cell leukemia subtype (HCL-V), chronic lymphocytic leukemia (CLL), mutagenic chronic lymphocytic leukemia (M-CLL), acute lymphoblastic leukemia (ALL), B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), activated B-cell diffuse large B-cell lymphoma Cellular lymphoma (ABC-DLBCL), germinal center B-cell diffuse large B-cell lymphoma (GBC-DLBCL), primary central nervous system (CNS) lymphoma, mantle cell lymphoma, autoimmune hemolytic anemia (AIHA), unclassified immunodeficiency (CVID), rheumatoid arthritis, systemic lupus erythematosus (SLE), lupus nephritis, CNS lupus, cold agglutinin disease (CAD), and Wiscott-Aldrich syndrome.

[0016]

[0018] In certain embodiments, methods for treating or preventing a disease or condition characterized by the expression of VH4-34 in a subject are described herein, the methods comprising the step of administering a VH4-34 antigen-binding molecule described herein, a CAR described herein, a nucleic acid or multiple nucleic acids described herein, an expression vector or multiple expression vectors described herein, or cells described herein to the subject. In some embodiments, the disease or condition characterized by the expression of VH4-34 is an autoimmune disease. In some embodiments, the autoimmune disease is selected from the group consisting of autoimmune hemolytic anemia (AIHA), unclassifiable immunodeficiency (CVID), rheumatoid arthritis, systemic lupus erythematosus (SLE), lupus nephritis, central nervous system (CNS) lupus, cold agglutinin disease (CAD), and Wiscott-Aldrich syndrome. In some embodiments, the autoimmune disease is cold agglutinin disease (CAD). In some embodiments, the autoimmune disease is systemic lupus erythematosus (SLE). In some embodiments, the disease or condition characterized by the expression of VH4-34 is cancer. In some embodiments, cancer is a B-cell malignancy. In some embodiments, B-cell malignancies are selected from the group consisting of: B-cell leukemia, Hairy cell leukemia (HCL), Hairy cell leukemia subtype (HCL-V), chronic lymphocytic leukemia (CLL), mutagenic chronic lymphocytic leukemia (M-CLL), acute lymphoblastic leukemia (ALL), B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), activated B-cell diffuse large B-cell lymphoma (ABC-DLBCL), germinal center B-cell diffuse large B-cell lymphoma (GBC-DLBCL), primary central nervous system (CNS) lymphoma, and mantle cell lymphoma. In some embodiments, B-cell malignancies are chronic lymphocytic leukemia (CLL).In some embodiments, the disease or condition characterized by the expression of VH4-34 is selected from: B-cell leukemia, Hairy cell leukemia (HCL), Hairy cell leukemia subtype (HCL-V), chronic lymphocytic leukemia (CLL), mutagenic chronic lymphocytic leukemia (M-CLL), acute lymphoblastic leukemia (ALL), B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), activated B-cell diffuse large B-cell lymphoma Cellular lymphoma (ABC-DLBCL), germinal center B-cell diffuse large B-cell lymphoma (GBC-DLBCL), primary central nervous system (CNS) lymphoma, mantle cell lymphoma, autoimmune hemolytic anemia (AIHA), unclassified immunodeficiency (CVID), rheumatoid arthritis, systemic lupus erythematosus (SLE), lupus nephritis, CNS lupus, cold agglutinin disease (CAD), and Wiscott-Aldrich syndrome.

[0017]

[0019] In certain embodiments, the use of a VH4-34 antigen-binding molecule described herein to promote the death of cells expressing VH4-34 is described herein.

[0020] In certain embodiments, an optionally isolated in vitro complex comprising a VH4-34 antigen-binding molecule described herein, conjugated to VH4-34, is described herein.

[0018]

[0021] In certain embodiments, a method for detecting VH4-34 in a sample is described herein, comprising the steps of contacting a sample containing or suspected to contain VH4-34 with a VH4-34 antigen-binding molecule described herein, and detecting the formation of a complex of the VH4-34 antigen-binding molecule with VH4-34.

[0019]

[0022] In certain embodiments, a method for selecting or stratifying subjects for treatment using a VH4-34-targeting agent is described herein, comprising the steps of contacting a sample derived from the subject in vitro with a VH4-34 antigen-binding molecule described herein, and detecting the formation of a complex of the VH4-34 antigen-binding molecule with VH4-34.

[0020]

[0023] In certain embodiments, the use of the VH4-34 antigen-binding molecules described herein as in vitro or in vivo diagnostic or prognostic agents is described herein.

[0021]

[0024] In certain embodiments, the use of the VH4-34 antigen-binding molecule described herein in a method for detecting, locating, or imaging a disease or condition characterized by VH4-34 expression is described herein, and optionally, the disease or condition characterized by VH4-34 expression is selected from: B-cell leukemia, Hairy cell leukemia (HCL), Hairy cell leukemia subtype (HCL-V), chronic lymphocytic leukemia (CLL), mutagenetic chronic lymphocytic leukemia (M-CLL), acute lymphoblastic leukemia Allergic disease (ALL), B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), activated B-cell diffuse large B-cell lymphoma (ABC-DLBCL), germinal center B-cell diffuse large B-cell lymphoma (GBC-DLBCL), primary central nervous system (CNS) lymphoma, mantle cell lymphoma, autoimmune hemolytic anemia (AIHA), unclassified immunodeficiency (CVID), rheumatoid arthritis, systemic lupus erythematosus (SLE), lupus nephritis, central nervous system (CNS) lupus, cold agglutinin disease (CAD), and Wiscott-Aldrich syndrome.

[0022]

[0025] Embodiments and experiments illustrating the principles of this disclosure are discussed below with reference to the accompanying drawings. [Brief explanation of the drawing]

[0023] [Figure 1]

[0026] Figures 1A and 1B show graphs and tables illustrating the dose-responsive binding of the anti-VH4-34 antibody D011-1E11p to a hydrophobic patch of VH4-34 on CHO cell lines overexpressing surface VH4-34 antibodies (Taba, Zano, 3A6), compared to negative control CHO cells expressing antibodies Zano FR1 and VSTB112, as well as non-transfected CHO (WT). Figure 1A shows the binding by D011-1E11p, and the EC50 values ​​are shown in the table in Figure 1B. [Figure 2]

[0027] Figures 2A and 2B are graphs and tables showing the dose-responsive binding of three anti-VH4-34 antibodies D011-7016, D011-6039, and D011-6040 to a hydrophobic patch of VH4-34 on CHO cell lines overexpressing surface VH4-34 antibodies (Taba, Zano, 3A6), compared to negative control CHO cells expressing antibodies Zano FR1, Taba FR1, and 3A6 FR1, VH4-34 negative cell lines 10D1F and / or VSTB112, and untransfected CHO (WT). Figure 2A shows the binding by the antibodies, and the EC50 values ​​are shown in the table in Figure 2B. [Figure 3]

[0028] Figures 3A and 3B show graphs and tables illustrating the dose-responsive binding of anti-VH4-34 antibodies D011-7016, D011-7016-LD1, and D011-7016-LD2 to a hydrophobic patch of VH4-34 on CHO cell lines overexpressing surface VH4-34 antibodies (Taba, Zano, 3A6), compared to negative control CHO cells expressing antibodies Zano FR1, Taba FR1, 3A6 FR1, and VSTB112, as well as non-transfected CHO (WT). Figure 3A shows the binding by the antibodies, and the EC50 values ​​are shown in the table in Figure 3B. [Figure 4]

[0029] Figures 4A and 4B are graphs and tables showing the dose-responsive binding of anti-VH4-34 antibodies D011-7016, D011-6039, and D011-6040 to VH4-34 expressing cell lines, OCI-Ly3. Pfeiffer cell lines are used as a negative control. Figure 4A shows antibody binding, and the EC50 values ​​are shown in the table in Figure 4B. [Figure 5]

[0030] Figures 5A and 5B show graphs and tables illustrating the dose-responsive binding of humanized clone D011-1E11-5T2 to cell lines endogenously expressing VH4-34-HBL-1 and GA-10, as well as to the negative control cell line MM.1S. Figure 5A shows antibody binding, and the EC50 values ​​are shown in the table in Figure 5B. [Figure 6]

[0031] Figures 6A–6D are graphs and tables showing ADCCs of CHO cells expressing tabarmab (Figures 6A, 6B) or zanolimumab (Figures 6C, 6D) induced by D011-7016.hIgG1. hIgG1 isotype control antibodies were used as negative controls. Figures 6A and 6C show antibody binding as a function of luminescence output of reporter cells, and the associated EC50 values ​​are shown in tables in Figures 6B and 6D. [Figure 7]

[0032] Figures 7A and 7B are graphs and tables showing ADCP induced by D011-7016.hIgG1. Rituximab IgG1 was used as a positive control, and an hIgG1 isotype control antibody was used as a negative control. Figure 7A shows antibody binding as a function of CFSE% in CD14+ cells, and EC50 values ​​are shown in the table in Figure 7B. [Figure 8A]

[0033] Figures 8A-8C are graphs and images showing that the anti-VH4-34 antibody D011-7016.hIgG1 blocks cold agglutination. (Figure 8A) ELISA conjugation of D011-7016.mIgG2a to IGM-55.5 and VH4-34 antibody. (Figure 8B) Antibody IGM-55.5 induces cold agglutination of red blood cells at 4°C. (Figure 8C) D011-7016.hIgG1 inhibits cold agglutination induced by antibody IGM-55.5 at 4°C. [Figure 8B] Figures 8A-8C are graphs and images showing that the anti-VH4-34 antibody D011-7016.hIgG1 blocks cold agglutination. (Figure 8A) ELISA conjugation of D011-7016.mIgG2a to IGM-55.5 and VH4-34 antibody. (Figure 8B) Antibody IGM-55.5 induces cold agglutination of red blood cells at 4°C. (Figure 8C) D011-7016.hIgG1 inhibits cold agglutination induced by antibody IGM-55.5 at 4°C. [Figure 8C] Figures 8A-8C are graphs and images showing that the anti-VH4-34 antibody D011-7016.hIgG1 blocks cold agglutination. (Figure 8A) ELISA conjugation of D011-7016.mIgG2a to IGM-55.5 and VH4-34 antibody. (Figure 8B) Antibody IGM-55.5 induces cold agglutination of red blood cells at 4°C. (Figure 8C) D011-7016.hIgG1 inhibits cold agglutination induced by antibody IGM-55.5 at 4°C. [Figure 9A]

[0034] Figures 9A-9C are graphs showing the clearance of the VH4-34 antibody zanorimumab by clone D011-7016.mIgG2a from mouse circulation. Figure 9A summarizes the serum zanorimumab concentrations at various time points shown in Figure 9B. [Figure 9B] Figures 9A-9C are graphs showing the clearance of the VH4-34 antibody zanorimumab by clone D011-7016.mIgG2a from mouse circulation. Figure 9A summarizes the serum zanorimumab concentrations at various time points shown in Figure 9B. [Figure 9C]Figures 9A-9C show graphs illustrating the clearance of the VH4-34 antibody zanorimumab by clone D011-7016.mIgG2a from mouse circulation. Figure 9C shows a graph of relative VH-34+ B cells detected in mice at the indicated time point after treatment with the control antibody or D011-7016. [Figure 10A]

[0035] Figure 10A is a graph showing the binding affinity of the humanized anti-VH4-34 antibody to the 3A6 antibody (which contains the VH4-34 antigen). [Figure 10B] Figure 10B is a graph showing the binding affinity of the humanized anti-VH4-34 antibody to (Figure 10B)3A6-FR1 (VH4-34 FR1 mutant). [Figure 10C] Figure 10C is a graph showing the binding affinity of the humanized anti-VH4-34 antibody to the VSTB112-negative control. [Figure 11A]

[0036] Figure 11A is a graph showing the binding percentage of the humanized anti-VH4-34 antibody to the VH4-34 expressing cell line OCI-Ly3 and the negative control Pfeiffer cell line. [Figure 11B] Figure 11B is a graph showing the mean fluorescence intensity (MFI) of the humanized anti-VH4-34 antibody against the VH4-34 expressing cell line OCI-Ly3 and the negative control Pfeiffer cell line. [Figure 11C] (Figure 11C) shows the EC50 values ​​in a table. [Figure 12]

[0037] Figure 12 is an image showing that the addition of humanized anti-VH4-34 antibodies (H0L0, H5L1, and H5L2) reverses the cold agglutination caused by antibody IGM-55.5 at 4°C and 10°C, rather than the control antibody. [Figure 13]

[0038] Figure 13 shows a volcano plot of differentially expressed IGHV4-34 genes. Studies analyzing differential expression of human IGHV4-34 in diseased samples compared to normal controls were analyzed. Log-2 scaling changes and Benjamin-Hochberg adjusted p-values ​​were determined and plotted on the volcano plot. [Modes for carrying out the invention]

[0024]

[0039] This disclosure provides VH4-34 antigen-binding molecules having a desirable combination of biophysical and / or functional properties. The VH4-34-binding antibodies of this disclosure may be used in the treatment and prevention of diseases characterized by VH4-34 expression. For example, the VH4-34 antigen-binding molecules described herein are useful for depleting VH4-34-containing immunoglobulins (e.g., IgG, IgM) from blood and for depleting VH4-34-expressing cells (e.g., plasmablasts, plasma cells, memory B cells) from a subject. The antibodies are highly selective and do not deplete immunoglobulin / immunoglobulin-expressing B cells that lack VH4-34.

[0025]

[0040] definition

[0041] To facilitate understanding of this disclosure, several terms and phrases are defined below.

[0026]

[0042] An "antigen-binding molecule" refers to a molecule that binds to a target antigen, and includes monoclonal antibodies, polyclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific antibodies, trispecific antibodies, etc.), and antibody fragments (e.g., Fv, scFv, Fab, scFab, F(ab')2, Fab2, diabody, triabody, scFv-Fc, minibody, single-domain antibody (e.g., VhH), etc.), insofar as they demonstrate binding to the associated target molecule(s).

[0027]

[0043] As used herein, unless otherwise specified, the term “antibody” is understood to mean an intact antibody (e.g., an intact monoclonal antibody) or a fragment thereof, e.g., an Fc fragment of an antibody (e.g., an Fc fragment of a monoclonal antibody) or an antigen-binding fragment of an antibody (e.g., an antigen-binding fragment of a monoclonal antibody), and includes intact antibodies, antigen-binding fragments, or Fc fragments that have been modified, manipulated, or chemically conjugated. Generally, an antibody is a multimeric protein containing four polypeptide chains. Two of the polypeptide chains are called immunoglobulin heavy chains (H chains), and two of the polypeptide chains are called immunoglobulin light chains (L chains). The immunoglobulin heavy and light chains are linked by interchain disulfide bonds. The immunoglobulin heavy chains are linked by interchain disulfide bonds. The light chains consist of one variable region (VL) and one constant region (CL). The heavy chain consists of one variable region (VH) and at least three constant regions (CH1, CH2, and CH3). The variable region determines the antibody binding specificity. Each variable region contains four relatively conserved regions known as framework regions (FRs) and three hypervariable regions known as complementarity-determining regions (CDRs) adjacent to each other. The ranges of the FRs and CDRs are defined (Kabat, E. et al. (1991) Sequences of Proteins of Immunological Interest, 5th edition, USD Department of Health and Human Services, NIH publication no. 91-3242 and Chothia, C. et al. (1987) J.Mol.Biol.196:901-917). The three CDRs, called CDR1, CDR2, and CDR3, contribute to antibody binding specificity. Naturally occurring antibodies are used as starting materials for modified antibodies, such as chimeric antibodies and humanized antibodies. Examples of antibody-based antigen-binding fragments include Fab, Fab', (Fab')2, Fv, single-chain antibodies (e.g., scFv), minibodies, and diabodies. Examples of modified or manipulated antibodies include chimeric antibodies, humanized antibodies, and multispecific antibodies (e.g., bispecific antibodies).An example of a chemically conjugated antibody is one in which the toxin portion is conjugated.

[0028]

[0044] The terms “variable domain” and “variable region” are used synonymously to refer to portions of an antibody or immunoglobulin domain that exhibit variability in their sequence and are involved in determining the specificity and binding affinity of a particular antibody. The variability is not evenly distributed across the antibody’s variable domain, but rather concentrated within the subdomains of the heavy and light chain variable regions, respectively. These subdomains are called “hypervariable regions” or “complementarity-determining regions” (CDRs). The more conserved (i.e., non-hypervariable) portions of the variable domain are called “framework” regions (FRMs or FRs), which provide a scaffold for six CDRs in three-dimensional space, forming an antigen-binding surface.

[0029]

[0045] The terms “recipient,” “individual,” “subject,” “host,” and “patient” are used synonymously herein and, in some embodiments, refer to any mammalian subject, in particular human, for whom diagnosis, treatment, or therapy is desired. “Mammal” for treatment means any animal classified as a mammal, including humans, domesticated and livestock, as well as experimental, zoo, sport, or pet animals, such as dogs, horses, cats, cattle, sheep, goats, pigs, mice, rats, rabbits, guinea pigs, and monkeys. In some embodiments, the mammal is human. None of these terms require the supervision of a medical professional.

[0030]

[0046] As used herein, the term “effective dose” means an amount of a compound (e.g., a compound of this disclosure) sufficient to achieve a beneficial or desired result. An effective dose may be administered in one or more doses, applications, or dosages and is not intended to be limited to a particular formulation or route of administration. As used herein, the term “to treat” includes any effect that results in improvement of a condition, disease, disorder, etc., such as reducing, mitigating, modulating, improving or eliminating, or improving the symptoms thereof.

[0031]

[0047] As used herein, the term “pharmaceutical composition” refers to a combination of an active agent with an inactive or active carrier that makes the composition particularly suitable for in vivo or ex vivo diagnostic or therapeutic use.

[0032]

[0048] As used herein, the term “pharmaceutically acceptable carrier” refers to any of the standard pharmaceutical carriers, e.g., phosphate-buffered saline, water, emulsions (e.g., oil / water or water / oil emulsions), and various types of wetting agents. The composition may also contain stabilizers and preservatives. For examples of carriers, stabilizers, and adjuvants, see, for example, Martin, Remington's Pharmaceutical Sciences, 15th edition, Mack Publ. Co., Easton, PA (1975).

[0033]

[0049] The terms “a” and “an” as used herein mean “one or more,” and include the plural form unless the context is appropriate.

[0050] As used herein, all numerical terms or numerical ranges include all integers within or encompassing such a range, as well as values ​​or fractions of integers within or encompassing such a range, unless the context explicitly indicates otherwise. Thus, for example, a reference to the range 90–100% includes 91%, 92%, 93%, 94%, 95%, 95%, 96%, 97%, etc., as well as 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, etc., 92.1%, 92.2%, 92.3%, 92.4%, 92.5%, etc., and others. In another example, references to the range of 1 to 5,000 times include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 times, as well as 1.1, 1.2, 1.3, 1.4, 1.5 times, 2.1, 2.2, 2.3, 2.4, 2.5 times, and others.

[0034]

[0051] When used herein, "approximately" a number refers to the range that includes that number and the range that extends from 10% below that number to 10% above it. The "approximately" range refers to the range from 10% below the lower limit of the range to 10% above the upper limit of the range.

[0035]

[0052] "Identity percentage (%)" refers to the degree to which two sequences (nucleotides or amino acids) have identical residues at the same position in the alignment. For example, "The amino acid sequence is X% identical to sequence number Y" refers to the identity percentage of the amino acid sequence with respect to sequence number Y, and details that X% of the residues in the amino acid sequence are identical to the residues in the sequence disclosed in sequence number Y. Generally, computer programs are used for such calculations. Exemplary programs for comparing and aligning pairs of sequences include ALIGN (Myers and Miller, 1988), FASTA (Pearson and Lipman, 1988; Pearson, 1990), and gapped BLAST (Altschul et al., 1997), BLASTP, BLASTN, or GCG (Devereux et al., 1984).

[0036]

[0053] Where a composition is described as having, including, or comprising certain components, or where a process and method is described as having, including, or comprising certain steps, it is further intended that there are compositions of the Disclosure that are essentially composed of or comprise the listed components, and processes and methods of the Disclosure that are essentially composed of or comprise the listed processing steps.

[0037]

[0054] In general, percentages of composition are given by weight unless otherwise specified. Furthermore, if a variable is not defined, the previous definition of the variable prevails.

[0055] Where nucleic acid sequences are disclosed herein, their reverse complements are also explicitly intended.

[0038]

[0056] The methods described herein may preferably be carried out in vitro. The term “in vitro” is intended to encompass procedures carried out using cells in culture, while the term “in vivo” is intended to encompass procedures using / in intact multicellular organisms. VH4-34

[0057] Immunoglobulin heavy chain variable 4-34 (VH4-34) is a protein encoded in humans by the IGHV4-34 gene, having the amino acid sequence shown in SEQ ID NO: 1 (UniProt KB:P06331-1, v1). The N-terminal 26 amino acids form a signal peptide (SEQ ID NO: 2), which is cleaved from the mature form of VH4-34 as shown in SEQ ID NO: 3.

[0039]

[0058] The biology of VH4-34 is described, for example, in Schickel et al., J Exp Med. (2017) 214(7): pp. 1991-2003 and Pugh-Bernard et al., J Clin Invest. (2001) 108(7): pp. 1061-70 (both of which are incorporated herein by reference in their entirety). VH4-34 is an endogenous autoreactive antibody heavy chain variable region (VH) subregion, specifically a VH subregion formed by heavy chain framework region 1 (HC-FR1), heavy chain complementarity determining region 1 (HC-CDR1), FR2, HC-CDR2, and FR-3 (i.e., [HC-FR1]-[HC-CDR1]-[HC-FR2]-[HC-CDR2]-[HC-FR3]). VH4-34 contains a hydrophobic patch in the first framework region (FR1) that recognizes I / i carbohydrates expressed by erythrocytes. A unique hydrophobic patch of autoreactive VH4-34 can be detected using the monoclonal rat anti-idiotype antibody 9G4 (see, for example, Stevenson et al., Blood (1986) 68:430, Potter et al., J Exp Med. (1993) 178:1419-1428, and Richardson et al., J Immunol (2013) 191(10):4926-4939).

[0040]

[0059] The FR1 of VH4-34 is shown in SEQ ID NO: 4. For autoreactivity, two motifs within FR1 are required: Q32-W33 and A49-V50-Y51 (amino acid residues numbered relative to SEQ ID NO: 1), which together form a hydrophobic patch. In other VH4 family members, these motifs are instead E32-S33 and T49-V50-S51. The hydrophobic patch of VH4-34 FR1 recognizes the linear lactosamine moiety of cell surface glycoproteins containing N-acetyllactosamine (see, e.g., Young et al. PNAS USA (2015) 112(44):13447-54).

[0041]

[0060] B cells expressing VH4-34 are common in the naive B cell repertoire, but B cell tolerance mechanisms normally prevent VH4-34-expressing B cells from entering germinal centers and switching to memory B cells or plasma cells; therefore, they are not typically found among memory and plasma B cells in healthy individuals. However, in diseases such as systemic lupus erythematosus (SLE) and cold agglutinin disease, autoreactive VH4-34-expressing B cells are overpresented in the memory and plasma cell compartments and are involved in disease pathogenesis.

[0042]

[0061] Bhat et al., J Rheumatol (2002) 29:21 pp. 14-21, found that 54% of SLE patients had elevated levels of VH4-34 IgG, and among those patients, 30% also had elevated levels of VH4-34 IgM. Patients with VH4-34 IgM were observed to have significantly fewer severe diseases than patients with only circulating VH4-34 IgG isotype, suggesting that isotype switching of the immunoglobulin encoding VH4-34 from IgM to IgG may correlate with disease progression in SLE.

[0043]

[0062] In this specification, references to "VH4-34" include human VH4-34 and its variants, isoforms, or fragments. In some embodiments, VH4-34 comprises or consists of an amino acid sequence having 70% or higher amino acid sequence identity with respect to the amino acid sequence of SEQ ID NO: 1, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity. In some embodiments, VH4-34 comprises or consists of an amino acid sequence having 70% or higher amino acid sequence identity with respect to the amino acid sequence of SEQ ID NO: 3, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity. In some embodiments, VH4-34 contains or consists of the amino acid sequence of SEQ ID NO: 3.

[0044]

[0063] Isoforms, fragments, and variants of human VH4-34 may be optionally characterized by having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to the amino acid sequence of immature or mature VH4-34 of a given species, e.g., human. Variants of human VH4-34 may be optionally characterized by having 70% or higher amino acid sequence identity with respect to the amino acid sequence of SEQ ID NO: 1 or 3, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher amino acid sequence identity.

[0045]

[0064] Isoforms, fragments, and variants of VH4-34 may, optionally, possess the functional properties / activities of human VH4-34, which can be determined, for example, by analysis using a suitable assay for functional properties / activities. For example, isoforms, fragments, or variants of human VH4-34 may form antibody:antigen complexes with the anti-idiotype antibody 9G4.

[0046]

[0065] In some embodiments, VH4-34 includes an amino acid sequence having 70% or higher amino acid sequence identity with respect to the amino acid sequence of SEQ ID NO: 4, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity. In some embodiments, VH4-34 includes the amino acid sequence of SEQ ID NO: 4.

[0047]

[0066] In some embodiments, VH4-34 includes Q at the position corresponding to position 32 of SEQ ID NO: 1. In some embodiments, VH4-34 includes W at the position corresponding to position 33 of SEQ ID NO: 1. In some embodiments, VH4-34 includes A at the position corresponding to position 49 of SEQ ID NO: 1. In some embodiments, VH4-34 includes V at the position corresponding to position 50 of SEQ ID NO: 1. In some embodiments, VH4-34 includes Y at the position corresponding to position 51 of SEQ ID NO: 1. In some embodiments, VH4-34 includes one or more of the following (for example, one, two, three, four, or all of the following): Q at the position corresponding to position 32 of SEQ ID NO: 1, W at the position corresponding to position 33, A at the position corresponding to position 49, V at the position corresponding to position 50, and Y at the position corresponding to position 51. In some embodiments, VH4-34 includes Q at the position corresponding to position 32 of SEQ ID NO: 1, W at the position corresponding to position 33, A at the position corresponding to position 49, V at the position corresponding to position 50, and Y at the position corresponding to position 51.

[0048]

[0067] In this specification, the location or region of an amino acid sequence that "corresponds" to a specific location / region of a reference amino acid sequence can be identified, for example, by sequence alignment of the target sequence to the reference sequence using sequence alignment software, such as ClustalOmega (Soding, J.2005, Bioinformatics21, pp. 951-960).

[0049]

[0068] In some embodiments, VH4-34 is provided as an isolated peptide / polypeptide. In some embodiments, VH4-34 is provided as a molecule containing VH4-34. In some embodiments, VH4-34 is provided as a polypeptide complex.

[0050]

[0069] In some embodiments, VH4-34 is provided as an antigen-binding molecule containing VH4-34. In some embodiments, VH4-34 is provided as a polypeptide complex formed between two or more polypeptides (i.e., one or more polypeptides of the polypeptide complex are or contain VH4-34). For example, VH4-34 is provided as an antigen-binding molecule formed from a polypeptide complex comprising one or more immunoglobulin light chains and one or more immunoglobulin heavy chains, wherein the immunoglobulin heavy chains comprise a heavy chain variable region (VH) containing VH4-34. Exemplarily, the monoclonal antibodies zanorimumab, patrizumab, and tavarumab described herein are each formed from a polypeptide complex comprising two immunoglobulin light chains and two immunoglobulin heavy chains comprising a VH region containing VH4-34.

[0051]

[0070] Cells encoding / containing / expressing VH4-34 and / or peptide / polypeptide / polypeptide complexes (e.g., antigen-binding molecules) containing VH4-34 may contain endogenous nucleic acids (e.g., genomic DNA) encoding VH4-34 and / or peptide / polypeptide / polypeptide complexes (e.g., antigen-binding molecules) containing VH4-34. Such cells may be called VH4-34 lineage cells.

[0052]

[0071] In some embodiments, cells encoding / containing / expressing VH4-34 and / or peptide / polypeptide / polypeptide complexes (e.g., antigen-binding molecules) containing VH4-34 are B cells or their precursors. In some embodiments, cells encoding / containing / expressing VH4-34 and / or peptide / polypeptide / polypeptide complexes (e.g., antigen-binding molecules) containing VH4-34 are B cells, naive B cells, plasmablasts, plasma B cells, memory B cells, or B cell precursors. In some embodiments, the cells are selected from plasmablasts, plasma B cells, and memory B cells.

[0053]

[0072] As used herein, “naive” B cells refer to mature B cells that have not encountered an antigen for which the B cell’s BCR is specific. Naive B cells may also be called mature naive B cells or mature B cells. Naive B cells may be characterized by the expression of one or more of the following (e.g., on the cell surface): CD19, CD20, CD24, CD40, CD38, CD45, CD21, MHC class II, IgM, and IgD. Naive B cells may be characterized by the absence of CD27 expression (e.g., on the cell surface).

[0054]

[0073] As used herein, “plasma” B cells refer to B cells expressing large amounts of soluble antibodies. Plasma B cells may be characterized by the expression of one or more of the following (e.g., on the cell surface): CD27, CD38, CD138, CD78, CD126, CXCR4, and BCMA. Plasma B cells may be characterized by the absence of CD20 and / or CD24 expression (e.g., on the cell surface). “Plasmablast” refers to a cell in the short-lived differentiation stage between post-germinal center B cells and mature plasma cells. Plasmablasts have high proliferative capacity and a nearly fully mature plasma cell phenotype.

[0055]

[0074] As used herein, “memory” B cells refer to B cells formed in germinal centers after the primary immune response. Memory B cells may be characterized by the expression of one or more of the following (e.g., on the cell surface): CD19, CD20, CD21, CD24, CD27, CD95, CD148, MHC class II, and TACI.

[0056]

[0075] As used herein, “B cell precursor” refers to the cell type upstream of naive B cells in the process of B cell development. B cell development is described, for example, in Pieper et al., J Allergy Clin Immunol (2013) 131(4): pp. 959-71, which is incorporated herein by reference in its entirety. In some embodiments, the precursor cells of naive B cells may be selected from stem cells, pro-B cells, early pro-B cells, late pro-B cells, pre-B cells, large pre-B cells, small pre-B cells, or immature B cells. Stem cells may be hematopoietic stem cells and may be characterized, for example, by the absence of CD34 expression (e.g., surface expression) and / or CD10 expression (e.g., surface expression). Early pro-B cells may be characterized by the expression (e.g., surface expression) of CD10, CD43, CD45 and / or MHC class II. Late pro-B cells may be characterized by the expression (e.g., surface expression) of CD19, CD40, CD43, CD45, and / or MHC class II. Large pre-B cells may be characterized by the expression (e.g., surface expression) of pre-BCR, CD19, CD40, CD43, CD45, and / or MHC class II. Small pre-B cells may be characterized by the expression (e.g., surface expression) of pre-BCR, CD19, CD40, CD45, and / or MHC class II. Immature B cells may be characterized by the absence of expression (e.g., surface expression) of CD10, CD19, CD20, CD24, CD38, CD40, CD45, IgM, and / or MHC class II, as well as / or CD27.

[0057]

[0076] In some embodiments, B cells are further characterized by referring to the class of immunoglobulin expressed by the cell. In some embodiments, B cells are B cells expressing IgM, IgD, IgG, IgE, or IgA.

[0058]

[0077] In some embodiments, the B cells are B cells expressing IgM and / or IgD. In some embodiments, the B cells are B cells expressing IgG, IgE, or IgA. In some embodiments, the B cells are B cells expressing IgM. In some embodiments, the B cells are B cells expressing IgG.

[0059]

[0078] In some embodiments, the VH4-34 binding molecule comprises isotype IgG and / or IgM immunoglobulins. In some embodiments, the VH4-34 binding molecule comprises isotype IgG immunoglobulins. In some embodiments, the VH4-34 binding molecule comprises isotype IgM immunoglobulins.

[0060]

[0079] In some embodiments, the peptide / polypeptide / polypeptide complex (e.g., antigen-binding molecule) containing VH4-34 is an immunoglobulin of isotype IgG and / or IgM. In some embodiments, the peptide / polypeptide / polypeptide complex (e.g., antigen-binding molecule) containing VH4-34 is an immunoglobulin of isotype IgG. In some embodiments, the peptide / polypeptide / polypeptide complex (e.g., antigen-binding molecule) containing VH4-34 is an immunoglobulin of isotype IgM.

[0061]

[0080] In some embodiments, cells encoding / containing / expressing VH4-34 and / or peptide / polypeptide / polypeptide complexes (e.g., antigen-binding molecules) containing VH4-34 are B cells encoding / containing / expressing IgG and / or IgM containing VH4-34. In some embodiments, cells encoding / containing / expressing VH4-34 and / or peptide / polypeptide / polypeptide complexes (e.g., antigen-binding molecules) containing VH4-34 are B cells encoding / containing / expressing IgG containing VH4-34. In some embodiments, cells encoding / containing / expressing VH4-34 and / or peptide / polypeptide / polypeptide complexes (e.g., antigen-binding molecules) containing VH4-34 are B cells encoding / containing / expressing IgM containing VH4-34.

[0062]

[0081] Polypeptide complexes containing VH4-34 (e.g., antigen-binding molecules) contain antibodies to which the anti-idiotype antibody 9G4 binds. Such antibodies are sometimes called "9G4-reactive" antibodies. Polypeptide complexes containing VH4-34 (e.g., antigen-binding molecules) include 9G4-reactive antibodies, such as zanorimumab (HuMAX-CD4; also known as CAS number 652153-01-0), patritumab (AMG-888; also known as CAS number 1262787-83-6), and tabarmab (LY-2127399; also known as CAS number 1143503-67-6). VH4-34 antigen binding molecule

[0082] This disclosure provides antigen-binding molecules that can bind to (i.e., bind to) VH4-34 and / or antigen-binding molecules containing VH4-34.

[0063]

[0083] The VH4-34 antigen-binding molecules of this disclosure include one or more VH4-34-binding moieties. In some embodiments, the VH4-34-binding moieties include antibody heavy chain variable regions (VH) and antibody light chain variable regions (VL) of an antibody that is specifically binding to VH4-34. The VH4-34-binding moieties may include, or consist of, aptamers capable of binding to a target antigen, such as nucleic acid aptamers (as outlined, for example, in Zhou and Rossi Nat Rev Drug Discov. 2017 16(3): pp. 181-202). In some embodiments, the portion may include, or may consist of, antigen-binding peptides / polypeptides, e.g., peptide aptamers, thioredoxins, monobodies, antikalin, Knitz domains, avimers, Nottin, finomers, atrimers, DARPin, afibodies, nanobodies (i.e., single-domain antibodies (sdAbs)), affilins, armadillo repeat proteins (ArmRPs), OBody, or fibronectin—for example, the whole of which is incorporated herein by reference in Reverdatto et al., Curr Top Med Chem. 2015;15(12):1082-1101 (see also, for example, Boersma et al., J Biol Chem (2011) 286:41273-85 and Emanuel et al., Mabs (2011) 3:38-48).

[0064]

[0084] As used herein, “peptide” refers to a chain of two or more amino acid monomers linked by peptide bonds. Peptides typically have a length of about 2 to 50 amino acids. “Polypeptide” is a polymer chain of two or more peptides. Polypeptides typically have a length of about 50 or more amino acids.

[0065]

[0085] The VH4-34 antigen-binding molecules of this disclosure generally include antigen-binding domains comprising VH and VL of an antibody specifically capable of binding to VH4-34. The antigen-binding domain formed by VH and VL may also be referred to herein as the Fv region.

[0066]

[0086] Antigen-binding molecules may be or contain antigen-binding polypeptides or antigen-binding polypeptide complexes. Antigen-binding molecules may contain more than one polypeptide that together form an antigen-binding domain. Polypeptides may associate covalently or non-covalently. In some embodiments, polypeptides form part of a larger polypeptide containing polypeptides (for example, in the case of scFv containing VH and VL, or in the case of scFab containing VH-CH1 and VL-CL).

[0067]

[0087] Antigen-binding molecules may refer to non-covalent or covalent complexes of more than one polypeptide (e.g., two, three, four, six, or eight polypeptides), such as IgG-like antigen-binding molecules containing two heavy-chain polypeptides and two light-chain polypeptides.

[0068]

[0088] The VH4-34 antigen-binding molecules of this disclosure may be designed and prepared using sequences of monoclonal antibodies (mAbs) capable of binding to VH4-34. Antigen-binding regions of antibodies, e.g., single-chain variable fragments (scFv), Fab, and F(ab')2 fragments, may also be used / provided. An "antigen-binding region" is any fragment of an antibody that binds to a target to which a given antibody is specific.

[0069]

[0089] Antibodies generally contain six complementarity-determining regions (CDRs): three heavy chain variable (VH) regions: HC-CDR1, HC-CDR2, and HC-CDR3, and three light chain variable (VL) regions: LC-CDR1, LC-CDR2, and LC-CDR3. The six CDRs together define the paratope of the antibody, which is part of the antibody that binds to the target antigen.

[0070]

[0090] The VH and VL regions each contain a framework region (FR) on either side of each CDR, which provides a scaffold for the CDR. From the N-terminus to the C-terminus, the VH region has the following structure: N-terminus-[HC-FR1]-[HC-CDR1]-[HC-FR2]-[HC-CDR2]-[HC-FR3]-[HC-CDR3]-[HC-FR4]-C-terminus, and the VL region has the following structure: N-terminus-[LC-FR1]-[LC-CDR1]-[LC-FR2]-[LC-CDR2]-[LC-FR3]-[LC-CDR3]-[LC-FR4]-C-terminus.

[0071]

[0091] There are several different conventions for defining antibody CDR and FR, such as those described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), Chothia et al., J.Mol.Biol.196:901-917 (1987), and VBASE2, as described in Retter et al., Nucl. Acids Res. (2005) 33 (Appendix 1):D671-D674. The CDRs and FRs of the VH and VL regions of the antibody clones described herein are defined according to the International IMGT (ImMunoGeneTics) Information System (LeFranc et al., Nucleic Acids Res. (2015) 43 (Database Issues): D413-22), which uses the IMGT V-domain numbering rules as described in Lefranc et al., Dev. Comp. Immunol. (2003) 27: pp. 55-77. In some embodiments, the CDRs and FRs of the antigen-binding molecules mentioned herein are defined according to the IMGT Information System, Kabat System, or Chothia System.

[0072]

[0092] In some embodiments, the VH4-34 antigen-binding molecule includes a CDR of the antigen-binding molecule that binds to VH4-34. In some embodiments, the VH4-34 antigen-binding molecule includes a FR of the antigen-binding molecule that binds to VH4-34. In some embodiments, the VH4-34 antigen-binding molecule includes both the CDR and FR of the antigen-binding molecule that binds to VH4-34. That is, in some embodiments, the VH4-34 antigen-binding molecule includes the VH region and / or VL region of the antigen-binding molecule that binds to VH4-34.

[0073]

[0093] In some embodiments, the VH4-34 antigen-binding molecule includes the CDR, FR, and / or VH and / or VL regions of the VH4-34-binding antibody clone described herein, or the CDR, FR, and / or VH and / or VL regions derived from those of the VH4-34-binding antibody clone described herein. In some embodiments, the VH4-34 binding antibody clones are D011-1E11p, D011-6039, D011-6040, D011-7016, D011-1E11-5T2, D011-7016-LD1, D011-7016-LD2, D011-7016-LD2_H0L0, D011-7016-LD2_H1L1, D011-7016-LD2_H1L2, D011-7016-LD2_H1L3, D011-7016-LD2_H1L4, and D011-7016-LD2_H3. Selected from L1, D011-7016-LD2_H3L2, D011-7016-LD2_H3L3, D011-7016-LD2_H3L4, D011-7016-LD2_H4L1, D011-7016-LD2_H4L2, D011-7016-LD2_H4L3, D011-7016-LD2_H4L4, D011-7016-LD2_H5L1, D011-7016-LD2_H5L2, D011-7016-LD2_H5L3, and D011-7016-LD2_H5L4.

[0074]

[0094] In some embodiments, the VH4-34 antigen-binding molecule includes a VH region that conforms to any one of the following (1) to (8): (1)(D011-1E11p;D011-1E11-5T2) HC-CDR1 having the amino acid sequence of SEQ ID NO: 6 HC-CDR2 having the amino acid sequence of SEQ ID NO: 7 HC-CDR3 having the amino acid sequence of SEQ ID NO: 8 VH region including Or a variant in which one or more amino acids in HC-CDR1, HC-CDR2, or HC-CDR3 are substituted with other amino acids, (2)(D011-6039) HC-CDR1 having the amino acid sequence of SEQ ID NO: 22 HC-CDR2 having the amino acid sequence of SEQ ID NO: 23 HC-CDR3 having the amino acid sequence of SEQ ID NO: 24 VH region including Or a variant in which one or more amino acids in HC-CDR1, HC-CDR2, or HC-CDR3 are substituted with other amino acids, (3)(D011-6040) HC-CDR1 having the amino acid sequence of SEQ ID NO: 38 HC-CDR2 having the amino acid sequence of SEQ ID NO: 39 HC-CDR3 having the amino acid sequence of SEQ ID NO: 40 VH region including Or a variant in which one or more amino acids in HC-CDR1, HC-CDR2, or HC-CDR3 are substituted with other amino acids, (4)(D011-7016) HC-CDR1 having the amino acid sequence of SEQ ID NO: 54 HC-CDR2 having the amino acid sequence of SEQ ID NO: 55 HC-CDR3 having the amino acid sequence of SEQ ID NO: 56 VH region including Or a variant in which one or more amino acids in HC-CDR1, HC-CDR2, or HC-CDR3 are substituted with other amino acids, (5)(D011-7016-LD1;D011-7016-LD2;D011-7016-LD2_H0) HC-CDR1 having the amino acid sequence of SEQ ID NO: 142 HC-CDR2 having the amino acid sequence of SEQ ID NO: 55 HC-CDR3 having the amino acid sequence of SEQ ID NO: 56 VH region including Or a variant in which one or more amino acids in HC-CDR1, HC-CDR2, or HC-CDR3 are substituted with other amino acids, (6) (D011-7016; D011-7016-LD1; D011-7016-LD2 consensus) HC-CDR1 having the amino acid sequence of SEQ ID NO: 152 HC-CDR2 having the amino acid sequence of SEQ ID NO: 55 HC-CDR3 having the amino acid sequence of SEQ ID NO: 56 VH region including Or a variant in which one or more amino acids in HC-CDR1, HC-CDR2, or HC-CDR3 are substituted with other amino acids, (7)(D011-7016-LD2_HCON) HC-CDR1 having the amino acid sequence of SEQ ID NO: 185 HC-CDR2 having the amino acid sequence of SEQ ID NO: 55 HC-CDR3 having the amino acid sequence of SEQ ID NO: 56 VH region including Or a variant in which one or more amino acids in HC-CDR1, HC-CDR2, or HC-CDR3 are substituted with other amino acids, (8)(D011-7016-LD2_H1;D011-7016-LD2_H3;D011-7016-LD2_H4;D011-7016-LD2_H5) HC-CDR1 having the amino acid sequence of SEQ ID NO: 172 HC-CDR2 having the amino acid sequence of SEQ ID NO: 55 HC-CDR3 having the amino acid sequence of SEQ ID NO: 56 VH region including Alternatively, a variant in which one or more amino acids in HC-CDR1, HC-CDR2, or HC-CDR3 are substituted with other amino acids.

[0075]

[0095] In some embodiments, the VH4-34 antigen-binding molecule includes a VH region that conforms to one of the following (9) to (21): (9)(D011-1E11p) HC-FR1 having the amino acid sequence of SEQ ID NO: 9 HC-FR2 having the amino acid sequence of SEQ ID NO: 10 HC-FR3 having the amino acid sequence of SEQ ID NO: 11 HC-FR4 having the amino acid sequence of SEQ ID NO: 12 VH region including Or, a variant in which one or more amino acids among HC-FR1, HC-FR2, HC-FR3, or HC-FR4 are substituted with other amino acids. (10)(D011-6039) HC-FR1 having the amino acid sequence of SEQ ID NO: 25 HC-FR2 having the amino acid sequence of SEQ ID NO: 26 HC-FR3 having the amino acid sequence of SEQ ID NO: 27 HC-FR4 having the amino acid sequence of SEQ ID NO: 28 VH region including Or, a variant in which one or more amino acids among HC-FR1, HC-FR2, HC-FR3, or HC-FR4 are substituted with other amino acids. (11)(D011-6040) HC-FR1 having the amino acid sequence of SEQ ID NO: 41 HC-FR2 having the amino acid sequence of SEQ ID NO: 42 HC-FR3 having the amino acid sequence of SEQ ID NO: 43 HC-FR4 having the amino acid sequence of SEQ ID NO: 44 VH region including Or, a variant in which one or more amino acids among HC-FR1, HC-FR2, HC-FR3, or HC-FR4 are substituted with other amino acids. (12)(D011-7016;D011-7016-LD1;D011-7016-LD2) HC-FR1 having the amino acid sequence of SEQ ID NO: 57 HC-FR2 having the amino acid sequence of SEQ ID NO: 58 HC-FR3 having the amino acid sequence of SEQ ID NO: 59 HC-FR4 having the amino acid sequence of SEQ ID NO: 60 VH region including Or, a variant in which one or more amino acids among HC-FR1, HC-FR2, HC-FR3, or HC-FR4 are substituted with other amino acids. (13)(D011-1E11-5T2) HC-FR1 having the amino acid sequence of SEQ ID NO: 126 HC-FR2 having the amino acid sequence of SEQ ID NO: 127 HC-FR3 having the amino acid sequence of SEQ ID NO: 128 HC-FR4 having the amino acid sequence of SEQ ID NO: 129 VH region including Or, a variant in which one or more amino acids among HC-FR1, HC-FR2, HC-FR3, or HC-FR4 are substituted with other amino acids. (14) (D011-1E11p; D011-1E11-5T2 consensus) HC-FR1 having the amino acid sequence of SEQ ID NO: 157 HC-FR2 having the amino acid sequence of SEQ ID NO: 158 HC-FR3 having the amino acid sequence of SEQ ID NO: 159 HC-FR4 having the amino acid sequence of SEQ ID NO: 160 VH region including Or, a variant in which one or more amino acids among HC-FR1, HC-FR2, HC-FR3, or HC-FR4 are substituted with other amino acids. (15)(D011-1E11p;D011-1E11-5T2) HC-FR1 having amino acid sequences of SEQ ID NOs. 9, 126, or 157 HC-FR2 having amino acid sequences of SEQ ID NOs. 10, 127, or 158 HC-FR3 having the amino acid sequence of SEQ ID NO: 11, 128, or 159 HC-FR4 having the amino acid sequence of SEQ ID NO: 12, 129, or 160 VH region including Or, a variant in which one or more amino acids among HC-FR1, HC-FR2, HC-FR3, or HC-FR4 are substituted with other amino acids. (16)(D011-7016-LD2 HCON) HC-FR1 having the amino acid sequence of SEQ ID NO: 186 HC-FR2 having the amino acid sequence of SEQ ID NO: 187 HC-FR3 having the amino acid sequence of SEQ ID NO: 188 HC-FR4 having the amino acid sequence of SEQ ID NO: 129 VH region including Or, a variant in which one or more amino acids among HC-FR1, HC-FR2, HC-FR3, or HC-FR4 are substituted with other amino acids. (17)(D011-7016-LD2_H0) HC-FR1 having the amino acid sequence of SEQ ID NO: 168 HC-FR2 having the amino acid sequence of SEQ ID NO: 169 HC-FR3 having the amino acid sequence of SEQ ID NO: 170 HC-FR4 having the amino acid sequence of SEQ ID NO: 129 VH region including Or, a variant in which one or more amino acids among HC-FR1, HC-FR2, HC-FR3, or HC-FR4 are substituted with other amino acids. (18)(D011-7016-LD2_H1) HC-FR1 having the amino acid sequence of SEQ ID NO: 173 HC-FR2 having the amino acid sequence of SEQ ID NO: 174 HC-FR3 having the amino acid sequence of SEQ ID NO: 175 HC-FR4 having the amino acid sequence of SEQ ID NO: 129 VH region including Or, a variant in which one or more amino acids among HC-FR1, HC-FR2, HC-FR3, or HC-FR4 are substituted with other amino acids. (19)(D011-7016-LD2_H3) HC-FR1 having the amino acid sequence of SEQ ID NO: 178 HC-FR2 having the amino acid sequence of SEQ ID NO: 179 HC-FR3 having the amino acid sequence of SEQ ID NO: 180 HC-FR4 having the amino acid sequence of SEQ ID NO: 129 VH region including Or, a variant in which one or more amino acids among HC-FR1, HC-FR2, HC-FR3, or HC-FR4 are substituted with other amino acids. (20)(D011-7016-LD2_H4) HC-FR1 having the amino acid sequence of SEQ ID NO: 168 HC-FR2 having the amino acid sequence of SEQ ID NO: 179 HC-FR3 having the amino acid sequence of SEQ ID NO: 170 HC-FR4 having the amino acid sequence of SEQ ID NO: 129 VH region including Or, a variant in which one or more amino acids among HC-FR1, HC-FR2, HC-FR3, or HC-FR4 are substituted with other amino acids. (21)(D011-7016-LD2_H5) HC-FR1 having the amino acid sequence of SEQ ID NO: 173 HC-FR2 having the amino acid sequence of SEQ ID NO: 183 HC-FR3 having the amino acid sequence of SEQ ID NO: 170 HC-FR4 having the amino acid sequence of SEQ ID NO: 129 VH region including Alternatively, a variant in which one, two, or three amino acids among HC-FR1, HC-FR2, HC-FR3, or HC-FR4 are substituted with other amino acids.

[0076]

[0096] In some embodiments, the VH4-34 antigen-binding molecule includes a VH region comprising a CDR according to one of (1) to (8) above, and an FR according to one of (9) to (21) above.

[0077]

[0097] In some embodiments, the VH4-34 antigen-binding molecule includes a VH region that conforms to one of the following (22) to (30): (22)(D011-1E11p)(1) and VH region including CDR and FR according to (9) or (11), (23)(D011-6039)(2) and VH region including CDR and FR according to (10), (24)(D011-6040)(3) and VH region including CDR and FR according to (11), (25)(D011-7016)(4) and VH region including CDR and (12) (26) VH region including CDR according to (D011-1E11-5T2)(1) and FR according to (13), (14) or (15), (27) VH region including CDR according to (5) and FR according to (12), (28) VH region including CDR according to (D011-7016-LD2 HCON)(7) and FR according to (16), (29) VH region including CDR according to (D011-7016-LD2_H0)(5) and FR according to (17), (30) A VH region containing a CDR conforming to (8) (D011-7016-LD2_H1;D011-7016-LD2_H3;D011-7016-LD2_H4;D011-7016-LD2_H5)(8) and an FR conforming to any one of (18), (19), (20), or (21).

[0078]

[0098] In some embodiments, the VH4-34 antigen-binding molecule includes a VH region that conforms to one of the following (31) to (46): (31) (D011-1E11P; D011-6040 consensus) A VH region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of Sequence ID No. 69, more preferably at least one of the following sequence identities: 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. (32) (D011-6093; D011-7016 consensus) A VH region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of Sequence ID No. 70, more preferably at least one of the following sequence identities: 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. (33) (D011-1E11p;D011-1E11-5T2 consensus) A VH region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO: 156, more preferably at least one of the following sequence identities: 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. (34) A VH region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO: 151 (D011-7016; D011-7016-LD1; D011-7016-LD2 consensus), more preferably one of the sequence identities of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. (35) A VH region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of (D011-7016-LD2 HCON) Sequence ID No. 184, more preferably at least one of the following sequence identities: 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. (36)(D011-1E11p) A VH region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of Sequence ID No. 5, more preferably at least one of the following sequence identities: 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. (37)(D011-6039) A VH region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO: 21, more preferably at least one of the following sequence identities: 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. (38)(D011-6040) A VH region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of Sequence ID No. 37, more preferably at least one of the following sequence identities: 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. (39)(D011-7016) A VH region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of Sequence ID No. 53, more preferably at least one of the following sequence identities: 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. (40)(D011-1E11-5T2) A VH region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of sequence number 125, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. (41) A VH region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of sequence number 141 (D011-7016-LD1;D011-7016-LD2), more preferably one of the sequence identities of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. (42)(D011-7016-LD2_H0) A VH region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of sequence number 167, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. (43) A VH region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of (D011-7016-LD2_H1) Sequence ID No. 171, more preferably at least one of the following sequence identities: 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. (44)(D011-7016-LD2_H3) A VH region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of sequence number 176, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. (45)(D011-7016-LD2_H4) A VH region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of sequence number 181, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. (46)(D011-7016-LD2_H5) A VH region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of sequence number 182, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

[0079]

[0099] In some embodiments, the VH4-34 antigen-binding molecule includes a VL region that conforms to one of the following (47) to (56): (47)(D011-1E11p) LC-CDR1 having the amino acid sequence of SEQ ID NO: 14 LC-CDR2 having amino acid sequence LVS LC-CDR3 having the amino acid sequence of SEQ ID NO: 16 VL area including, Or a variant in which one or more amino acids in LC-CDR1, LC-CDR2, or LC-CDR3 are substituted with other amino acids, (48)(D011-6039) LC-CDR1 having the amino acid sequence of SEQ ID NO: 30 LC-CDR2 having amino acid sequence YTS LC-CDR3 having the amino acid sequence of SEQ ID NO: 32 VL area including, Or a variant in which one or more amino acids in LC-CDR1, LC-CDR2, or LC-CDR3 are substituted with other amino acids, (49)(D011-6040) LC-CDR1 having the amino acid sequence of SEQ ID NO: 46 LC-CDR2 having amino acid sequence AAS LC-CDR3 having the amino acid sequence of SEQ ID NO: 48 VL area including, Or a variant in which one or more amino acids in LC-CDR1, LC-CDR2, or LC-CDR3 are substituted with other amino acids, (50)(D011-7016) LC-CDR1 having the amino acid sequence of SEQ ID NO: 62 LC-CDR2 having the amino acid sequence YAS LC-CDR3 having the amino acid sequence of SEQ ID NO: 64 VL area including, Or a variant in which one or more amino acids in LC-CDR1, LC-CDR2, or LC-CDR3 are substituted with other amino acids, (51)(D011-1E11-5T2) LC-CDR1 having the amino acid sequence of SEQ ID NO: 131 LC-CDR2 having amino acid sequence LVS LC-CDR3 having the amino acid sequence of SEQ ID NO: 16 VL area including, Or a variant in which one or more amino acids in LC-CDR1, LC-CDR2, or LC-CDR3 are substituted with other amino acids, (52)(D011-7016-LD1;D011-7016-LD2;D011-7016-LD2 LCON;D011-7016-LD2_L0;D011-7016-LD2_L1;D011-7016-LD2_L2;D011-7016-LD2_L3;D011-7016-LD2_L4) LC-CDR1 having the amino acid sequence of SEQ ID NO: 144 LC-CDR2 having the amino acid sequence YAS LC-CDR3 having the amino acid sequence of SEQ ID NO: 64 VL area including, Or a variant in which one or more amino acids in LC-CDR1, LC-CDR2, or LC-CDR3 are substituted with other amino acids, (53) (D011-1E11p; D011-1E11-5T2 consensus) LC-CDR1 having the amino acid sequence of SEQ ID NO: 162 LC-CDR2 having amino acid sequence LVS LC-CDR3 having the amino acid sequence of SEQ ID NO: 16 VL area including, Or a variant in which one or more amino acids in LC-CDR1, LC-CDR2, or LC-CDR3 are substituted with other amino acids, (54)(D011-1E11p;D011-1E11-5T2) LC-CDR1 having amino acid sequences of SEQ ID NOs. 14, 131, or 162 LC-CDR2 having amino acid sequence LVS LC-CDR3 having the amino acid sequence of SEQ ID NO: 16 VL area including, Or a variant in which one or more amino acids in LC-CDR1, LC-CDR2, or LC-CDR3 are substituted with other amino acids, (55) (D011-7016; D011-7016-LD1; D011-7016-LD2 consensus) LC-CDR1 having the amino acid sequence of SEQ ID NO: 154 LC-CDR2 having the amino acid sequence YAS LC-CDR3 having the amino acid sequence of SEQ ID NO: 64 VL area including, Or a variant in which one or more amino acids in LC-CDR1, LC-CDR2, or LC-CDR3 are substituted with other amino acids, (56)(D011-7016;D011-7016-LD1;D011-7016-LD2) LC-CDR1 having amino acid sequences of SEQ ID NOs. 62, 144, or 154 LC-CDR2 having the amino acid sequence YAS LC-CDR3 having the amino acid sequence of SEQ ID NO: 64 VL area including, Alternatively, a variant of LC-CDR1, LC-CDR2, or LC-CDR3 in which one or more amino acids are substituted with other amino acids.

[0080]

[0100] In some embodiments, the VH4-34 antigen-binding molecule includes a VL region that conforms to one of the following (57) to (73): (57)(D011-1E11p) LC-FR1 having the amino acid sequence of SEQ ID NO: 17 LC-FR2 having the amino acid sequence of SEQ ID NO: 18 LC-FR3 having the amino acid sequence of SEQ ID NO: 19 LC-FR4 having the amino acid sequence of SEQ ID NO: 20 VL area including, Or a variant in which one or more amino acids among LC-FR1, LC-FR2, LC-FR3, or LC-FR4 are substituted with other amino acids, (58)(D011-6039) LC-FR1 having the amino acid sequence of SEQ ID NO: 33 LC-FR2 having the amino acid sequence of SEQ ID NO: 34 LC-FR3 having the amino acid sequence of SEQ ID NO: 35 LC-FR4 having the amino acid sequence of SEQ ID NO: 36 VL area including, Or a variant in which one or more amino acids among LC-FR1, LC-FR2, LC-FR3, or LC-FR4 are substituted with other amino acids, (59)(D011-6040) LC-FR1 having the amino acid sequence of SEQ ID NO: 49 LC-FR2 having the amino acid sequence of SEQ ID NO: 50 LC-FR3 having the amino acid sequence of SEQ ID NO: 51 LC-FR4 having the amino acid sequence of SEQ ID NO: 52 VL area including, Or a variant in which one or more amino acids among LC-FR1, LC-FR2, LC-FR3, or LC-FR4 are substituted with other amino acids, (60)(D011-7016) LC-FR1 having the amino acid sequence of SEQ ID NO: 65 LC-FR2 having the amino acid sequence of SEQ ID NO: 66 LC-FR3 having the amino acid sequence of SEQ ID NO: 67 LC-FR4 having the amino acid sequence of SEQ ID NO: 68 VL area including, Or a variant in which one or more amino acids among LC-FR1, LC-FR2, LC-FR3, or LC-FR4 are substituted with other amino acids, (61)(D011-1E11-5T2) LC-FR1 having the amino acid sequence of SEQ ID NO: 132 LC-FR2 having the amino acid sequence of SEQ ID NO: 133 LC-FR3 having the amino acid sequence of SEQ ID NO: 134 LC-FR4 having the amino acid sequence of SEQ ID NO: 135 VL area including, Or a variant in which one or more amino acids among LC-FR1, LC-FR2, LC-FR3, or LC-FR4 are substituted with other amino acids, (62)(D011-7016-LD1) LC-FR1 having the amino acid sequence of SEQ ID NO: 145 LC-FR2 having the amino acid sequence of SEQ ID NO: 66 LC-FR3 having the amino acid sequence of SEQ ID NO: 67 LC-FR4 having the amino acid sequence of SEQ ID NO: 68 VL area including, Or a variant in which one or more amino acids among LC-FR1, LC-FR2, LC-FR3, or LC-FR4 are substituted with other amino acids, (63)(D011-7016-LD2) LC-FR1 having the amino acid sequence of SEQ ID NO: 148 LC-FR2 having the amino acid sequence of SEQ ID NO: 66 LC-FR3 having the amino acid sequence of SEQ ID NO: 67 LC-FR4 having the amino acid sequence of SEQ ID NO: 68 VL area including, Or a variant in which one or more amino acids among LC-FR1, LC-FR2, LC-FR3, or LC-FR4 are substituted with other amino acids, (64) (D011-1E11p; D011-1E11-5T2 consensus) LC-FR1 having the amino acid sequence of SEQ ID NO: 163 LC-FR2 having the amino acid sequence of SEQ ID NO: 164 LC-FR3 having the amino acid sequence of SEQ ID NO: 165 LC-FR4 having the amino acid sequence of SEQ ID NO: 166 VL area including, Or a variant in which one or more amino acids among LC-FR1, LC-FR2, LC-FR3, or LC-FR4 are substituted with other amino acids, (65)(D011-1E11p;D011-1E11-5T2) LC-FR1 having amino acid sequences of SEQ ID NOs. 17, 132, or 163 LC-FR2 having the amino acid sequence of SEQ ID NOs. 18, 133, or 164 LC-FR3 having the amino acid sequence of SEQ ID NO: 19, 134, or 165 LC-FR4 having the amino acid sequence of SEQ ID NOs. 20, 135, or 166 VL area including, Or a variant in which one or more amino acids among LC-FR1, LC-FR2, LC-FR3, or LC-FR4 are substituted with other amino acids, (66) (D011-7016; D011-7016-LD1; D011-7016-LD2 consensus) LC-FR1 having the amino acid sequence of SEQ ID NO: 155 LC-FR2 having the amino acid sequence of SEQ ID NO: 66 LC-FR3 having the amino acid sequence of SEQ ID NO: 67 LC-FR4 having the amino acid sequence of SEQ ID NO: 68 VL area including, Or a variant in which one or more amino acids among LC-FR1, LC-FR2, LC-FR3, or LC-FR4 are substituted with other amino acids, (67)(D011-7016;D011-7016-LD1;D011-7016-LD2) LC-FR1 having amino acid sequences of SEQ ID NOs. 65, 145, 148, or 155 LC-FR2 having the amino acid sequence of SEQ ID NO: 66 LC-FR3 having the amino acid sequence of SEQ ID NO: 67 LC-FR4 having the amino acid sequence of SEQ ID NO: 68 VL area including, Or a variant in which one or more amino acids among LC-FR1, LC-FR2, LC-FR3, or LC-FR4 are substituted with other amino acids, (68)(D011-7016-LD2 LCON) LC-FR1 having the amino acid sequence of SEQ ID NO: 210 LC-FR2 having the amino acid sequence of SEQ ID NO: 211 LC-FR3 having the amino acid sequence of SEQ ID NO: 212 LC-FR4 having the amino acid sequence of SEQ ID NO: 213 VL area including, Or a variant in which one or more amino acids among LC-FR1, LC-FR2, LC-FR3, or LC-FR4 are substituted with other amino acids, (69)(D011-7016-LD2_L0) LC-FR1 having the amino acid sequence of SEQ ID NO: 190 LC-FR2 having the amino acid sequence of SEQ ID NO: 191 LC-FR3 having the amino acid sequence of SEQ ID NO: 192 LC-FR4 having the amino acid sequence of SEQ ID NO: 135 VL area including, Or a variant in which one or more amino acids among LC-FR1, LC-FR2, LC-FR3, or LC-FR4 are substituted with other amino acids, (70)(D011-7016-LD2_L1) LC-FR1 having the amino acid sequence of SEQ ID NO: 194 LC-FR2 having the amino acid sequence of SEQ ID NO: 195 LC-FR3 having the amino acid sequence of SEQ ID NO: 196 LC-FR4 having the amino acid sequence of SEQ ID NO: 197 VL area including, Or a variant in which one or more amino acids among LC-FR1, LC-FR2, LC-FR3, or LC-FR4 are substituted with other amino acids, (71)(D011-7016-LD2_L2) LC-FR1 having the amino acid sequence of SEQ ID NO: 199 LC-FR2 having the amino acid sequence of SEQ ID NO: 195 LC-FR3 having the amino acid sequence of SEQ ID NO: 200 LC-FR4 having the amino acid sequence of SEQ ID NO: 197 VL area including, Or a variant in which one or more amino acids among LC-FR1, LC-FR2, LC-FR3, or LC-FR4 are substituted with other amino acids, (72)(D011-7016-LD2_L3) LC-FR1 having the amino acid sequence of SEQ ID NO: 202 LC-FR2 having the amino acid sequence of SEQ ID NO: 203 LC-FR3 having the amino acid sequence of SEQ ID NO: 204 LC-FR4 having the amino acid sequence of SEQ ID NO: 197 VL area including, Or a variant in which one or more amino acids among LC-FR1, LC-FR2, LC-FR3, or LC-FR4 are substituted with other amino acids, (73)(D011-7016-LD2_L4) LC-FR1 having the amino acid sequence of SEQ ID NO: 206 LC-FR2 having the amino acid sequence of SEQ ID NO: 207 LC-FR3 having the amino acid sequence of SEQ ID NO: 208 LC-FR4 having the amino acid sequence of SEQ ID NO: 68 VL area including, Alternatively, a variant in which one or more amino acids among LC-FR1, LC-FR2, LC-FR3, or LC-FR4 are substituted with other amino acids.

[0081]

[0101] In some embodiments, the VH4-34 antigen-binding molecule includes a VL region comprising a CDR according to one of (47) to (56) above, and an FR according to one of (57) to (73) above.

[0082]

[0102] In some embodiments, the VH4-34 antigen-binding molecule includes a VL region that conforms to one of the following (74) to (85): VL area including CDR according to (74)(D011-1E11p)(47) and FR according to (57), VL area including CDR according to (75)(D011-6039)(48) and FR according to (58), VL area including CDR according to (76)(D011-6040)(49) and FR according to (59), VL area including CDR according to (77)(D011-7016)(50) and FR according to (60), VL area including CDR according to (78)(D011-1E11-5T2)(51) and FR according to (61), VL area including CDR according to (79)(D011-7016-LD1)(52) and FR according to (62), VL area including CDR according to (80)(D011-7016-LD2)(52) and FR according to (63), (81)(D011-1E11p;D011-1E11-5T2 consensus)(53) and VL area including CDR and (64) (82)(D011-1E11p;D011-1E11-5T2)(47), (51) or (53) CDR and VL area including FR according to (57), (61) or (64), (83) (D011-7016;D011-7016-LD1;D011-7016-LD2 consensus) (55) and VL areas including FRs according to (66), (84)(D011-7016;D011-7016-LD1;D011-7016-LD2)VL area including CDR in accordance with (54), (52), (55) or (56) and FR in accordance with (60), (62), (63), (66) or (67), (85) A VL area containing a CDR conforming to (52) (D011-7016-LD2 LCON;D011-7016-LD2_L0;D011-7016-LD2_L1;D011-7016-LD2_L2;D011-7016-LD2_L3;D011-7016-LD2_L4)(52) and an FR conforming to any one of (68), (69), (70), (71), (72) or (73).

[0083]

[0103] In some embodiments, the VH4-34 antigen-binding molecule includes a VL region that conforms to one of the following (86) to (100): (86)(D011-1E11p) A VL region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of Sequence ID No. 13, more preferably at least one of the following sequence identities: 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. (87)(D011-6039) A VL region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of Sequence ID No. 29, more preferably at least one of the following sequence identities: 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. (88)(D011-6040) A VL region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO: 45, more preferably at least one of the following sequence identities: 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. (89)(D011-7016) A VL region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of Sequence ID No. 61, more preferably at least one of the following sequence identities: 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. (90)(D011-1E11-5T2) A VL region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of sequence number 130, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. (91)(D011-7016-LD1) A VL region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of Sequence ID No. 143, more preferably at least one of the following sequence identities: 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. (92)(D011-7016-LD2) A VL region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of Sequence ID No. 147, more preferably at least one of the following sequence identities: 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. (93)(D011-1E11p;D011-1E11-5T2 consensus) A VL region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of Sequence ID No. 161, more preferably at least one of the following sequence identities: 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. (94)(D011-7016;D011-7016-LD1;D011-7016-LD2 consensus) A VL region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of Sequence ID No. 153, more preferably at least one of the following sequence identities: 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. (95)(D011-7016-LD2 LCON) A VL region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of sequence number 209, more preferably at least one of the following sequence identities: 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. (96)(D011-7016-LD2_L0) A VL region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of sequence number 189, more preferably at least one of the following sequence identities: 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. (97)(D011-7016-LD2_L1) A VL region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of sequence number 193, more preferably at least one of the following sequence identities: 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. (98)(D011-7016-LD2_L2) A VL region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of sequence number 198, more preferably at least one of the following sequence identities: 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. (99)(D011-7016-LD2_L3) A VL region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of Sequence ID No. 201, more preferably at least one of the following sequence identities: 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. (100)(D011-7016-LD2_L4) A VL region comprising an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of sequence number 205, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

[0084]

[0104] In some embodiments, the VH4-34 antigen-binding molecule includes a VH region comprising HC-CDR1 having the amino acid sequence of SEQ ID NO: 6, HC-CDR2 having the amino acid sequence of SEQ ID NO: 7, and HC-CDR3 having the amino acid sequence of SEQ ID NO: 8, or a variant thereof in which one, two, or three amino acids in one or more of HC-CDR1, HC-CDR2, or HC-CDR3 are substituted with other amino acids, and a VL region comprising LC-CDR1 having the amino acid sequence of SEQ ID NO: 14, LC-CDR2 having the amino acid sequence LVS, and LC-CDR3 having the amino acid sequence of SEQ ID NO: 16, or a variant thereof in which one, two, or three amino acids in one or more of LC-CDR1, LC-CDR2, or LC-CDR3 are substituted with other amino acids.

[0085]

[0105] In some embodiments, the VH4-34 antigen-binding molecule includes a VH region comprising HC-CDR1 having the amino acid sequence of SEQ ID NO: 22, HC-CDR2 having the amino acid sequence of SEQ ID NO: 23, and HC-CDR3 having the amino acid sequence of SEQ ID NO: 24, or a variant thereof in which one, two, or three amino acids in one or more of HC-CDR1, HC-CDR2, or HC-CDR3 are substituted with other amino acids; a VL region comprising LC-CDR1 having the amino acid sequence of SEQ ID NO: 30, LC-CDR2 having the amino acid sequence YTS, and LC-CDR3 having the amino acid sequence of SEQ ID NO: 32, or a variant thereof in which one, two, or three amino acids in one or more of LC-CDR1, LC-CDR2, or LC-CDR3 are substituted with other amino acids.

[0086]

[0106] In some embodiments, the VH4-34 antigen-binding molecule includes a VH region comprising HC-CDR1 having the amino acid sequence of SEQ ID NO: 38, HC-CDR2 having the amino acid sequence of SEQ ID NO: 39, and HC-CDR3 having the amino acid sequence of SEQ ID NO: 40, or a variant thereof in which one, two, or three amino acids in one or more of HC-CDR1, HC-CDR2, or HC-CDR3 are substituted with other amino acids, and a VL region comprising LC-CDR1 having the amino acid sequence of SEQ ID NO: 46, LC-CDR2 having the amino acid sequence AAS, and LC-CDR3 having the amino acid sequence of SEQ ID NO: 48, or a variant thereof in which one, two, or three amino acids in one or more of LC-CDR1, LC-CDR2, or LC-CDR3 are substituted with other amino acids.

[0087]

[0107] In some embodiments, the VH4-34 antigen-binding molecule includes a VH region comprising HC-CDR1 having the amino acid sequence of SEQ ID NO: 54, HC-CDR2 having the amino acid sequence of SEQ ID NO: 55, and HC-CDR3 having the amino acid sequence of SEQ ID NO: 56, or a variant thereof in which one, two, or three amino acids in one or more of HC-CDR1, HC-CDR2, or HC-CDR3 are substituted with other amino acids; and a VL region comprising LC-CDR1 having the amino acid sequence of SEQ ID NO: 62, LC-CDR2 having the amino acid sequence YAS, and LC-CDR3 having the amino acid sequence of SEQ ID NO: 64, or a variant thereof in which one, two, or three amino acids in one or more of LC-CDR1, LC-CDR2, or LC-CDR3 are substituted with other amino acids.

[0088]

[0108] In some embodiments, the VH4-34 antigen-binding molecule includes a VH region comprising HC-CDR1 having the amino acid sequence of SEQ ID NO: 6, HC-CDR2 having the amino acid sequence of SEQ ID NO: 7, and HC-CDR3 having the amino acid sequence of SEQ ID NO: 8, or a variant thereof in which one, two, or three amino acids in one or more of HC-CDR1, HC-CDR2, or HC-CDR3 are substituted with other amino acids, and a VL region comprising LC-CDR1 having the amino acid sequence of SEQ ID NO: 131, LC-CDR2 having the amino acid sequence LVS, and LC-CDR3 having the amino acid sequence of SEQ ID NO: 16, or a variant thereof in which one, two, or three amino acids in one or more of LC-CDR1, LC-CDR2, or LC-CDR3 are substituted with other amino acids.

[0089]

[0109] In some embodiments, the VH4-34 antigen-binding molecule includes a VH region comprising HC-CDR1 having the amino acid sequence of SEQ ID NO: 142, HC-CDR2 having the amino acid sequence of SEQ ID NO: 55, and HC-CDR3 having the amino acid sequence of SEQ ID NO: 56, or a variant thereof in which one, two, or three amino acids in one or more of HC-CDR1, HC-CDR2, or HC-CDR3 are substituted with other amino acids; and a VL region comprising LC-CDR1 having the amino acid sequence of SEQ ID NO: 144, LC-CDR2 having the amino acid sequence YAS, and LC-CDR3 having the amino acid sequence of SEQ ID NO: 64, or a variant thereof in which one, two, or three amino acids in one or more of LC-CDR1, LC-CDR2, or LC-CDR3 are substituted with other amino acids.

[0090]

[0110] In some embodiments, the VH4-34 antigen-binding molecule includes a VH region comprising HC-CDR1 having the amino acid sequence of SEQ ID NO: 6, HC-CDR2 having the amino acid sequence of SEQ ID NO: 7, and HC-CDR3 having the amino acid sequence of SEQ ID NO: 8, or a variant thereof in which one, two, or three amino acids in one or more of HC-CDR1, HC-CDR2, or HC-CDR3 are substituted with other amino acids, and a VL region comprising LC-CDR1 having the amino acid sequence of SEQ ID NO: 162, LC-CDR2 having the amino acid sequence LVS, and LC-CDR3 having the amino acid sequence of SEQ ID NO: 16, or a variant thereof in which one, two, or three amino acids in one or more of LC-CDR1, LC-CDR2, or LC-CDR3 are substituted with other amino acids.

[0091]

[0111] In some embodiments, the VH4-34 antigen-binding molecule includes a VH region comprising HC-CDR1 having the amino acid sequence of SEQ ID NO: 152, HC-CDR2 having the amino acid sequence of SEQ ID NO: 55, and HC-CDR3 having the amino acid sequence of SEQ ID NO: 56, or a variant thereof in which one, two, or three amino acids in one or more of HC-CDR1, HC-CDR2, or HC-CDR3 are substituted with other amino acids; and a VL region comprising LC-CDR1 having the amino acid sequence of SEQ ID NO: 154, LC-CDR2 having the amino acid sequence YAS, and LC-CDR3 having the amino acid sequence of SEQ ID NO: 64, or a variant thereof in which one, two, or three amino acids in one or more of LC-CDR1, LC-CDR2, or LC-CDR3 are substituted with other amino acids.

[0092]

[0112] In some embodiments, the VH4-34 antigen-binding molecule includes a VH region comprising HC-CDR1 having the amino acid sequence of SEQ ID NO: 185, HC-CDR2 having the amino acid sequence of SEQ ID NO: 55, and HC-CDR3 having the amino acid sequence of SEQ ID NO: 56, or a variant thereof in which one, two, or three amino acids in one or more of HC-CDR1, HC-CDR2, or HC-CDR3 are substituted with other amino acids, and a VL region comprising LC-CDR1 having the amino acid sequence of SEQ ID NO: 144, LC-CDR2 having the amino acid sequence YAS, and LC-CDR3 having the amino acid sequence of SEQ ID NO: 64, or a variant thereof in which one, two, or three amino acids in one or more of LC-CDR1, LC-CDR2, or LC-CDR3 are substituted with other amino acids.

[0093]

[0113] In some embodiments, the VH4-34 antigen-binding molecule comprises a VH region having the amino acid sequence of SEQ ID NO: 172 for HC-CDR1, the amino acid sequence of SEQ ID NO: 55 for HC-CDR2, and the amino acid sequence of SEQ ID NO: 56 for HC-CDR3, or a variant thereof in which one or two or three amino acids in one or more of HC-CDR1, HC-CDR2 or HC-CDR3 are substituted with another amino acid, and a VL region having the amino acid sequence of SEQ ID NO: 144 for LC-CDR1, the amino acid sequence YAS for LC-CDR2, and the amino acid sequence of SEQ ID NO: 64 for LC-CDR3, or a variant thereof in which one or two or three amino acids in one or more of LC-CDR1, LC-CDR2 or LC-CDR3 are substituted with another amino acid.

[0094]

[0114] In some embodiments, the VH4-34 antigen-binding molecule comprises a VH region according to any one of (1)-(46) above, and a VL region according to any one of (47)-(100) above.

[0095]

[0115] In some embodiments, the VH4-34 antigen-binding molecule comprises a heavy chain variable region (VH) comprising an amino acid sequence that is at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to the heavy chain variable region (VH) of the VH4-34 antigen-binding molecule disclosed in Table C, and a light chain variable region (VL) comprising an amino acid sequence that is at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to the light chain variable region (VL) of the same VH4-34 antigen-binding molecule disclosed in Table C.

[0096]

[0116] In some embodiments, the VH4-34 antigen-binding molecule includes a heavy chain variable region containing an amino acid sequence having at least 80% sequence identity with an amino acid sequence following any one of SEQ ID NOs: 5, 21, 37, 53, 125, 141, 156, 151, 184, 167, 171, 176, 181, and 182, and a light chain variable region containing an amino acid sequence having at least 80% sequence identity with an amino acid sequence following any one of SEQ ID NOs: 13, 29, 45, 61, 130, 143, 147, 161, 153, 209, 189, 193, 198, 201, and 205. In some embodiments, the VH4-34 antigen-binding molecule includes a heavy chain variable region containing an amino acid sequence having at least 85% sequence identity with an amino acid sequence following any one of SEQ ID NOs: 75, 21, 37, 53, 125, 141, 156, 151, 184, 167, 171, 176, 181, and 182, and a light chain variable region containing an amino acid sequence having at least 85% sequence identity with an amino acid sequence following any one of SEQ ID NOs: 13, 29, 45, 61, 130, 143, 147, 161, 153, 209, 189, 193, 198, 201, and 205. In some embodiments, the VH4-34 antigen-binding molecule includes a heavy chain variable region containing an amino acid sequence having at least 90% sequence identity with an amino acid sequence following any one of SEQ ID NOs: 75, 21, 37, 53, 125, 141, 156, 151, 184, 167, 171, 176, 181, and 182, and a light chain variable region containing an amino acid sequence having at least 90% sequence identity with an amino acid sequence following any one of SEQ ID NOs: 13, 29, 45, 61, 130, 143, 147, 161, 153, 209, 189, 193, 198, 201, and 205.In some embodiments, the VH4-34 antigen-binding molecule includes a heavy chain variable region containing an amino acid sequence having at least 95% sequence identity with an amino acid sequence following any one of SEQ ID NOs: 75, 21, 37, 53, 125, 141, 156, 151, 184, 167, 171, 176, 181, and 182, and a light chain variable region containing an amino acid sequence having at least 95% sequence identity with an amino acid sequence following any one of SEQ ID NOs: 13, 29, 45, 61, 130, 143, 147, 161, 153, 209, 189, 193, 198, 201, and 205. In some embodiments, the VH4-34 antigen-binding molecule includes a heavy chain variable region containing an amino acid sequence having at least 96% sequence identity with an amino acid sequence following any one of SEQ ID NOs: 75, 21, 37, 53, 125, 141, 156, 151, 184, 167, 171, 176, 181, and 182, and a light chain variable region containing an amino acid sequence having at least 96% sequence identity with an amino acid sequence following any one of SEQ ID NOs: 13, 29, 45, 61, 130, 143, 147, 161, 153, 209, 189, 193, 198, 201, and 205. In some embodiments, the VH4-34 antigen-binding molecule includes a heavy chain variable region containing an amino acid sequence having at least 97% sequence identity with an amino acid sequence following any one of SEQ ID NOs: 75, 21, 37, 53, 125, 141, 156, 151, 184, 167, 171, 176, 181, and 182, and a light chain variable region containing an amino acid sequence having at least 97% sequence identity with an amino acid sequence following any one of SEQ ID NOs: 13, 29, 45, 61, 130, 143, 147, 161, 153, 209, 189, 193, 198, 201, and 205.In some embodiments, the VH4-34 antigen-binding molecule includes a heavy chain variable region containing an amino acid sequence having at least 98% sequence identity with an amino acid sequence following any one of SEQ ID NOs: 75, 21, 37, 53, 125, 141, 156, 151, 184, 167, 171, 176, 181, and 182, and a light chain variable region containing an amino acid sequence having at least 98% sequence identity with an amino acid sequence following any one of SEQ ID NOs: 13, 29, 45, 61, 130, 143, 147, 161, 153, 209, 189, 193, 198, 201, and 205. In some embodiments, the VH4-34 antigen-binding molecule includes a heavy chain variable region containing an amino acid sequence having at least 99% sequence identity with an amino acid sequence following any one of SEQ ID NOs: 75, 21, 37, 53, 125, 141, 156, 151, 184, 167, 171, 176, 181, and 182, and a light chain variable region containing an amino acid sequence having at least 99% sequence identity with an amino acid sequence following any one of SEQ ID NOs: 13, 29, 45, 61, 130, 143, 147, 161, 153, 209, 189, 193, 198, 201, and 205. In some embodiments, the VH4-34 antigen-binding molecule includes a heavy chain variable region containing an amino acid sequence following any one of SEQ ID NOs: 75, 21, 37, 53, 125, 141, 156, 151, 184, 167, 171, 176, 181, and 182, and a light chain variable region containing an amino acid sequence following any one of SEQ ID NOs: 13, 29, 45, 61, 130, 143, 147, 161, 153, 209, 189, 193, 198, 201, and 205.

[0097]

[0117] In some embodiments, the VH4-34 antigen-binding molecule comprises a heavy-chain variable region (VH) comprising an amino acid sequence having at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identity to the amino acid sequence according to SEQ ID NO: 5, and a light-chain variable region (VL) comprising an amino acid sequence having at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identity to the amino acid sequence according to SEQ ID NO: 13.

[0098]

[0118] In some embodiments, the VH4-34 antigen-binding molecule comprises a heavy-chain variable region (VH) comprising an amino acid sequence having at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identity to the amino acid sequence according to SEQ ID NO: 21, and a light-chain variable region (VL) comprising an amino acid sequence having at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identity to the amino acid sequence according to SEQ ID NO: 29.

[0099]

[0119] In some embodiments, the VH4-34 antigen-binding molecule includes a heavy chain variable region (VH) comprising an amino acid sequence having at least 60% identity (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with the amino acid sequence according to SEQ ID NO: 37, and a light chain variable region (VL) comprising an amino acid sequence having at least 60% identity (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with the amino acid sequence according to SEQ ID NO: 45.

[0100]

[0120] In some embodiments, the VH4-34 antigen-binding molecule includes a heavy chain variable region (VH) comprising an amino acid sequence having at least 60% identity (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with the amino acid sequence according to SEQ ID NO: 53, and a light chain variable region (VL) comprising an amino acid sequence having at least 60% identity (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with the amino acid sequence according to SEQ ID NO: 61.

[0101]

[0121] In some embodiments, the VH4-34 antigen-binding molecule includes a heavy chain variable region (VH) comprising an amino acid sequence having at least 60% identity (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with the amino acid sequence according to SEQ ID NO: 125, and a light chain variable region (VL) comprising an amino acid sequence having at least 60% identity (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with the amino acid sequence according to SEQ ID NO: 130.

[0102]

[0122] In some embodiments, the VH4-34 antigen-binding molecule includes a heavy chain variable region (VH) comprising an amino acid sequence having at least 60% identity (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with the amino acid sequence according to SEQ ID NO: 141, and a light chain variable region (VL) comprising an amino acid sequence having at least 60% identity (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with the amino acid sequence according to SEQ ID NO: 143.

[0103]

[0123] In some embodiments, the VH4-34 antigen-binding molecule includes a heavy chain variable region (VH) comprising an amino acid sequence having at least 60% identity (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with the amino acid sequence according to SEQ ID NO: 141, and a light chain variable region (VL) comprising an amino acid sequence having at least 60% identity (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with the amino acid sequence according to SEQ ID NO: 147.

[0104]

[0124] In some embodiments, the VH4-34 antigen-binding molecule includes a heavy chain variable region (VH) comprising an amino acid sequence having at least 60% identity (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with the amino acid sequence according to SEQ ID NO: 156, and a light chain variable region (VL) comprising an amino acid sequence having at least 60% identity (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with the amino acid sequence according to SEQ ID NO: 161.

[0105]

[0125] In some embodiments, the VH4-34 antigen-binding molecule includes a heavy chain variable region (VH) comprising an amino acid sequence having at least 60% identity (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with the amino acid sequence according to SEQ ID NO: 151, and a light chain variable region (VL) comprising an amino acid sequence having at least 60% identity (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with the amino acid sequence according to SEQ ID NO: 153.

[0106]

[0126] In some embodiments, the VH4-34 antigen-binding molecule includes a heavy chain variable region (VH) comprising an amino acid sequence having at least 60% identity (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with the amino acid sequence according to SEQ ID NO: 184, and a light chain variable region (VL) comprising an amino acid sequence having at least 60% identity (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with the amino acid sequence according to SEQ ID NO: 209.

[0107]

[0127] In some embodiments, the VH4-34 antigen-binding molecule includes a heavy chain variable region (VH) comprising an amino acid sequence having at least 60% identity (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with the amino acid sequence according to SEQ ID NO: 184, and a light chain variable region (VL) comprising an amino acid sequence having at least 60% identity (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with the amino acid sequence according to SEQ ID NO: 189.

[0108]

[0128] In some embodiments, the VH4-34 antigen-binding molecule includes a heavy chain variable region (VH) comprising an amino acid sequence having at least 60% identity (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with the amino acid sequence according to SEQ ID NO: 184, and a light chain variable region (VL) comprising an amino acid sequence having at least 60% identity (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with the amino acid sequence according to SEQ ID NO: 193.

[0109]

[0129] In some embodiments, the VH4-34 antigen-binding molecule includes a heavy chain variable region (VH) comprising an amino acid sequence having at least 60% identity (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with the amino acid sequence according to SEQ ID NO: 184, and a light chain variable region (VL) comprising an amino acid sequence having at least 60% identity (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with the amino acid sequence according to SEQ ID NO: 198.

[0110]

[0130] In some embodiments, the VH4-34 antigen-binding molecule includes a heavy chain variable region (VH) comprising an amino acid sequence having at least 60% identity (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with the amino acid sequence according to SEQ ID NO: 184, and a light chain variable region (VL) comprising an amino acid sequence having at least 60% identity (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with the amino acid sequence according to SEQ ID NO: 201.

[0111]

[0131] In some embodiments, the VH4-34 antigen-binding molecule includes a heavy chain variable region (VH) comprising an amino acid sequence having at least 60% identity (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with the amino acid sequence according to SEQ ID NO: 184, and a light chain variable region (VL) comprising an amino acid sequence having at least 60% identity (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with the amino acid sequence according to SEQ ID NO: 205.

[0112]

[0132] In some embodiments, the VH4-34 antigen-binding molecule includes a heavy chain variable region (VH) comprising an amino acid sequence having at least 60% identity (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with the amino acid sequence according to SEQ ID NO: 167, and a light chain variable region (VL) comprising an amino acid sequence having at least 60% identity (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with the amino acid sequence according to SEQ ID NO: 209.

[0113]

[0133] In some embodiments, the VH4-34 antigen-binding molecule includes a heavy chain variable region (VH) comprising an amino acid sequence having at least 60% identity (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with the amino acid sequence according to SEQ ID NO: 167, and a light chain variable region (VL) comprising an amino acid sequence having at least 60% identity (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with the amino acid sequence according to SEQ ID NO: 189.

[0114]

[0134] In some embodiments, the VH4-34 antigen-binding molecule includes a heavy chain variable region (VH) comprising an amino acid sequence having at least 60% identity (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with the amino acid sequence according to SEQ ID NO: 167, and a light chain variable region (VL) comprising an amino acid sequence having at least 60% identity (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with the amino acid sequence according to SEQ ID NO: 193.

[0115]

[0135] In some embodiments, the VH4-34 antigen-binding molecule includes a heavy chain variable region (VH) comprising an amino acid sequence having at least 60% identity (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with the amino acid sequence according to SEQ ID NO: 167, and a light chain variable region (VL) comprising an amino acid sequence having at least 60% identity (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with the amino acid sequence according to SEQ ID NO: 198.

[0116]

[0136] In some embodiments, the VH4-34 antigen-binding molecule includes a heavy chain variable region (VH) comprising an amino acid sequence having at least 60% identity (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with the amino acid sequence according to SEQ ID NO: 167, and a light chain variable region (VL) comprising an amino acid sequence having at least 60% identity (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with the amino acid sequence according to SEQ ID NO: 201.

[0117]

[0137] In some embodiments, the VH4-34 antigen-binding molecule includes a heavy chain variable region (VH) comprising an amino acid sequence having at least 60% identity (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with the amino acid sequence according to SEQ ID NO: 167, and a light chain variable region (VL) comprising an amino acid sequence having at least 60% identity (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with the amino acid sequence according to SEQ ID NO: 205.

[0118]

[0138] In some embodiments, the VH4-34 antigen-binding molecule includes a heavy chain variable region (VH) comprising an amino acid sequence having at least 60% identity (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with the amino acid sequence according to SEQ ID NO: 171, and a light chain variable region (VL) comprising an amino acid sequence having at least 60% identity (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with the amino acid sequence according to SEQ ID NO: 209.

[0119]

[0139] In some embodiments, the VH4-34 antigen-binding molecule comprises a heavy chain variable region (VH) comprising an amino acid sequence having at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identity to the amino acid sequence according to SEQ ID NO: 171, and a light chain variable region (VL) comprising an amino acid sequence having at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identity to the amino acid sequence according to SEQ ID NO: 189.

[0120]

[0140] In some embodiments, the VH4-34 antigen-binding molecule comprises a heavy chain variable region (VH) comprising an amino acid sequence having at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identity to the amino acid sequence according to SEQ ID NO: 171, and a light chain variable region (VL) comprising an amino acid sequence having at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identity to the amino acid sequence according to SEQ ID NO: 193.

[0121]

[0141] In some embodiments, the VH4-34 antigen-binding molecule includes a heavy chain variable region (VH) comprising an amino acid sequence having at least 60% identity (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with the amino acid sequence according to SEQ ID NO: 171, and a light chain variable region (VL) comprising an amino acid sequence having at least 60% identity (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with the amino acid sequence according to SEQ ID NO: 198.

[0122]

[0142] In some embodiments, the VH4-34 antigen-binding molecule includes a heavy chain variable region (VH) comprising an amino acid sequence having at least 60% identity (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with the amino acid sequence according to SEQ ID NO: 171, and a light chain variable region (VL) comprising an amino acid sequence having at least 60% identity (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with the amino acid sequence according to SEQ ID NO: 201.

[0123]

[0143] In some embodiments, the VH4-34 antigen-binding molecule includes a heavy chain variable region (VH) comprising an amino acid sequence having at least 60% identity (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with the amino acid sequence according to SEQ ID NO: 171, and a light chain variable region (VL) comprising an amino acid sequence having at least 60% identity (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with the amino acid sequence according to SEQ ID NO: 205.

[0124]

[0144] In some embodiments, the VH4-34 antigen-binding molecule includes a heavy chain variable region (VH) comprising an amino acid sequence having at least 60% identity (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with the amino acid sequence according to SEQ ID NO: 176, and a light chain variable region (VL) comprising an amino acid sequence having at least 60% identity (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with the amino acid sequence according to SEQ ID NO: 209.

[0125]

[0145] In some embodiments, the VH4-34 antigen-binding molecule includes a heavy chain variable region (VH) comprising an amino acid sequence having at least 60% identity (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with the amino acid sequence according to SEQ ID NO: 176, and a light chain variable region (VL) comprising an amino acid sequence having at least 60% identity (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with the amino acid sequence according to SEQ ID NO: 189.

[0126]

[0146] In some embodiments, the VH4-34 antigen-binding molecule includes a heavy chain variable region (VH) comprising an amino acid sequence having at least 60% identity (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with the amino acid sequence according to SEQ ID NO: 176, and a light chain variable region (VL) comprising an amino acid sequence having at least 60% identity (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with the amino acid sequence according to SEQ ID NO: 193.

[0127]

[0147] In some embodiments, the VH4-34 antigen-binding molecule includes a heavy chain variable region (VH) comprising an amino acid sequence having at least 60% identity (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with the amino acid sequence according to SEQ ID NO: 176, and a light chain variable region (VL) comprising an amino acid sequence having at least 60% identity (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with the amino acid sequence according to SEQ ID NO: 198.

[0128]

[0148] In some embodiments, the VH4-34 antigen-binding molecule includes a heavy chain variable region (VH) comprising an amino acid sequence having at least 60% identity (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with the amino acid sequence according to SEQ ID NO: 176, and a light chain variable region (VL) comprising an amino acid sequence having at least 60% identity (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with the amino acid sequence according to SEQ ID NO: 201.

[0129]

[0149] In some embodiments, the VH4-34 antigen-binding molecule includes a heavy chain variable region (VH) comprising an amino acid sequence having at least 60% identity (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with the amino acid sequence according to SEQ ID NO: 176, and a light chain variable region (VL) comprising an amino acid sequence having at least 60% identity (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with the amino acid sequence according to SEQ ID NO: 205.

[0130]

[0150] In some embodiments, the VH4-34 antigen-binding molecule includes a heavy chain variable region (VH) comprising an amino acid sequence having at least 60% identity (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with the amino acid sequence according to SEQ ID NO: 181, and a light chain variable region (VL) comprising an amino acid sequence having at least 60% identity (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with the amino acid sequence according to SEQ ID NO: 209.

[0131]

[0151] In some embodiments, the VH4-34 antigen-binding molecule includes a heavy chain variable region (VH) comprising an amino acid sequence having at least 60% identity (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with the amino acid sequence according to SEQ ID NO: 181, and a light chain variable region (VL) comprising an amino acid sequence having at least 60% identity (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with the amino acid sequence according to SEQ ID NO: 189.

[0132]

[0152] In some embodiments, the VH4-34 antigen-binding molecule includes a heavy chain variable region (VH) comprising an amino acid sequence having at least 60% identity (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with the amino acid sequence according to SEQ ID NO: 181, and a light chain variable region (VL) comprising an amino acid sequence having at least 60% identity (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with the amino acid sequence according to SEQ ID NO: 193.

[0133]

[0153] In some embodiments, the VH4-34 antigen-binding molecule includes a heavy chain variable region (VH) comprising an amino acid sequence having at least 60% identity (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with the amino acid sequence according to SEQ ID NO: 181, and a light chain variable region (VL) comprising an amino acid sequence having at least 60% identity (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with the amino acid sequence according to SEQ ID NO: 198.

[0134]

[0154] In some embodiments, the VH4-34 antigen-binding molecule includes a heavy chain variable region (VH) comprising an amino acid sequence having at least 60% identity (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with the amino acid sequence according to SEQ ID NO: 181, and a light chain variable region (VL) comprising an amino acid sequence having at least 60% identity (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with the amino acid sequence according to SEQ ID NO: 201.

[0135]

[0155] In some embodiments, the VH4-34 antigen-binding molecule includes a heavy chain variable region (VH) comprising an amino acid sequence having at least 60% identity (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with the amino acid sequence according to SEQ ID NO: 181, and a light chain variable region (VL) comprising an amino acid sequence having at least 60% identity (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with the amino acid sequence according to SEQ ID NO: 205.

[0136]

[0156] In some embodiments, the VH4-34 antigen-binding molecule includes a heavy chain variable region (VH) comprising an amino acid sequence having at least 60% identity (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with the amino acid sequence according to SEQ ID NO: 182, and a light chain variable region (VL) comprising an amino acid sequence having at least 60% identity (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with the amino acid sequence according to SEQ ID NO: 209.

[0137]

[0157] In some embodiments, the VH4-34 antigen-binding molecule includes a heavy chain variable region (VH) comprising an amino acid sequence having at least 60% identity (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with the amino acid sequence according to SEQ ID NO: 182, and a light chain variable region (VL) comprising an amino acid sequence having at least 60% identity (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with the amino acid sequence according to SEQ ID NO: 189.

[0138]

[0158] In some embodiments, the VH4-34 antigen-binding molecule includes a heavy chain variable region (VH) comprising an amino acid sequence having at least 60% identity (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with the amino acid sequence according to SEQ ID NO: 182, and a light chain variable region (VL) comprising an amino acid sequence having at least 60% identity (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with the amino acid sequence according to SEQ ID NO: 193.

[0139]

[0159] In some embodiments, the VH4-34 antigen-binding molecule includes a heavy chain variable region (VH) comprising an amino acid sequence having at least 60% identity (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with the amino acid sequence according to SEQ ID NO: 182, and a light chain variable region (VL) comprising an amino acid sequence having at least 60% identity (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with the amino acid sequence according to SEQ ID NO: 198.

[0140]

[0160] In some embodiments, the VH4-34 antigen-binding molecule includes a heavy chain variable region (VH) comprising an amino acid sequence having at least 60% identity (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with the amino acid sequence according to SEQ ID NO: 182, and a light chain variable region (VL) comprising an amino acid sequence having at least 60% identity (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with the amino acid sequence according to SEQ ID NO: 201.

[0141]

[0161] In some embodiments, the VH4-34 antigen-binding molecule includes a heavy chain variable region (VH) comprising an amino acid sequence having at least 60% identity (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with the amino acid sequence according to SEQ ID NO: 182, and a light chain variable region (VL) comprising an amino acid sequence having at least 60% identity (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with the amino acid sequence according to SEQ ID NO: 205.

[0142]

[0162] In some embodiments, the VH4-34 antigen-binding molecule comprises a heavy chain containing an amino acid sequence having at least 70% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity with an amino acid sequence following any one of SEQ ID NOs. 86-89, 136, 139, and 214-219. In some embodiments, the VH4-34 antigen-binding molecule comprises a heavy chain containing an amino acid sequence having at least 80% sequence identity with an amino acid sequence following any one of SEQ ID NOs. 5, 21, 37, 53, 125, 141, 156, 151, 184, 167, 171, 176, 181, and 182, and a light chain containing an amino acid sequence having at least 80% sequence identity with an amino acid sequence following any one of SEQ ID NOs. 86-89, 136, 139, and 214-219. In some embodiments, the VH4-34 antigen-binding molecule comprises a heavy chain containing an amino acid sequence having at least 80% sequence identity with an amino acid sequence following any one of SEQ ID NOs. 5, 21, 37, 53, 125, 141, 156, 151, 184, 167, 171, 176, 181, and 182, and a light chain containing an amino acid sequence having at least 80% sequence identity with an amino acid sequence following any one of SEQ ID NOs. 86-89, 136, 139, and 214-219. In some embodiments, the VH4-34 antigen-binding molecule comprises a heavy chain containing an amino acid sequence having at least 85% sequence identity with an amino acid sequence following any one of SEQ ID NOs: 90-93, 137, 140, 146 and 220-225, and a light chain containing an amino acid sequence having at least 85% sequence identity with an amino acid sequence following any one of SEQ ID NOs: 86-89, 136, 139 and 214-219.In some embodiments, the VH4-34 antigen-binding molecule comprises a heavy chain containing an amino acid sequence having at least 90% sequence identity with an amino acid sequence following any one of SEQ ID NOs: 90-93, 137, 140, 146 and 220-225, and a light chain containing an amino acid sequence having at least 90% sequence identity with an amino acid sequence following any one of SEQ ID NOs: 86-89, 136, 139 and 214-219. In some embodiments, the VH4-34 antigen-binding molecule comprises a heavy chain containing an amino acid sequence having at least 95% sequence identity with an amino acid sequence following any one of SEQ ID NOs: 90-93, 137, 140, 146 and 220-225, and a light chain containing an amino acid sequence having at least 95% sequence identity with an amino acid sequence following any one of SEQ ID NOs: 86-89, 136, 139 and 214-219. In some embodiments, the VH4-34 antigen-binding molecule includes a heavy chain containing an amino acid sequence having at least 96% sequence identity with an amino acid sequence following any one of SEQ ID NOs: 90-93, 137, 140, 146 and 220-225, and a light chain containing an amino acid sequence having at least 96% sequence identity with an amino acid sequence following any one of SEQ ID NOs: 86-89, 136, 139 and 214-219. In some embodiments, the VH4-34 antigen-binding molecule includes a heavy chain containing an amino acid sequence having at least 97% sequence identity with an amino acid sequence following any one of SEQ ID NOs: 90-93, 137, 140, 146 and 220-225, and a light chain containing an amino acid sequence having at least 97% sequence identity with an amino acid sequence following any one of SEQ ID NOs: 86-89, 136, 139 and 214-219. In some embodiments, the VH4-34 antigen-binding molecule comprises a heavy chain containing an amino acid sequence having at least 98% sequence identity with an amino acid sequence following any one of SEQ ID NOs: 90-93, 137, 140, 146 and 220-225, and a light chain containing an amino acid sequence having at least 98% sequence identity with an amino acid sequence following any one of SEQ ID NOs: 86-89, 136, 139 and 214-219.In some embodiments, the VH4-34 antigen-binding molecule comprises a heavy chain containing an amino acid sequence having at least 99% sequence identity with an amino acid sequence following one of the sequence codes 90-93, 137, 140, 146, and 220-225, and a light chain containing an amino acid sequence having at least 99% sequence identity with an amino acid sequence following one of the sequence codes 86-89, 136, 139, and 214-219. See Table D for combinations of heavy and light chains.

[0143]

[0163] In some embodiments, the VH4-34 antigen-binding molecule includes a heavy chain comprising an amino acid sequence having at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity with the amino acid sequence according to SEQ ID NO: 215, and a light chain comprising an amino acid sequence having at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity with the amino acid sequence according to SEQ ID NO: 221.

[0144]

[0164] In some embodiments, the VH4-34 antigen-binding molecule includes a heavy chain comprising an amino acid sequence having at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity with the amino acid sequence according to SEQ ID NO: 230, and a light chain comprising an amino acid sequence having at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity with the amino acid sequence according to SEQ ID NO: 221.

[0145]

[0165] In some embodiments, the VH4-34 antigen-binding molecule includes a heavy chain comprising an amino acid sequence having at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity with the amino acid sequence according to SEQ ID NO: 219, and a light chain comprising an amino acid sequence having at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity with the amino acid sequence according to SEQ ID NO: 222.

[0146]

[0166] In some embodiments, the VH4-34 antigen-binding molecule includes a heavy chain comprising an amino acid sequence having at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity with the amino acid sequence according to SEQ ID NO: 234, and a light chain comprising an amino acid sequence having at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity with the amino acid sequence according to SEQ ID NO: 222.

[0147]

[0167] In some embodiments, the VH4-34 antigen-binding molecule includes a heavy chain comprising an amino acid sequence having at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity with the amino acid sequence according to SEQ ID NO: 219, and a light chain comprising an amino acid sequence having at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity with the amino acid sequence according to SEQ ID NO: 223.

[0148]

[0168] In some embodiments, the VH4-34 antigen-binding molecule includes a heavy chain comprising an amino acid sequence having at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity with the amino acid sequence according to SEQ ID NO: 234, and a light chain comprising an amino acid sequence having at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity with the amino acid sequence according to SEQ ID NO: 223.

[0149]

[0169] In embodiments of this disclosure in which one or more amino acids are substituted with other amino acids, the substitution may be a conservative substitution, for example, according to the following table. In some embodiments, amino acids in the same block in the middle column are substituted. In some embodiments, amino acids in the same row in the far right column are substituted:

[0150] [Table 1]

[0170] In some embodiments, the substitution(s) are functionally conserved. That is, in some embodiments, the substitution may not affect (or substantially affect) one or more functional properties (e.g., target binding) of the VH4-34 antigen-binding molecule containing the substitution compared to an equivalent unsubstituted molecule.

[0151]

[0171] The VH and VL regions of the antigen-binding domain of an antibody together constitute the Fv region. In some embodiments, the VH4-34 antigen-binding molecule includes, or consists of, an Fv region that binds to VH4-34. In some embodiments, the VH and VL regions of Fv are provided as a single polypeptide, i.e., a single-chain Fv (scFv), linked by a linker region.

[0152]

[0172] The VL and light chain constant (CL) regions, as well as the VH region and heavy chain constant 1 (CH1) region of the antibody's antigen-binding region, together constitute a Fab region. In some embodiments, the VH4-34 antigen-binding molecule includes a Fab region comprising VH, CH1, VL, and CL (e.g., Cκ or Cλ). In some embodiments, the Fab region comprises a polypeptide comprising VH and CH1 (e.g., a VH-CH1 fusion polypeptide), as well as a polypeptide comprising VL and CL (e.g., a VL-CL fusion polypeptide). In some embodiments, the Fab region comprises a polypeptide comprising VH and CL (e.g., a VH-CL fusion polypeptide), as well as a polypeptide comprising VL and CH (e.g., a VL-CH1 fusion polypeptide); that is, in some embodiments, the Fab region is a CrossFab region. In some embodiments, the VH, CH1, VL, and CL regions of the Fab or CrossFab are provided as a single polypeptide linked by a linker region, i.e., as a single-chain Fab (scFab) or single-chain CrossFab (scCrossFab).

[0153]

[0173] In some embodiments, the VH4-34 antigen-binding molecule of this disclosure includes, or comprises, a Fab region that binds to VH4-34.

[0174] In some embodiments, the VH4-34 antigen-binding molecules described herein include, or consist of, a whole antibody that binds to VH4-34. As used herein, “whole antibody” means an antibody having a structure substantially similar to that of an immunoglobulin (Ig). Various types of immunoglobulins and their structures are described, for example, in Schroeder and Cavacini J Allergy Clin Immunol. (2010) 125(202):S41–S52, which are incorporated herein by reference in their entirety.

[0154]

[0175] G-type immunoglobulins (i.e., IgG) are glycoproteins of approximately 150 kDa containing two heavy chains and two light chains. From the N-terminus to the C-terminus, the heavy chain contains VH, followed by a heavy chain constant region containing three constant domains (CH1, CH2, and CH3), and similarly the light chain contains VL, followed by CL. Depending on the heavy chain, immunoglobulins can be classified as IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgA (e.g., IgA1, IgA2), IgD, IgE, or IgM. The light chain can be kappa (κ) or lambda (λ).

[0155]

[0176] In some embodiments, the VH4-34 antigen-binding molecules described herein include, or consist of, IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgA (e.g., IgA1, IgA2), IgD, IgE, or IgM that bind to VH4-34.

[0156]

[0177] In some embodiments, the VH4-34 antigen-binding molecule of this disclosure comprises one or more regions of an immunoglobulin heavy chain constant sequence (e.g., CH1, CH2, CH3, etc.). In some embodiments, the immunoglobulin heavy chain constant sequence is or is derived from the heavy chain constant sequence of IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgA (e.g., IgA1, IgA2), IgD, IgE, or IgM, for example, human IgG (e.g., hIgG1, hIgG2, hIgG3, hIgG4), hIgA (e.g., hIgA1, hIgA2), hIgD, hIgE, or hIgM. In some embodiments, the immunoglobulin heavy chain constant sequence is or is derived from the heavy chain constant sequence of a human IgG1 allotype (e.g., G1m1, G1m2, G1m3, or G1m17).

[0157]

[0178] In some embodiments, the VH4-34 antigen-binding molecule includes an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO: 71 or 76, more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

[0158]

[0179] In some embodiments, the VH4-34 antigen-binding molecule includes a CH1 region containing an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO: 72 or 77, more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the VH4-34 antigen-binding molecule includes a hinge region containing an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 73 or 85, more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the VH4-34 antigen-binding molecule includes a CH2 region containing an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO: 74, more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the VH4-34 antigen-binding molecule includes a CH3 region containing an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO: 75 or 78, more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

[0159]

[0180] It will be acknowledged that the CH2 and / or CH3 regions may be provided with further substitutions in accordance with modifications to the Fc region of a VH4-34 antigen-binding molecule as described herein.

[0160]

[0181] In some embodiments, the VH4-34 antigen-binding molecule of this disclosure comprises one or more regions of an immunoglobulin light chain constant sequence. In some embodiments, the immunoglobulin light chain constant sequence is human immunoglobulin kappa constant (IGKC; Cκ). In some embodiments, the immunoglobulin light chain constant sequence is human immunoglobulin lambda constant (IGLC; Cλ), for example, IGLC1, IGLC2, IGLC3, IGLC6, or IGLC7.

[0161]

[0182] In some embodiments, the VH4-34 antigen-binding molecule includes an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO: 79, more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the VH4-34 antigen-binding molecule includes an amino acid sequence having at least 70% sequence identity with respect to one of the amino acid sequences from SEQ ID NOs: 80 to 84, more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

[0162]

[0183] In some embodiments, the VH4-34 antigen-binding molecule is or contains a fully human antibody / antibody fragment. The fully human antibody / antibody fragment may be encoded by a human nucleic acid sequence(s). The fully human antibody / antibody fragment may not contain a non-human amino acid sequence. Techniques commonly used for the production of fully human antibodies include (i) phage display, in which the human antibody gene is expressed in a phage display library, and (ii) antibody production in transgenic mice engineered to have the human antibody gene (as described by Park and Smolen, Advances in Protein Chemistry (2001) 56:369-421). Briefly, in the human antibody gene-phage display technique, the genes encoding the VH and VL chains can be produced from "naive" human lymphocytes by PCR amplification and cloning, assembled into a library, and expressed therefrom as either disulfide-linked Fab fragments or single-chain Fv(scFv) fragments. Genes encoding Fab or scFv can be fused to the surface coat protein of a filamentous bacteriophage, and Fab or scFv capable of binding to the target of interest can then be identified by screening the library with the antigen. The affinity of Fab / scFv fragments can be enhanced using molecular evolution or affinity maturation procedures. In transgenic mouse technology, mice in which the endogenous mouse lg gene locus is replaced with its human homolog by homologous recombination are immunized with the antigen, and monoclonal antibodies are prepared using conventional hybridoma technology to obtain fully human monoclonal antibodies.

[0163]

[0184] In some embodiments, the VH4-34 antigen-binding molecule of this disclosure is a mouse antibody / antibody fragment. In some embodiments, the antibody / antibody fragment is obtained from phage display using a human naive antibody gene library.

[0164]

[0185] In some embodiments, the VH4-34 antigen-binding molecule is a mouse / human chimeric antibody / antibody fragment (i.e., an antigen-binding molecule containing a mouse antibody variable domain and a human antibody constant region). In some embodiments, the VH4-34 antigen-binding molecule is a humanized antibody / antibody fragment. In some embodiments, the VH4-34 antigen-binding molecule includes a mouse antibody CDR and a human antibody framework and constant region. In some embodiments, the VH4-34 antigen-binding molecule includes a human antibody CDR and a human antibody framework and constant region.

[0165]

[0186] Mouse / human chimeric antigen-binding molecules can be prepared from mouse antibodies, for example, by a chimeric process as described in Chapter 8, particularly Section 3 of Chapter 8, of *Human Monoclonal Antibodies: Methods and Protocols*, edited by Michael Steinitz, *Methods in Molecular Biology*, Springer Protocols, Humana Press (2014).

[0166]

[0187] Humanized antigen-binding molecules can be designed and prepared from mouse antibodies by humanization processes, for example, as described in Chapter 7, specifically Section 3.1 of Chapter 7 titled "Antibody Humanization," in *Human Monoclonal Antibodies: Methods and Protocols*, edited by Michael Steinitz, *Methods in Molecular Biology*, Springer Protocols, Humana Press (2014). Techniques for antibody humanization are also described, for example, in *Safdari et al.*, *Biotechnol Genet Eng Rev* (2013) 29: pp. 175-1786.

[0167]

[0188] Aspects of this disclosure relate to multispecific antigen-binding molecules. "Multispecific" means that the VH4-34 antigen-binding molecule specifically binds to more than one target. In some embodiments, the VH4-34 antigen-binding molecule is a bispecific antigen-binding molecule. In some embodiments, the VH4-34 antigen-binding molecule comprises at least two different antigen-binding domains (i.e., at least two antigen-binding domains, for example, including non-identical VH and VL).

[0168]

[0189] In some embodiments, the VH4-34 antigen-binding molecule binds to both VH4-34 and another target (e.g., an antigen other than VH4-34), and is therefore at least bispecific. The term "bispecific" means that the VH4-34 antigen-binding molecule can specifically bind to at least two distinct antigenic determinants.

[0169]

[0190] It can be acknowledged that antigen-binding molecules (e.g., multispecific antigen-binding molecules) may include antigen-binding molecules that can bind to targets specific to the VH4-34 antigen-binding molecule. For example, antigen-binding molecules that bind to VH4-34 and antigens other than VH4-34 may include (i) antigen-binding molecules that bind to VH4-34, and (ii) antigen-binding molecules that bind to antigens other than VH4-34.

[0170]

[0191] It can also be acknowledged that antigen-binding molecules (e.g., multispecific antigen-binding molecules) may include antigen-binding polypeptides or antigen-binding polypeptide complexes that are capable of binding to targets specific to the VH4-34 antigen-binding molecule.

[0171]

[0192] In some embodiments, the antigen-binding component of a larger antigen-binding molecule (e.g., a multispecific antigen-binding molecule) may be referred to, for example, as the "antigen-binding domain" or "antigen-binding region" of the larger antigen-binding molecule.

[0172]

[0193] In some embodiments, the antigen other than VH4-34 in the multispecific antigen-binding molecule is an immune cell surface molecule. In some embodiments, the antigen is a cancer cell antigen. In some embodiments, the antigen is a receptor molecule, such as a cell surface receptor. In some embodiments, the antigen is a cell signaling molecule, such as a cytokine, chemokine, interferon, interleukin, or lymphokine. In some embodiments, the antigen is a growth factor or hormone.

[0173]

[0194] Cancer cell antigens are antigens expressed or overexpressed by cancer cells. Cancer cell antigens can be any peptide / polypeptide, glycoprotein, lipoprotein, glycan, glycolipid, lipid or fragment thereof. The expression of cancer cell antigens may be associated with cancer. Cancer cell antigens may be abnormally expressed by cancer cells (e.g., cancer cell antigens may be expressed with abnormal localization) or with abnormal structures by cancer cells. Cancer cell antigens may be able to induce an immune response. In some embodiments, the antigen is expressed on the cell surface of cancer cells (i.e., cancer cell antigens are cancer cell surface antigens). In some embodiments, some of the antigens bound by the VH4-34 antigen-binding molecules described herein are presented on the outer surface of cancer cells (i.e., extracellular). Cancer cell antigens may be cancer-associated antigens. In some embodiments, cancer cell antigens are antigens whose expression is associated with the development, progression, or severity of cancer. Cancer-associated antigens may be associated with the cause or pathology of cancer, or may be abnormally expressed as a result of cancer. In some embodiments, cancer cell antigens are antigens whose expression is upregulated by cancer cells (e.g., at the RNA and / or protein level) compared to the level of expression by equivalent non-cancerous cells (e.g., non-cancerous cells from the same tissue / cell type). In some embodiments, cancer-associated antigens are preferentially expressed by cancer cells and not expressed by equivalent non-cancerous cells (e.g., non-cancerous cells from the same tissue / cell type). In some embodiments, cancer-associated antigens are products of mutated oncogenes or mutated tumor suppressor genes. In some embodiments, cancer-associated antigens are products of overexpressed cellular proteins, cancer antigens produced by oncogenic viruses, carcinoembryonic antigens, or cell surface glycolipids or glycoproteins.

[0174]

[0195] Immune cell surface molecules may be any peptide / polypeptide, glycoprotein, lipoprotein, glycan, glycolipid, lipid or fragment thereof expressed on or on the cell surface of an immune cell. In some embodiments, some of the immune cell surface molecules bound by the VH4-34 antigen-binding molecule of this disclosure are located on the outer surface of the immune cell (i.e., extracellular). Immune cell surface molecules may be expressed on the cell surface of any immune cell. In some embodiments, immune cells are cells of hematopoietic origin, such as neutrophils, eosinophils, basophils, dendritic cells, lymphocytes, or monocytes. Lymphocytes may be, for example, T cells, B cells, natural killer (NK) cells, NKT cells, or innate lymphoid cells (ILCs) or their precursors (e.g., thymocytes or pre-B cells).

[0175]

[0196] In some embodiments, the antigen is selected from BCMA, TACI, CD47, CD33, CD123, Wilms oncoprotein (WT1), CD13, CD15, CD30, CD45, type C lectin-like molecule 1 (CLL1), Fms-like tyrosine kinase 3 (FLT-3), VEGF, and angiopoietin-2 (Ang-2). In some embodiments, the antigen is a CD3 polypeptide (e.g., CD3ε, CD3δ, CD3γ, or CD3ζ).

[0176]

[0197] In some embodiments, the multispecific antigen-binding molecules described herein exhibit at least monovalent binding to VH4-34 and at least monovalent binding to antigens other than VH4-34.

[0177]

[0198] In some embodiments, the VH4-34 antigen-binding molecule includes antigen-binding regions (e.g., polypeptides, Fv, Fab, or antibodies) capable of binding to antigens other than VH4-34. In some embodiments, the VH4-34 antigen-binding molecule includes VH and VL of antibodies capable of binding to VH4-34, and VH and VL of antibodies capable of binding to antigens other than VH4-34. The titer refers to the number of binding sites in the antigen-binding molecule for a given antigenic determinant.

[0178]

[0199] In some embodiments, the VH4-34 antigen-binding molecule is an immune cell engager. Immune cell engagers are outlined, for example, in Goebeler and Bargou, Nat. Rev. Clin. Oncol. (2020) 17: pp. 418–434 and Ellerman, Methods (2019) 154: pp. 102–117, both of which are incorporated herein by reference in their entirety.

[0179]

[0200] Immune cell engager molecules contain an antigen-binding region for a target antigen of interest, and an antigen-binding region for mobilizing / engaging the target immune cells. Immune cell engagers mobilize / engage immune cells via antigen-binding regions specific to immune cell surface molecules.

[0180]

[0201] The most studied immune cell engagers are bispecific T cell engagers (BiTEs), which contain a target antigen-binding domain and a CD3 polypeptide (usually CD3ε)-binding domain, through which BiTEs recruit T cells. The binding of BiTEs to their target antigen and to the CD3 polypeptide expressed by T cells results in T cell activation, ultimately directing T cell effector activity toward cells expressing the target antigen. Other types of immune cell engagers are well known in the art and include natural killer cell engagers, such as bispecific killer engagers (BiKEs), which recruit and activate NK cells.

[0181]

[0202] In some embodiments, the immune cells involved by the immune cell engager are T cells or NK cells. In some embodiments, the immune cell engager is a T cell engager.

[0182]

[0203] Multispecific antigen-binding molecules may be provided in any suitable form, e.g., the form described in Brinkmann and Kontermann, MAbs(2017)9(2):182-212, which is incorporated herein by reference in its entirety. Suitable forms are shown in Figure 2 of Brinkmann and Kontermann, MAbs(2017)9(2):182-212: antibody conjugates, e.g., IgG2, F(ab')2, or CovX-Body; IgG or IgG-like molecules, e.g., IgG, chimeric IgG, κλ-body common HC; CH1 / CL fusion proteins, e.g., scFv2-CH1 / CL, VHH2-CH1 / CL; bispecific antigen-binding molecules of "variable domain only". Child, e.g., tandem scFv (taFV), triple body, dia body (Db), dsDb, Db (kih), DART, scDB, dsFv-dsFv, tandAbs, triple head, tandem dAb / VHH, tetravalent (tertravalent) dAb.VHH; non-Ig fusion proteins, e.g., scFv2-albumin, scDb-albumin, taFv-albumin, taFv-toxin, mini-antibody, DNL-Fab2, DNL-Fab2-scFv, D NL-Fab2-IgG-cytokine2, ImmTAC(TCR-scFv); modified Fc and CH3 fusion proteins, e.g., scFv-Fc(kih), scFv-Fc(CH3 charge pair), scFv-Fc(EW-RVT), scFv-fc(HA-TF), scFv-Fc(SEED body), taFv-Fc(kih), scFv-Fc(kih)-Fv, Fab-Fc(kih)-scFv, Fab-scFv-Fc(kih), Fab-scFv- Fc(BEAT), Fab-scFv-Fc(SEED body), DART-Fc, scFv-CH3(kih), TriFabs; Fc fusions, e.g., Di-diabody, scDb-Fc, taFv-Fc, scFv-Fc-scFv, HCAb-VHH, Fab-scFv-Fc, scFv4-Ig, scFv2-Fcab; CH3 fusions, e.g., Dia-diabody, scDb-CH3; IgE / IgM CH2 fusions, e.g., scFv-EHD2-scFv, scFvMHD2-scFv;Fab fusion proteins, e.g., Fab-scFv (bibody), Fab-scFv2 (tribody), Fab-Fv, Fab-dsFv, Fab-VHH, orthogonal Fab-Fab; non-Ig fusion proteins, e.g., DNL-Fab3, DNL-Fab2-scFv, DNL-Fab2-IgG-cytokine2; asymmetric IgG or IgG-like molecules, e.g., IgG(kih), IgG(kih) common LC, ZW1 IgG common LC, Biclonics common LC, CrossMab, CrossMab(kih), scFab-IgG(kih), Fab-scFab-IgG(kih), orthogonal Fab IgG(kih), DuetMab, CH3 charge pair + CH1 / CL charge pair, hinge / CH3 charge pair, SEED-body, Duobody, four-in-one-CrossMab(kih), LUZ-Y common LC; LUZ-Y scFab-IgG, FcFc*; IgG with added Fc modification, e.g., IgG(kih)-Fv, IgG HA-TF-Fv, IgG(kih)scFab, scFab-Fc(kih)-scFv2, scFab-Fc(kih)-scFv, Half DVD-Ig, DVI-Ig (Four-in-One), CrossMab-Fab; Modified Fc and CH3 fusion proteins, e.g., Fab-Fc(kih)-scFv, Fab-scFv-Fc(kih), Fab-scFv-Fc(BEAT), Fab-scFv-Fc-SEED body, TriFab; Added IgG-HC fusions, e.g., IgG-HC, scFv, IgG-dAb, IgG-taFV, IgG-CrossFab, IgG-Orthogonal Fab, IgG-(CαCβ)Fab, scFv-HC-IgG, Tandem Fab-IgG(Orthogonal Fab), Fab-IgG(CαCβ) Fab), Fab-IgG(CR3), Fab-Hinge-IgG(CR3); added IgG-LC fusions, e.g., IgG-scFv(LC), scFv(LC)-IgG, dAb-IgG; added IgG-HC and LC fusions, e.g., DVD-Ig, TVD-Ig, CODV-Ig, scFv4-IgG, Zybody; Fc fusions, e.g., Fab-scFv-Fc, scFv4-Ig; F(ab')2 fusions, e.g., F(ab')2-scFv2;CH1 / CL fusion proteins, e.g., scFv2-CH1-hinge / CL; modified IgG, e.g., DAF (two-in-one-IgG), DutaMab, Mab; 2 ; and non-Ig fusions, such as DNL-Fab4-IgG.

[0183]

[0204] Those skilled in the art can design and prepare bispecific antigen-binding molecules. Methods for generating bispecific antigen-binding molecules include, for example, the step of chemically crosslinking an antigen-binding molecule or antibody fragment using, for example, a reducible disulfide or a non-reducible thioether bond, as described in Segal and Bast, 2001. Production of Bispecific Antigen-binding molecules. Current Protocols in Immunology. 14:IV:2.13:2.13.1~2.13.16, which is incorporated herein by reference in its entirety. For example, N-succinimidyl-3-(-2-pyridyldithio)-propionate (SPDP) can be used to chemically crosslink a Fab fragment, for example, with an SH group via a hinge region to create a disulfide-bonded bispecific F(ab)2 heterodimer.

[0184]

[0205] Other methods for generating bispecific antigen-binding molecules include, for example, the step of fusing antibody-producing hybridomas with, for example, polyethylene glycol to generate quadroma cells capable of secreting bispecific antibodies, as described in DM and Bast, BJ2001. Production of Bispecific Antigen-binding molecules. Current Protocols in Immunology. 14:IV:2.13:2.13.1~2.13.16.

[0185]

[0206] Bispecific antigen-binding molecules can also be produced by recombination, for example, by expression from nucleic acid constructs encoding the polypeptide of a VH4-34 antigen-binding molecule, as described in Chapter 40 of Antibody Engineering: Methods and Protocols, Second Edition (Humana Press, 2012), both of which are incorporated herein by reference: Production of Bispecific Antigen-binding molecules: Diabodies and Tandem scFv (Hornig and Farber-Schwarz) or French, How to make bispecific antigen-binding molecules, Methods Mol. Med. 2000; 40: pp. 333-339.

[0186]

[0207] For example, a DNA construct containing sequences encoding the light and heavy chain variable domains of two antigen-binding fragments (i.e., light and heavy chain variable domains of an antigen-binding fragment capable of binding to VH4-34, and light and heavy chain variable domains of an antigen-binding fragment capable of binding to another target protein), and containing sequences encoding a suitable linker or dimerization domain between the antigen-binding fragments, can be prepared by molecular cloning techniques. Recombinant bispecific antibodies can then be produced (e.g., in vitro) by expressing a suitable construct in a suitable host cell (e.g., a mammalian host cell), and the expressed recombinant bispecific antibodies can then be optionally purified. Fc area

[0208] In some embodiments, the VH4-34 antigen-binding molecule of this disclosure includes an Fc region.

[0187]

[0209] The Fc region consists of CH2 and CH3 regions derived from one polypeptide and CH2 and CH3 regions derived from another polypeptide. The CH2 and CH3 regions derived from the two polypeptides together form the Fc region.

[0188]

[0210] Fc-mediated functions include Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), complement-dependent cell-mediated cytotoxicity (CDC), membrane invasion complex (MAC) formation, cell degranulation, cytokine and / or chemokine production, and antigen processing and presentation.

[0189]

[0211] Modifications of antibody Fc regions that affect Fc-mediated function are known in the art, for example, those described in their entirety by reference in Wang et al., Protein Cell (2018) 9(1): pp. 63-73. Exemplary Fc region modifications known to affect antibody effector function are summarized in Table 1 of Wang et al., Protein Cell (2018) 9(1): pp. 63-73.

[0190]

[0212] In some embodiments, the VH4-34 antigen-binding molecules of this disclosure include an Fc region that has been modified to increase or decrease Fc-mediated function compared to an antigen-binding molecule that includes a corresponding unmodified Fc region.

[0191]

[0213] The substitution combination F243L / R292P / Y300L / V305I / P396L is described in Stavenhagen et al., Cancer Res. (2007), and increases binding to FcγRIIIa, thereby enhancing ADCC. The substitution combination S239D / I332E or S239D / I332E / A330L is described in Lazar et al., Proc Natl Acad Sci USA. (2006) 103:4005~4010, and increases binding to FcγRIIIa, thereby enhancing ADCC. The substitution combination S239D / I332E / A330L is also described, and decreases binding to FcγRIIb, thereby enhancing ADCC. The substitution combination S298A / E333A / K334A is described by Shields et al. in J Biol Chem. (2001) 276: pp. 6591-6604, and increases binding to FcγRIIIa, thereby increasing ADCC. The substitution combination L234Y / L235Q / G236W / S239M / H268D / D270E / S298A in one heavy chain, and the substitution combination D270E / K326D / A330M / K334E in the other heavy chain are described by Mimoto et al. in MAbs. (2013): 5: pp. 229-236, and increases binding to FcγRIIIa, thereby increasing ADCC. The substitution combination G236A / S239D / I332E is described by Richards et al. in Mol Cancer Ther. (2008) 7:2517-2527, and it increases binding to FcγRIIa and FcγRIIIa, thereby increasing ADCP.

[0192]

[0214] The substitution combination K326W / E333S is described by Idusogie et al. J Immunol. (2001) 166(4): pp. 2571-255, and increases binding to C1q, thereby increasing CDC. The substitution combination S267E / H268F / S324T is described by Moore et al. MAbs. (2010) 2(2): pp. 181-189, and increases binding to C1q, thereby increasing CDC. The substitution combination described by Natsume et al. Cancer Res. (2008) 68(10): pp. 3863-372 has been reported to increase binding to C1q, thereby increasing CDC. The substitution combination E345R / E430G / S440Y is described by Diebolder et al. in Science (2014) 343(6176): pp. 1260-123, and increases hexamerization, thereby increasing CDC.

[0193]

[0215] The substitution combination M252Y / S254T / T256E is described by Dall'Acqua et al. J Immunol. (2002) 169: pp. 5171-5180, and increases binding to FcRn at pH 6.0, thereby increasing the half-life of the antigen-binding molecule. The substitution combination M428L / N434S is described by Zalevsky et al. Nat Biotechnol. (2010) 28: pp. 157-159, and increases binding to FcRn at pH 6.0, thereby increasing the half-life of the antigen-binding molecule.

[0194]

[0216] When a heavy chain steady region / Fc region / CH2-CH3 region / CH2 region / CH3 region is described herein as containing a position(s) / substitution(s) that "corresponds" to a reference position(s) / substitution(s)(s), then equivalent positions(s) / substitution(s) in the homologous heavy chain steady region / Fc region / CH2-CH3 region / CH2 region / CH3 region are intended.

[0195]

[0217] When an Fc region is described as containing specific positions / substitutions, those positions / substitutions may be present on one or both of the polypeptide chains that together form the Fc region.

[0196]

[0218] Unless otherwise specified, positions in this specification refer to positions in the amino acid sequences of the constant region of human immunoglobulins numbered according to the EU numbering system, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, 1991. For example, the substitutions L242C and K334C in human IgG1 correspond to the L>C substitution at position 125 and the K>C substitution at position 217 of the constant region of human IgG1, as numbered according to Sequence ID No. 71.

[0197]

[0219] The homologous heavy chain constant region is a heavy chain constant region containing an amino acid sequence having at least 60%, preferably 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to the heavy chain constant region of human IgG1 (i.e., the amino acid sequence shown in SEQ ID NO: 71). The homologous Fc region is an Fc region composed of a polypeptide containing an amino acid sequence having at least 60%, preferably 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to the CH2-CH3 region of human IgG1 (i.e., the amino acid sequences shown in SEQ ID NOs: 74 and 75). The homologous CH2 region is a CH2 region containing an amino acid sequence that has at least 60%, preferably 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with the CH2 region of human IgG1 (i.e., the amino acid sequence shown in SEQ ID NO: 74). The homologous CH3 region is a CH3 region containing an amino acid sequence that has at least 60%, preferably 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with the CH3 region of human IgG1 (i.e., the amino acid sequence shown in SEQ ID NO: 75).

[0198]

[0220] The corresponding positions for those identified in human IgG1 can be identified by sequence alignment, which can be performed using, for example, sequence alignment software such as ClustalOmega (Soding, J.2005, Bioinformatics 21, pp. 951-960).

[0199]

[0221] In some embodiments, the VH4-34 antigen-binding molecule of this disclosure includes an Fc region that includes modifications to enhance Fc-mediated function. In some embodiments, the Fc region includes modifications to enhance ADCC. In some embodiments, the Fc region includes modifications to enhance ADCP. In some embodiments, the Fc region includes modifications to enhance CDC. Antigen-binding molecules including an Fc region that includes modifications to enhance Fc-mediated function (e.g., ADCC, ADCP, CDC) induce an increased level of the relevant effector function compared to antigen-binding molecules including the corresponding unmodified Fc region.

[0200]

[0222] In some embodiments, the VH4-34 antigen-binding molecule of this disclosure includes an Fc region that includes modifications to increase affinity to one or more Fc receptors (e.g., FcγRIIa, FcγRIIIa). Modifications that increase affinity to Fc receptors may increase Fc-mediated effector function, such as antibody-dependent cell-mediated cytotoxicity (ADCC) and / or antibody-dependent cell-mediated phagocytosis (ADCP). In some embodiments, the VH4-34 antigen-binding molecule of this disclosure includes an Fc region that includes modifications to reduce affinity to C1q, and such modifications that reduce complement-dependent cell-mediated cytotoxicity (CDC) may be desirable. In some embodiments, the VH4-34 antigen-binding molecule of this disclosure includes an Fc region that includes modifications to increase hexamer formation. Modifications to the Fc region that can increase affinity for one or more Fc receptors, decrease affinity for C1q, and / or increase hexamer formation are described, for example, in Saxena and Wu Front Immunol. (2016) 7:580, which are incorporated herein by reference in their entirety. In some embodiments, the VH4-34 antigen-binding molecules of this disclosure include an Fc region comprising a CH2 / CH3 containing one or more of the substitutions shown in Table 1 of Saxena and Wu Front Immunol. (2016) 7:580.

[0201]

[0223] In some embodiments, the VH4-34 antigen-binding molecule of this disclosure includes an Fc region that has been modified to increase binding to the Fc receptor. In some embodiments, the Fc region includes a modification that increases binding to the Fcγ receptor. In some embodiments, the Fc region includes a modification that increases binding to one or more of FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb. In some embodiments, the Fc region includes a modification that increases binding to FcγRIIIa. In some embodiments, the Fc region includes a modification that increases binding to FcγRIIa. In some embodiments, the Fc region includes a modification that increases binding to FcγRIIb. In some embodiments, the Fc region includes a modification that increases binding to FcRn. In some embodiments, the Fc region includes a modification that increases binding to complement proteins. In some embodiments, the Fc region includes a modification that increases or decreases binding to C1q. In some embodiments, the Fc region includes modifications that promote hexamerization of the VH4-34 antigen-binding molecule. In some embodiments, the Fc region includes modifications that increase the serum half-life of the antigen-binding molecule. In some embodiments, the Fc region includes modifications that increase co-engagement.

[0202]

[0224] In this specification, “Fcγ receptor” may originate from any species and includes isoforms, fragments, variants (including mutants) or homologs of any species. Similarly, “FcγRI,” “FcγRIIa,” “FcγRIIb,” “FcγRIIc,” “FcγRIIIa,” and “FcγRIIIb” respectively refer to FcγRI / FcγRIIa / FcγRIIb / FcγRIIc / FcγRIIIa / FcγRIIIb of any species and include isoforms, fragments, variants (including mutants) or homologs of any species. Humans have six distinct classes of Fcγ receptors (mouse homologous species are shown in parentheses): FcγRI (mFcγRI), FcγRIIa (mFcγRIII), FcγRIIb (mFcγRIIb), FcγRIIc, FcγRIIIa (mFcγRIV), and FcγRIIIb.

[0203]

[0225] Variant Fcγ receptors include, for example, the 158V and 158F polymorphs of human FcγRIIIa, and the 167H and 167R polymorphs of human FcγRIIa.

[0204]

[0226] In some embodiments, the VH4-34 antigen-binding molecule of the present disclosure includes an Fc region (e.g., a heavy chain constant region, or another polypeptide including a CH2-CH3 region) containing one or more of the following (e.g., one, two, three, four, five, six, seven, or eight): C at position 242; C at position 334; A at position 236; D at position 239; E at position 332; L at position 330; K at position 345; and G at position 430.

[0205]

[0227] In some embodiments, the VH4-34 antigen-binding molecule of the present disclosure comprises an Fc region (e.g., a heavy chain constant region, or another polypeptide comprising a CH2-CH3 region) containing one or more of the following substitutions (or corresponding substitutions) (e.g., one, two, three, four, five, six, seven, or eight): L242C, K334C, G236A, S239D, I332E, A330L, E345K, and E430G.

[0206]

[0228] In some embodiments, the VH4-34 antigen-binding molecule includes an Fc region (e.g., another polypeptide including a heavy chain constant region, a CH2-CH3 region, or a CH2 region) containing C at the position corresponding to position 242. In some embodiments, the Fc region (e.g., another polypeptide including a heavy chain constant region, a CH2-CH3 region, or a CH2 region) contains C at the position corresponding to position 334. In some embodiments, the Fc region (e.g., another polypeptide including a heavy chain constant region, a CH2-CH3 region, or a CH2 region) contains C at the position corresponding to position 242 and C at the position corresponding to position 334.

[0207]

[0229] In some embodiments, the VH4-34 antigen-binding molecule includes an Fc region (e.g., another polypeptide including a heavy chain constant region, a CH2-CH3 region, or a CH2 region) containing A at the position corresponding to 236. In some embodiments, the Fc region (e.g., another polypeptide including a heavy chain constant region, a CH2-CH3 region, or a CH2 region) contains D at the position corresponding to 239. In some embodiments, the Fc region (e.g., another polypeptide including a heavy chain constant region, a CH2-CH3 region, or a CH2 region) contains A at the position corresponding to 236 and D at the position corresponding to 239.

[0208]

[0230] In some embodiments, the VH4-34 antigen-binding molecule includes an Fc region (e.g., another polypeptide including a heavy chain constant region, a CH2-CH3 region, or a CH2 region) with E at the position corresponding to 332. In some embodiments, the Fc region (e.g., another polypeptide including a heavy chain constant region, a CH2-CH3 region, or a CH2 region) includes A at the position corresponding to 236, D at the position corresponding to 239, and E at the position corresponding to 332.

[0209]

[0231] In some embodiments, the VH4-34 antigen-binding molecule includes an Fc region (e.g., another polypeptide including a heavy chain constant region, a CH2-CH3 region, or a CH2 region) with L at the position corresponding to 330. In some embodiments, the Fc region (e.g., another polypeptide including a heavy chain constant region, a CH2-CH3 region, or a CH2 region) includes A at the position corresponding to 236, D at the position corresponding to 239, E at the position corresponding to 332, and L at the position corresponding to 330.

[0210]

[0232] In some embodiments, the VH4-34 antigen-binding molecule includes an Fc region (e.g., another polypeptide including a heavy chain constant region, a CH2-CH3 region, or a CH3 region) containing K at the position corresponding to 345. In some embodiments, the Fc region (e.g., another polypeptide including a heavy chain constant region, a CH2-CH3 region, or a CH3 region) contains G at the position corresponding to 430. In some embodiments, the Fc region (e.g., another polypeptide including a heavy chain constant region, a CH2-CH3 region, or a CH2 region) contains K at the position corresponding to 345 and G at the position corresponding to 430.

[0211]

[0233] In some embodiments, the VH4-34 antigen-binding molecule includes an Fc region (e.g., including a heavy chain constant region, a CH2-CH3 region, or another polypeptide including a CH2 region) with C at position 242, C at position 334, A at position 236, and D at position 239.

[0212]

[0234] In some embodiments, the VH4-34 antigen-binding molecule includes an Fc region (e.g., including a heavy chain constant region, a CH2-CH3 region, or another polypeptide including a CH2 region) with C at position 242, C at position 334, A at position 236, D at position 239, and E at position 332.

[0213]

[0235] In some embodiments, the VH4-34 antigen-binding molecule includes an Fc region (e.g., including a heavy chain constant region, a CH2-CH3 region, or another polypeptide including a CH2 region) with C at position 242, C at position 334, A at position 236, D at position 239, E at position 332, and L at position 330.

[0214]

[0236] In some embodiments, the VH4-34 antigen-binding molecule includes an Fc region (e.g., another polypeptide including a heavy chain constant region or a CH2-CH3 region) containing C at position 242, C at position 334, K at position 345, and G at position 430.

[0215]

[0237] In some embodiments, the VH4-34 antigen-binding molecule includes an Fc region (e.g., another polypeptide including a heavy chain constant region, a CH2-CH3 region, or a CH2 region) containing a substituted L242C (or equivalent substitution). In some embodiments, the Fc region (e.g., another polypeptide including a heavy chain constant region, a CH2-CH3 region, or a CH2 region) includes a substituted K334C (or equivalent substitution). In some embodiments, the Fc region (e.g., another polypeptide including a heavy chain constant region, a CH2-CH3 region, or a CH2 region) includes a substituted L242C (or equivalent substitution) and a substituted K334C (or equivalent substitution).

[0216]

[0238] In some embodiments, the VH4-34 antigen-binding molecule includes an Fc region (e.g., another polypeptide including a heavy chain constant region, a CH2-CH3 region, or a CH2 region) containing substitution G236A (or an equivalent substitution). In some embodiments, the Fc region (e.g., another polypeptide including a heavy chain constant region, a CH2-CH3 region, or a CH2 region) includes substitution S239D (or an equivalent substitution). In some embodiments, the Fc region (e.g., another polypeptide including a heavy chain constant region, a CH2-CH3 region, or a CH2 region) includes substitution G236A (or an equivalent substitution) and substitution S239D (or an equivalent substitution).

[0217]

[0239] In some embodiments, the VH4-34 antigen-binding molecule includes an Fc region (e.g., another polypeptide including a heavy chain constant region, a CH2-CH3 region, or a CH2 region) containing substitution I332E (or an equivalent substitution). In some embodiments, the Fc region (e.g., another polypeptide including a heavy chain constant region, a CH2-CH3 region, or a CH2 region) includes substitution G236A (or an equivalent substitution), substitution S239D (or an equivalent substitution), and substitution I332E (or an equivalent substitution).

[0218]

[0240] In some embodiments, the VH4-34 antigen-binding molecule includes an Fc region (e.g., another polypeptide including a heavy chain constant region, a CH2-CH3 region, or a CH2 region) containing substitution A330L (or an equivalent substitution). In some embodiments, the Fc region (e.g., another polypeptide including a heavy chain constant region, a CH2-CH3 region, or a CH2 region) includes substitution G236A (or an equivalent substitution), substitution S239D (or an equivalent substitution), substitution I332E (or an equivalent substitution), and substitution A330L (or an equivalent substitution).

[0219]

[0241] In some embodiments, the VH4-34 antigen-binding molecule includes an Fc region (e.g., another polypeptide including a heavy chain constant region, a CH2-CH3 region, or a CH3 region) containing substitution E345K (or an equivalent substitution). In some embodiments, the Fc region (e.g., another polypeptide including a heavy chain constant region, a CH2-CH3 region, or a CH3 region) includes substitution E430G (or an equivalent substitution). In some embodiments, the Fc region (e.g., another polypeptide including a heavy chain constant region, a CH2-CH3 region, or a CH2 region) includes substitution E345K (or an equivalent substitution) and substitution E430G (or an equivalent substitution).

[0220]

[0242] In some embodiments, the VH4-34 antigen-binding molecule includes an Fc region (e.g., another polypeptide including a heavy chain constant region, a CH2-CH3 region, or a CH2 region) containing substitutions L242C (or equivalent substitutions), K334C (or equivalent substitutions), G236A (or equivalent substitutions), and S239D (or equivalent substitutions).

[0221]

[0243] In some embodiments, the VH4-34 antigen-binding molecule includes an Fc region (e.g., including a heavy chain constant region, a CH2-CH3 region, or another polypeptide including a CH2 region) containing substitutions L242C (or equivalent substitutions), K334C (or equivalent substitutions), G236A (or equivalent substitutions), S239D (or equivalent substitutions), and I332E (or equivalent substitutions).

[0222]

[0244] In some embodiments, the VH4-34 antigen-binding molecule includes an Fc region (e.g., another polypeptide including a heavy chain constant region, a CH2-CH3 region, or a CH2 region) containing substitutions L242C (or equivalent substitutions), K334C (or equivalent substitutions), G236A (or equivalent substitutions), S239D (or equivalent substitutions), I332E (or equivalent substitutions), and A330L (or equivalent substitutions).

[0223]

[0245] In some embodiments, the VH4-34 antigen-binding molecule includes an Fc region (for example, another polypeptide including a heavy chain constant region or a CH2-CH3 region) containing substitutions L242C (or equivalent substitutions), K334C (or equivalent substitutions), E345K (or equivalent substitutions), and E430G (or equivalent substitutions).

[0224]

[0246] In some embodiments, the VH4-34 antigen-binding molecule includes an Fc region (e.g., another polypeptide including a heavy chain constant region or a CH2-CH3 region) containing one or more of the following (e.g., one, two, three, four, five, six, seven, eight, nine, ten, 11, or 12): L at the position corresponding to 243, P at the position corresponding to 292, L at the position corresponding to 300, I at the position corresponding to 305 and L at the position corresponding to 396; D at the position corresponding to 239 and E at the position corresponding to 332; D at the position corresponding to 239, E at the position corresponding to 332 and L at the position corresponding to 330; A at the position corresponding to 298, A at the position corresponding to 333 and A at the position corresponding to 334; Y at the position corresponding to 234, Q at the position corresponding to 235, and the position corresponding to 236 W is located at position 239, M at position 268, D at position 270, and A at position 298; E at position 270, D at position 326, M at position 330, and E at position 334; A at position 236, D at position 239, and E at position 332; W at position 326 and S at position 333; E at position 267, F at position 268, and T at position 324; R at position 345, G at position 430, and Y at position 440; Y at position 252, T at position 254, and E at position 256; and L at position 428 and S at position 434.

[0225]

[0247] In some embodiments, the VH4-34 antigen-binding molecule includes an Fc region (e.g., another polypeptide including a heavy chain constant region or a CH2-CH3 region) containing one or more of the following combinations of substitutions (or corresponding substitutions) (e.g., one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve): F243L / R292P / Y300L / V305I / P396L;S239D / I332E;S239 D / I332E / A330L;S298A / E333A / K334A;L234Y / L235Q / G236W / S239M / H268D / D270E / S298A;D270E / K326D / A330M / K334E; G236A / S239D / I332E; K326W / E333S; S267E / H268F / S324T; E345R / E430G / S440Y; M252Y / S254T / T256E; and M428L / N434S.

[0226]

[0248] In some embodiments, the VH4-34 antigen-binding molecule of this disclosure includes an Fc region containing modifications in one or more of the CH2 and CH3 regions that promote the association of the Fc region. Recombinant co-expression and subsequent association of the component polypeptides of the antigen-binding molecule leads to several possible combinations. To improve the yield of desired combinations of polypeptides in the antigen-binding molecule in recombinant generation, it is advantageous to introduce modifications(s) in the Fc region that promote the association of desired combinations of heavy-chain polypeptides. Modifications may, for example, promote hydrophobic and / or electrostatic interactions between the CH2 and / or CH3 regions of different polypeptide chains. Suitable modifications are described, for example, in Ha et al., Front.Immnol (2016) 7:394, which is incorporated herein by reference in its entirety.

[0227]

[0249] In some embodiments, the antigen-binding molecule of this disclosure includes an Fc region containing a pair substitution in the CH3 region of the Fc region, as shown in Table 1 of Ha et al., Front.Immnol (2016) 7:394: KiH, KiH s-s , HA-TF, ZW1, 7.8.60, DD-KK, EW-RVT, EW-RVTs-s , SEED or A107. Polypeptide

[0250] This disclosure also provides polypeptide components of antigen-binding molecules. Polypeptides may be provided in isolated or substantially purified forms.

[0228]

[0251] The VH4-34 antigen-binding molecule of this disclosure may be or may comprise a polypeptide complex.

[0252] In this specification, when a polypeptide contains more than one domain or region, it will be acknowledged that multiple domains / regions are preferably present within the same polypeptide chain. That is, a polypeptide containing more than one domain or region is a fusion polypeptide containing domains / regions.

[0229]

[0253] In some embodiments, the polypeptide comprises or consists of VH as described herein. In some embodiments, the polypeptide comprises or consists of VL as described herein.

[0230]

[0254] In some embodiments, the polypeptide further comprises one or more antibody heavy chain constant regions (CH). In some embodiments, the polypeptide further comprises one or more antibody light chain constant regions (CL). In some embodiments, the polypeptide comprises the CH1, CH2, and / or CH3 regions of immunoglobulin (Ig).

[0231]

[0255] In some embodiments, the polypeptide comprises one or more regions of the constant sequence of an immunoglobulin heavy chain. In some embodiments, the polypeptide comprises a CH1 region as described herein. In some embodiments, the polypeptide comprises a CH1-CH2 hinge region as described herein. In some embodiments, the polypeptide comprises a CH2 region as described herein. In some embodiments, the polypeptide comprises a CH3 region as described herein.

[0232]

[0256] In some embodiments, the polypeptide comprises one or more regions of the constant sequence of an immunoglobulin light chain. In some embodiments, the polypeptide comprises a CL region as described herein.

[0233]

[0257] In some embodiments, the polypeptide includes a structure that follows one of the following from the N-terminus to the C-terminus: (i)VH (ii) VL (iii) VH-CH1 (iv) VL-CL (v)VL-CH1 (vi) VH-CL (vii) VH-CH1-CH2-CH3 (viii)VL-CL-CH2-CH3 (ix)VL-CH1-CH2-CH3 (x)VH-CL-CH2-CH3

[0258] Furthermore, antigen-binding molecules composed of the polypeptides of this disclosure are also provided by this disclosure. In some embodiments, the VH4-34 antigen-binding molecule of this disclosure comprises one of the following combinations of polypeptides: (A)VH+VL (B)VH-CH1+VL-CL (C)VL-CH1+VH-CL (D)VH-CH1-CH2-CH3+VL-CL (E)VH-CL-CH2-CH3+VL-CH1 (F)VL-CH1-CH2-CH3+VH-CL (G)VL-CL-CH2-CH3+VH-CH1 (H)VH-CH1-CH2-CH3+VL-CL-CH2-CH3 (I)VH-CL-CH2-CH3+VL-CH1-CH2-CH3

[0259] In some embodiments, the VH4-34 antigen-binding molecule comprises more polypeptides than the combinations shown in (A) to (I) above. For example, in relation to (D) above, in some embodiments, the VH4-34 antigen-binding molecule comprises two polypeptides having the structure VH-CH1-CH2-CH3 and two polypeptides having the structure VL-CL.

[0234]

[0260] In some embodiments, the VH4-34 antigen-binding molecule of this disclosure comprises one of the following combinations of polypeptides: (J)VH (anti-VH4-34) + VL (anti-VH4-34) (K)VH(anti-VH4-34)-CH1+VL(anti-VH4-34)-CL (L)VL(anti-VH4-34)-CH1+VH(anti-VH4-34)-CL (M)VH(anti-VH4-34)-CH1-CH2-CH3+VL(anti-VH4-34)-CL (N)VH(anti-VH4-34)-CL-CH2-CH3+VL(anti-VH4-34)-CH1 (O)VL(anti-VH4-34)-CH1-CH2-CH3+VH(anti-VH4-34)-CL (P)VL(anti-VH4-34)-CL-CH2-CH3+VH(anti-VH4-34)-CH1 (Q)VH(Anti-VH4-34)-CH1-CH2-CH3+VL(Anti-VH4-34)-CL-CH2-CH3 (R)VH(Anti-VH4-34)-CL-CH2-CH3+VL(Anti-VH4-34)-CH1-CH2-CH3

[0261] Here, "VH (anti-VH4-34)" refers to VH, an antigen-binding molecule capable of binding to VH4-34, as defined in this specification, for example, by one of (1) to (31). "VL (anti-VH4-34)" refers to VL, an antigen-binding molecule capable of binding to VH4-34, as defined in this specification, for example, by one of (32) to (66).

[0235]

[0262] In some embodiments, the VH4-34 antigen-binding molecules of the present disclosure include an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to any one of SEQ ID NOs. 5, 21, 37, 53, 69, 70, 125, 141, 151, 156, 167, 171, 176, 181, 182, or 184, or a polypeptide comprising such an amino acid sequence.

[0236]

[0263] In some embodiments, the VH4-34 antigen-binding molecules of this disclosure include an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to any one of SEQ ID NOs: 13, 29, 45, 61, 130, 143, 147, 153, 161, 189, 193, 198, 201, 205, or 209, or a polypeptide comprising such an amino acid sequence.

[0237]

[0264] In some embodiments, the VH4-34 antigen-binding molecules of the present disclosure include an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to any one of SEQ ID NOs. 86-89, 136, 139, or 214-219, or a polypeptide comprising such an amino acid sequence.

[0238]

[0265] In some embodiments, the VH4-34 antigen-binding molecules of the present disclosure include an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to any one of SEQ ID NOs. 90-93, 137, 140, 146, or 220-225, or a polypeptide comprising such an amino acid sequence.

[0239]

[0266] In some embodiments, the VH4-34 antigen-binding molecules of the present disclosure include an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to any one of SEQ ID NOs. 95-99 or 138, or a polypeptide comprising such an amino acid sequence.

[0240]

[0267] In some embodiments, the VH4-34 antigen-binding molecules of this disclosure include an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to any one of SEQ ID NOs. 101-105, 149, or 150, or a polypeptide comprising such an amino acid sequence.

[0241]

[0268] In some embodiments, the VH4-34 antigen-binding molecule of this disclosure is (i) One or more (e.g., one, two) polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to SEQ ID NO: 156, and (ii) One or more (e.g., one, two) polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to SEQ ID NO: 13 or 130. Includes.

[0242] In some embodiments, the VH4-34 antigen-binding molecule of this disclosure is (i) One or more (e.g., one, two) polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to SEQ ID NO: 5 or 125, and (ii) One or more (e.g., one, two) polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to SEQ ID NO: 161. Includes.

[0243] In some embodiments, the VH4-34 antigen-binding molecule of this disclosure is (i) One or more (e.g., one, two) polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to SEQ ID NO: 151, and (ii) One or more (e.g., one, two) polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to SEQ ID NOs. 61, 143, or 147. Includes.

[0244] In some embodiments, the VH4-34 antigen-binding molecule of this disclosure is (i) One or more (e.g., one, two) polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to SEQ ID NO: 53 or 141, and (ii) One or more (e.g., one, two) polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to SEQ ID NO: 153. Includes. Specific exemplary embodiments of VH4-34 antigen-binding molecules

[0269] In some embodiments, the VH4-34 antigen-binding molecule of the present disclosure comprises one or more polypeptides comprising a VH region containing a heavy chain CDR and a VL region containing a light chain CDR, selected from clones shown in Table A herein. That is, in some embodiments, the VH4-34 antigen-binding molecule comprises one or more polypeptides comprising (i) a VH region containing HC-CDR1, HC-CDR2, and HC-CDR3 as shown in column A of Table A, and (ii) a VL region containing LC-CDR1, LC-CDR2, and LC-CDR3 as shown in column B of Table A, where the sequences in columns A and B are selected from the same row of Table A.

[0245]

[0270] In some embodiments, the VH4-34 antigen-binding molecule of the present disclosure comprises one or more polypeptides of a clone selected from those shown in Table B herein, comprising a VH region containing a heavy chain FR and a VL region containing a light chain FR. That is, in some embodiments, the VH4-34 antigen-binding molecule comprises one or more polypeptides comprising (i) a VH region containing HC-FR1, HC-FR2, HC-FR3 and HC-FR4 as shown in column A of Table B and (ii) a VL region containing LC-FR1, LC-FR2, LC-FR3 and LC-FR4 as shown in column B of Table B, where the sequences in columns A and B are selected from the same row of Table B.

[0246]

[0271] In some embodiments, the VH4-34 antigen-binding molecules of the present disclosure include (i) a polypeptide (one or more) comprising an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to the amino acid sequence shown in column A of Table C, and (ii) an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to the amino acid sequence shown in column B of Table C, wherein the sequences in columns A and B are selected from the same row of Table C.

[0247]

[0272] In some embodiments, the VH4-34 antigen-binding molecule of this disclosure comprises one or more polypeptides containing the VH and VL regions of a clone selected from those shown in Table C herein. That is, in some embodiments, the VH4-34 antigen-binding molecule comprises one or more polypeptides containing (i) the amino acid sequence shown in column A of Table C and (ii) the amino acid sequence shown in column B of Table C, wherein the sequences in columns A and B are selected from the same row of Table C.

[0248]

[0273] In some embodiments, the VH4-34 antigen-binding molecules of the present disclosure include (i) a polypeptide comprising or having an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to the amino acid sequence shown in column A of Table D, and (ii) a polypeptide comprising or having an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to the amino acid sequence shown in column B of Table D, wherein the sequences in columns A and B are selected from the same row of Table D.

[0249]

[0274] In some embodiments, the VH4-34 antigen-binding molecule of this disclosure comprises a polypeptide of an antigen-binding molecule according to Table D herein. That is, in some embodiments, the VH4-34 antigen-binding molecule comprises (i) a polypeptide comprising or consisting thereof an amino acid sequence shown in column A of Table D and (ii) a polypeptide comprising or consisting thereof an amino acid sequence shown in column B of Table D, wherein the sequences in columns A and B are selected from the same row of Table D.

[0250]

[0275] In some embodiments, the VH4-34 antigen-binding molecule of this disclosure includes: (i) One or more (e.g., one, two) polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to SEQ ID NO: 86, and (ii) One or more polypeptides (e.g., one or two) comprising an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to SEQ ID NO: 90.

[0251]

[0276] In some embodiments, the VH4-34 antigen-binding molecule of this disclosure includes: (i) One or more (e.g., one, two) polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to SEQ ID NO: 87, and (ii) One or more polypeptides (e.g., one or two) comprising an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to SEQ ID NO: 91.

[0252]

[0277] In some embodiments, the VH4-34 antigen-binding molecule of this disclosure includes: (i) One or more (e.g., one, two) polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to SEQ ID NO: 88, and (ii) One or more polypeptides (e.g., one or two) comprising an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to SEQ ID NO: 92.

[0253]

[0278] In some embodiments, the VH4-34 antigen-binding molecule of this disclosure includes: (i) One or more (e.g., one, two) polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to SEQ ID NO: 89, and (ii) One or more polypeptides (e.g., one or two) comprising an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to SEQ ID NO: 93.

[0254]

[0279] In some embodiments, the VH4-34 antigen-binding molecule of this disclosure includes: (i) One or more (e.g., one, two) polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to SEQ ID NO: 136, and (ii) One or more polypeptides (e.g., one or two) comprising an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to SEQ ID NO: 137.

[0255]

[0280] In some embodiments, the VH4-34 antigen-binding molecule of this disclosure includes: (i) One or more (e.g., one, two) polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to SEQ ID NO: 139, and (ii) One or more polypeptides (e.g., one or two) comprising an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to SEQ ID NO: 140.

[0256]

[0281] In some embodiments, the VH4-34 antigen-binding molecule of this disclosure includes: (i) One or more (e.g., one, two) polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to SEQ ID NO: 139, and (ii) One or more polypeptides (e.g., one or two) comprising an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to SEQ ID NO: 146.

[0257]

[0282] In some embodiments, the VH4-34 antigen-binding molecule of this disclosure includes: (i) One or more (e.g., one, two) polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to SEQ ID NO: 95, and (ii) One or more polypeptides (e.g., one or two) comprising an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to SEQ ID NO: 90.

[0258]

[0283] In some embodiments, the VH4-34 antigen-binding molecule of this disclosure includes: (i) One or more (e.g., one, two) polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to SEQ ID NO: 96, and (ii) One or more polypeptides (e.g., one or two) comprising an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to SEQ ID NO: 91.

[0259]

[0284] In some embodiments, the VH4-34 antigen-binding molecule of this disclosure includes: (i) One or more (e.g., one, two) polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to SEQ ID NO: 97, and (ii) One or more polypeptides (e.g., one or two) comprising an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to SEQ ID NO: 92.

[0260]

[0285] In some embodiments, the VH4-34 antigen-binding molecule of this disclosure includes: (i) One or more (e.g., one, two) polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to SEQ ID NO: 98, and (ii) One or more polypeptides (e.g., one or two) comprising an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to SEQ ID NO: 93.

[0261]

[0286] In some embodiments, the VH4-34 antigen-binding molecule of this disclosure includes: (i) One or more (e.g., one, two) polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to SEQ ID NO: 99, and (ii) One or more polypeptides (e.g., one or two) comprising an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to SEQ ID NO: 137.

[0262]

[0287] In some embodiments, the VH4-34 antigen-binding molecule of this disclosure includes: (i) One or more (e.g., one, two) polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to SEQ ID NO: 138, and (ii) One or more polypeptides (e.g., one or two) comprising an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to SEQ ID NO: 140.

[0263]

[0288] In some embodiments, the VH4-34 antigen-binding molecule of this disclosure includes: (i) One or more (e.g., one, two) polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to SEQ ID NO: 139, and (ii) One or more polypeptides (e.g., one or two) comprising an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to SEQ ID NO: 146.

[0264]

[0289] In some embodiments, the VH4-34 antigen-binding molecule of this disclosure includes: (i) One or more (e.g., one, two) polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to SEQ ID NO: 228, and (ii) One or more polypeptides (e.g., one or two) comprising an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to SEQ ID NOs. 90, 137, or 229.

[0265]

[0290] In some embodiments, the VH4-34 antigen-binding molecule of this disclosure includes: (i) One or more (e.g., one, two) polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to SEQ ID NOs. 86, 136, or 228, and (ii) One or more polypeptides (e.g., one or two) comprising an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to SEQ ID NO: 229.

[0266]

[0291] In some embodiments, the VH4-34 antigen-binding molecule of this disclosure includes: (i) One or more (e.g., one, two) polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to SEQ ID NO: 226, and (ii) One or more (e.g., one, two) polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to SEQ ID NOs. 93, 140, 146, or 227.

[0267]

[0292] In some embodiments, the VH4-34 antigen-binding molecule of this disclosure includes: (i) One or more (e.g., one, two) polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to SEQ ID NOs. 89, 139, or 226, and (ii) One or more polypeptides (e.g., one, two) comprising an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to SEQ ID NO: 227.

[0268]

[0293] In some embodiments, the VH4-34 antigen-binding molecule of this disclosure includes: (i) One or more (e.g., one, two) polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to SEQ ID NO: 214, and (ii) One or more (e.g., one, two) polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to SEQ ID NOs. 220, 221, 222, 223, 224, or 225.

[0269]

[0294] In some embodiments, the VH4-34 antigen-binding molecule of this disclosure includes: (i) One or more (e.g., one, two) polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to SEQ ID NO: 215, and (ii) One or more (e.g., one, two) polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to SEQ ID NOs. 220, 221, 222, 223, 224, or 225.

[0270]

[0295] In some embodiments, the VH4-34 antigen-binding molecule of this disclosure includes: (i) One or more (e.g., one, two) polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to SEQ ID NO: 216, and (ii) One or more (e.g., one, two) polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to SEQ ID NOs. 220, 221, 222, 223, 224, or 225.

[0271]

[0296] In some embodiments, the VH4-34 antigen-binding molecule of this disclosure includes: (i) One or more (e.g., one, two) polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to SEQ ID NO: 217, and (ii) One or more (e.g., one, two) polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to SEQ ID NOs. 220, 221, 222, 223, 224, or 225.

[0272]

[0297] In some embodiments, the VH4-34 antigen-binding molecule of this disclosure includes: (i) One or more (e.g., one, two) polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to SEQ ID NO: 218, and (ii) One or more (e.g., one, two) polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to SEQ ID NOs. 220, 221, 222, 223, 224, or 225.

[0273]

[0298] In some embodiments, the VH4-34 antigen-binding molecule of this disclosure includes: (i) One or more (e.g., one, two) polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to SEQ ID NO: 219, and (ii) One or more (e.g., one, two) polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to SEQ ID NOs. 220, 221, 222, 223, 224, or 225.

[0274]

[0299] In some embodiments, it may be acknowledged that the antigen-binding molecule may include polypeptides comprising polypeptides according to (i) and (ii) as defined in the preceding paragraph. For example, in embodiments in which the VH4-34 antigen-binding molecule comprises or consists of a single chain Fv, the polypeptides according to (i) and (ii) are provided in tandem within the same polypeptide, linked, for example, by a linker sequence. Linker and additional arrays

[0300] In some embodiments, the VH4-34 antigen-binding molecules and polypeptides of this disclosure include one or more linker sequences between amino acid sequences. The linker sequences may be provided at one or more of the VH, VL, CH1-CH2 hinge region, CH2 region, and CH3 region, or at both ends, of the VH4-34 antigen-binding molecule / polypeptide.

[0275]

[0301] Linker sequences are known to those skilled in the art and are described, for example, in Chen et al., Adv Drug Deliv Rev (2013) 65(10): pp. 1357-1369, which are incorporated herein by reference in their entirety. In some embodiments, the linker sequence is a mobile linker sequence. A mobile linker sequence allows for the relative movement of the amino acid sequence linked by the linker sequence. Mobile linkers are known to those skilled in the art and some are identified in Chen et al., Adv Drug Deliv Rev (2013) 65(10): pp. 1357-1369. Mobile linker sequences often contain a high proportion of glycine and / or serine residues.

[0276]

[0302] In some embodiments, the linker sequence comprises at least one glycine residue and / or at least one serine residue. In some embodiments, the linker sequence consists of a glycine and a serine residue. In some embodiments, the linker sequence comprises one or more copies (e.g., in tandem) of the sequence motif G4S. In some embodiments, the linker sequence has a length of 1-2, 1-3, 1-4, 1-5, 1-10, 1-15, 1-20, 1-25, or 1-30 amino acids.

[0277]

[0303] The VH4-34 antigen-binding molecules and polypeptides of this disclosure may further comprise additional amino acids or sequences of amino acids. For example, the VH4-34 antigen-binding molecules and polypeptides may comprise amino acid sequences(s) to facilitate the expression, folding, transport, processing, purification, or detection of the VH4-34 antigen-binding molecule / polypeptide. For example, the VH4-34 antigen-binding molecule / polypeptide may optionally comprise a sequence encoding His (e.g., 6×His (SEQ ID NO: 236)), Myc, GST, MBP, FLAG, HA, E, or a biotin tag at the N-terminus or C-terminus of the VH4-34 antigen-binding molecule / polypeptide. In some embodiments, the VH4-34 antigen-binding molecule / polypeptide comprises a detectable portion, e.g., fluorescent, luminescent, immunodetectable, radioactive, chemical, nucleic acid, or enzymatic labeling.

[0278]

[0304] The VH4-34 antigen-binding molecules and polypeptides of this disclosure may further comprise a signal peptide (also known as a leader sequence or signal sequence). A signal peptide typically consists of a sequence of 5 to 30 hydrophobic amino acids forming a single alpha-helix. Secretory proteins and proteins expressed on the cell surface often contain signal peptides.

[0279]

[0305] Signal peptides may be present at the N-terminus of VH4-34 antigen-binding molecules / polypeptides, or in newly synthesized antigen-binding molecules / polypeptides. Signal peptides facilitate the efficient transport and secretion of VH4-34 antigen-binding molecules / polypeptides. Signal peptides are often removed by cleavage and are therefore not present in mature antigen-binding molecules / polypeptides secreted from cells expressing VH4-34 antigen-binding molecules / polypeptides.

[0280]

[0306] Signal peptides are known for many proteins and are recorded in databases such as GenBank, UniProt, Swiss-Prot, TrEMBL, Protein Information Resource, Protein Data Bank, Ensembl, and InterPro, and / or can be identified / predicted using amino acid sequence analysis tools such as SignalP (Petersen et al., 2011 Nature Methods 8:785 - 786) or Signal-BLAST (Frank and Sippl, 2008 Bioinformatics 24:2172 - 2176). Labels and conjugates

[0307] In some embodiments, the VH4-34 antigen-binding molecules of the present disclosure further comprise a detectable moiety.

[0281]

[0308] In some embodiments, the VH4-34 antigen-binding molecule comprises a detectable moiety, such as a fluorescent label, a phosphorescent label, a luminescent label, an immunodetectable label (e.g., an epitope tag), a radiolabel, a chemical, nucleic acid or enzyme label. The VH4-34 antigen-binding molecule can be labeled covalently or non-covalently using the detectable moiety.

[0282]

[0309] Examples of fluorescent labels include fluorescein, rhodamine, allophycocyanin, eosin, and NDB, green fluorescent protein (GFP), chelates of rare earths such as europium (Eu), terbium (Tb), and samarium (Sm), tetramethylrhodamine, Texas Red, 4-methylumbelliferone, 7-amino-4-methylcoumarin, Cy3, and Cy5. Examples of radiolabels include radioisotopes such as iodine 123 , iodine 125 , iodine 126 , iodine 131 , iodine 133 , bromine 77 , technetium 99m , indium 111 , indium 113m , gallium 67,gallium 68 ,ruthenium 95 ,ruthenium 97 ,ruthenium 103 ,ruthenium 105 ,mercury 207 ,mercury 203 ,rhenium 99m ,rhenium 101 ,rhenium 105 ,scandium 47 ,tellurium 121m ,tellurium 122m ,tellurium 125m ,thulium 165 ,thulium 167 ,thulium 168 ,copper 67 fluorine 18 ,yttrium 90 ,palladium 100 bismuth 217 and antimony 211 This includes: Luminescent labels include emissive, chemiluminescent (e.g., acridinium esters, luminols, isoluminols) and bioluminescent labels. Immunodetectable labels include haptens, peptides / polypeptides, antibodies, receptors and ligands, e.g., biotin, avidin, streptavidin or digoxigenin. Nucleic acid labels include aptamers. Enzyme labels include, for example, peroxidase, alkaline phosphatase, glucose oxidase, beta-galactosidase and luciferase.

[0283]

[0310] In some embodiments, the VH4-34 antigen-binding molecule of this disclosure is conjugated to a chemical portion. The chemical portion may be a portion that provides a therapeutic effect. The VH4-34 antigen-binding molecule may be provided as an antibody-drug conjugate. Antibody-drug conjugates are outlined, for example, in Parslow et al., Biomedicines. 2016 Sept; 4(3): 14 (which is incorporated herein by reference in its entirety). In some embodiments, the chemical portion is a drug portion (e.g., a cytotoxic portion). In some embodiments, the drug portion is a chemotherapeutic agent. Functional properties of the VH4-34 antigen-binding molecule

[0311] The VH4-34 antigen-binding molecules described herein can be characterized by referring to certain functional properties. In some embodiments, the VH4-34 antigen-binding molecules described herein have one or more of the following properties: Binds to VH4-34 and / or peptide / polypeptide / polypeptide complexes (e.g., antigen-binding molecules) containing VH4-34. Cells expressing VH4-34 and / or a peptide / polypeptide / polypeptide complex (e.g., an antigen-binding molecule) containing VH4-34, Inhibits the interaction between VH4-34 and / or a peptide / polypeptide / polypeptide complex containing VH4-34 (e.g., an antigen-binding molecule) and VH4-34's interaction partner (e.g., I / i carbohydrates, glycoproteins with N-acetyllactosamine (e.g., CD45 isoform B220), lactosamine moiety and / or antibody 9G4). To reduce the number / percentage of cells (e.g., B cells) expressing VH4-34 and / or peptide / polypeptide / polypeptide complexes (e.g., antigen-binding molecules) containing VH4-34, To increase cell death in cells (e.g., B cells) expressing VH4-34 and / or peptide / polypeptide / polypeptide complexes (e.g., antigen-binding molecules) containing VH4-34, To increase cell death in cells expressing VH4-34 and / or VH4-34-containing peptide / polypeptide / polypeptide complexes (e.g., antigen-binding molecules) via antibody-dependent cell-mediated cytotoxicity (ADCC), To increase cell death in cells expressing VH4-34 and / or VH4-34-containing peptide / polypeptide / polypeptide complexes (e.g., antigen-binding molecules) via antibody-dependent cell-mediated phagocytosis (ADCP), To reduce the levels of VH4-34 and / or VH4-34-containing peptides / polypeptides / polypeptide complexes (e.g., antigen-binding molecules) in blood / serum / plasma, and / or Reduce the number / percentage of cells expressing VH4-34 and / or VH4-34-containing peptides / polypeptides / polypeptide complexes (e.g., antigen-binding molecules) in blood / serum / plasma.

[0284]

[0312] It can be acknowledged that a given antigen-binding molecule may exhibit more than one of the properties listed in the previous paragraph. A given antigen-binding molecule can be evaluated for the properties listed in the previous paragraph using a suitable assay. The assay may be an in vitro assay, which may be a cell-free or cell-based assay. Alternatively, the assay may be an in vivo assay, i.e., one performed in a non-human animal.

[0285]

[0313] If the assay is a cell-based assay, it may involve contacting cells with a given antigen-binding molecule to determine whether the VH4-34 antigen-binding molecule exhibits one or more of the listed properties. The assay may use species labeled with a detectable entity to facilitate its detection. The assay may involve evaluating the listed properties after treating cells individually with a range of amounts / concentrations (e.g., a dilution series) of the antigen-binding molecule. The cells will preferably be cells expressing VH4-34, e.g., B cells expressing an antigen-binding molecule containing VH4-34.

[0286]

[0314] Analysis of the results of such assays may involve determining the concentration at which 50% of the maximum level of the relevant activity is achieved. The concentration of the antigen-binding molecule at which 50% of the maximum level of the relevant activity is achieved may be called the “semi-maximal effective concentration” of the VH4-34 antigen-binding molecule with respect to the relevant activity, and this is also known as the “EC 50 It is sometimes referred to as "[...]". As an example, consider the EC of a given antigen-binding molecule regarding its binding to VH4-34. 50 This concentration may be such that 50% of the maximum level of binding to the related species is achieved.

[0287]

[0315] Depending on the characteristics, EC50 Also, "half-maximal inhibitory concentration" or "IC 50 This is sometimes referred to as the IC50, which is the concentration of an antigen-binding molecule at which 50% of the maximum level of inhibition is observed. As an example, the IC50 of a given antigen-binding molecule to inhibit the interaction between VH4-34 and a glycoprotein containing N-acetyllactosamine (e.g., CD45 isoform B220). 50 This can be the concentration at which 50% of the maximum level of inhibition is achieved.

[0288]

[0316] The VH4-34 antigen-binding molecules described herein bind to VH4-34. In some embodiments, the VH4-34 antigen-binding molecules exhibit specific binding to VH4-34. As used herein, “specific binding” refers to binding that is selective for an antigen and can be distinguished from nonspecific binding to a non-target antigen. Antigen-binding molecules that specifically bind to VH4-34 preferably bind to VH4-34 with greater affinity and / or for a longer duration than they would to other, non-target molecules.

[0289]

[0317] The ability of a given polypeptide to specifically bind to a given molecule can be determined by analysis according to methods known in the art, for example, by ELISA, surface plasmon resonance (SPR; see, e.g., Hearty et al., Methods Mol Biol (2012) 907:411-442), biolayer interferometry (see, e.g., Lad et al., (2015) J Biomol Screen 20(4):498-507), flow cytometry, or radiolabeled antigen binding assay (RIA) enzyme-linked immunosorbent assay. Such analyses allow for the measurement and quantification of binding to a given molecule. In some embodiments, binding is a response detected in a given assay.

[0290]

[0318] In some embodiments, the degree of binding of the VH4-34 antigen-binding molecule to non-target molecules is less than 10% of the binding of the antibody to the target molecule, as measured, for example, by ELISA, SPR, biolayer interferometry, or RIA. Alternatively, the binding specificity is such that the VH4-34 antigen-binding molecule binds to non-target molecules. D At least 0.1 digits greater than (i.e., 0.1 × 10⁻¹⁰) n (In the formula, n is an integer representing the digit)) Large dissociation constant (K D This can be reflected in terms of binding affinity when binding with ). This can be at least one of 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, or 2.0, at any choice.

[0291]

[0319] Binding to VH4-34 can be determined, for example, by ELISA, as described in the examples of this disclosure.

[0320] In some embodiments, the antigen-binding molecule binds to VH4-34. In some embodiments, the antigen-binding molecule binds to a peptide or polypeptide containing VH4-34. In some embodiments, the antigen-binding molecule binds to the variable region of an immunoglobulin heavy chain (i.e., VH) containing VH4-34. In some embodiments, the antigen-binding molecule binds to a peptide / polypeptide containing VH containing VH4-34. In some embodiments, the antigen-binding molecule binds to an immunoglobulin heavy chain containing VH4-34. In some embodiments, the antigen-binding molecule binds to the VH of zanorimumab, patritumab, and / or tabarmab. In some embodiments, the antigen-binding molecule binds to the heavy chain of zanorimumab, patritumab, and / or tabarmab.

[0292]

[0321] In some embodiments, the antigen-binding molecule binds to a peptide or polypeptide complex containing VH4-34. In some embodiments, the antigen-binding molecule binds to a peptide or polypeptide complex containing a peptide or polypeptide containing VH4-34. In some embodiments, the antigen-binding molecule binds to a peptide or polypeptide complex containing a peptide / polypeptide containing VH4-34. In some embodiments, the antigen-binding molecule binds to an immunoglobulin containing an immunoglobulin heavy chain containing VH4-34. In some embodiments, the antigen-binding molecule binds to an immunoglobulin formed from an immunoglobulin heavy chain and light chain, and the VH4-34 antigen-binding molecule includes an immunoglobulin heavy chain containing VH4-34. In some embodiments, the antigen-binding molecule binds to zanorimumab, patritumab, and / or tabarmab.

[0293]

[0322] Zanolimumab contains the heavy chain of SEQ ID NO: 106 and the light chain of SEQ ID NO: 109. Patrizumab contains the heavy chain of SEQ ID NO: 107 and the light chain of SEQ ID NO: 110. Tavarumab contains the heavy chain of SEQ ID NO: 108 and the light chain of SEQ ID NO: 111.

[0294]

[0323] In some embodiments, the antigen-binding molecule has an affinity in the micromolar range, i.e., K D = 9.9 × 10 -4 ~1 × 10 -6 It binds to VH4-34 at M. In some embodiments, the antigen-binding molecule has an affinity of less than a micromolar, i.e., K D <1×10 -6 It binds to VH4-34 at M. In some embodiments, the antigen-binding molecule has an affinity in the nanomolar range, i.e., K D = 9.9 × 10 -7 ~1 × 10 -9 It binds to VH4-34 at M. In some embodiments, the antigen-binding molecule has an affinity of less than nanomolar, i.e., K D <1×10 -9 It binds to VH4-34 at M. In some embodiments, the antigen-binding molecule has an affinity in the picomolar range, i.e., K D = 9.9 × 10-10 ~1 × 10 -12 It binds to VH4-34 at M. In some embodiments, the antigen-binding molecule has a sub-picomolelic affinity, i.e., K D <1×10 -12 It binds to VH4-34 via M.

[0295]

[0324] In some embodiments, the antigen-binding molecule has a K content of 10 μM or less, preferably one of the following: ≤5 μM, ≤2 μM, ≤1 μM, ≤500 nM, ≤100 nM, ≤75 nM, ≤50 nM, ≤40 nM, ≤30 nM, ≤20 nM, ≤15 nM, ≤12.5 nM, ≤10 nM, ≤9 nM, ≤8 nM, ≤7 nM, ≤6 nM, ≤5 nM, ≤4 nM, ≤3 nM, ≤2 nM, ≤1 nM, ≤500 pM, ≤400 pM, ≤300 pM, ≤200 pM, ≤100 pM, ≤50 pM, ≤40 pM, ≤30 pM, ≤20 pM, ≤10 pM, or ≤1 pM. D It then binds to VH4-34.

[0296]

[0325] In some embodiments, the antigen-binding molecule is 0.1 μg / ml or less, preferably one of the following ECs: ≤0.05 μg / ml, ≤0.04 μg / ml, ≤0.03 μg / ml, ≤0.02 μg / ml, ≤0.01 μg / ml, ≤0.009 μg / ml, ≤0.008 μg / ml, ≤0.007 μg / ml, ≤0.006 μg / ml, ≤0.005 μg / ml, ≤0.004 μg / ml, ≤0.003 μg / ml, ≤0.002 μg / ml, or ≤0.001 μg / ml. 50 The VH4-34 is bound by (for example, ELISA, as determined by ELISA as described in the examples of this disclosure).

[0297]

[0326] The VH4-34 antigen-binding molecules of this disclosure can bind to a specific region of VH4-34 of interest. The antigen-binding domain of the antigen-binding molecule can bind to a linear epitope of VH4-34 consisting of a continuous sequence of amino acids (i.e., a primary amino acid sequence). In some embodiments, the antigen-binding domain of the antigen-binding molecule can bind to a conformational epitope of VH4-34 consisting of a discontinuous sequence of amino acids in the amino acid sequence.

[0298]

[0327] The region of the peptide / polypeptide to which the antigen-binding molecule binds can be determined by those skilled in the art using various methods known in the art, including X-ray cocrystallographic analysis of antibody-antigen complexes, peptide scanning, mutagenesis mapping, hydrogen-deuterium exchange analysis by mass spectrometry, phage display, competitive ELISA, and proteolysis-based "protection" methods. Such methods are described, for example, by Gershoni et al., BioDrugs, 2007, 21(3):145-156, which are incorporated herein by reference in their entirety.

[0299]

[0328] In some embodiments, the antigen-binding molecule binds to the VH4-34 region corresponding to the region shown in SEQ ID NO: 3. In some embodiments, the antigen-binding molecule binds to a polypeptide containing or consisting of the amino acid sequence shown in SEQ ID NO: 3.

[0300]

[0329] In some embodiments, the antigen-binding molecule binds to the region of VH4-34 corresponding to the region shown in SEQ ID NO: 4. In some embodiments, the epitope of the antigen-binding molecule contains one or more amino acids in the region shown in SEQ ID NO: 4. In some embodiments, the antigen-binding molecule binds to VH4-34 via contact with one or more amino acids in the region shown in SEQ ID NO: 4. In some embodiments, the antigen-binding molecule binds to a polypeptide containing or consisting of the amino acid sequence shown in SEQ ID NO: 4.

[0301]

[0330] In some embodiments, the VH4-34 antigen-binding molecule can inhibit the interaction between VH4-34 and an antigen, such as a molecule containing an N-acetyllactosamine moiety.

[0331] In some embodiments, the antigen-binding molecule binds to the region of VH4-34 where VH4-34 binds to an I / i carbohydrate. In some embodiments, the antigen-binding molecule binds to the region of VH4-34 where VH4-34 binds to a glycoprotein having N-acetyllactosamine. In some embodiments, the antigen-binding molecule binds to the region of VH4-34 where VH4-34 binds to the lactosamine moiety.

[0302]

[0332] In some embodiments, the epitope of the VH4-34 antigen-binding molecule includes one or more (e.g., one, two, three, four, or all) of the following in SEQ ID NO: 1: Q at position 32, W at position 33, A at position 49, V at position 50, and Y at position 51. In some embodiments, the antigen-binding molecule binds to VH4-34 via contact with one or more (e.g., one, two, three, four, or all) of the following in SEQ ID NO: 1: Q at position 32, W at position 33, A at position 49, V at position 50, and Y at position 51.

[0303]

[0333] In some embodiments, the VH4-34 antigen-binding molecule binds to a polypeptide containing or comprising the amino acid sequence shown in SEQ ID NO: 3 with a greater affinity than the affinity with which the antigen-binding molecule binds to a polypeptide containing or comprising the amino acid sequence shown in SEQ ID NO: 112. In some embodiments, the level of binding of the antigen-binding molecule to a polypeptide containing or comprising the amino acid sequence shown in SEQ ID NO: 112 is less than 50% of the level of binding to a polypeptide containing or comprising the amino acid sequence shown in SEQ ID NO: 3. In some embodiments, the antigen-binding molecule shows substantially no binding to a polypeptide containing or comprising the amino acid sequence shown in SEQ ID NO: 112.

[0304]

[0334] In some embodiments, the antigen-binding molecule of this disclosure binds to an epitope of VH4-34 that is not identical to the epitope of VH4-34 bound by the anti-VH4-34 antibody 9G4 (for example, described in Stevenson et al. Blood (1986) 68:430, which is incorporated herein by reference in its entirety).

[0305]

[0335] The ability of antigen-binding molecules to bind to a given peptide / polypeptide can be analyzed by methods well known to those skilled in the art, including analysis by ELISA, immunoblotting (e.g., Western blotting), immunoprecipitation, surface plasmon resonance, and biolayer interferometry.

[0306]

[0336] In some embodiments, when VH4-34 (for example, present in a VH4-34-containing peptide / polypeptide or polypeptide complex) is expressed on the cell surface (i.e., in or on the cell membrane) of cells expressing VH4-34 or a VH4-34-containing peptide / polypeptide or polypeptide complex, the antigen-binding molecule binds to VH4-34 in a region accessible to the antigen-binding molecule (i.e., the extracellular antigen-binding molecule). In some embodiments, the antigen-binding molecule binds to cells expressing VH4-34 or a VH4-34-containing peptide / polypeptide or polypeptide complex. Examples of cells expressing VH4-34 or a VH4-34-containing peptide / polypeptide or polypeptide complex include B cell lineages encoding / expressing VH-containing immunoglobulins containing VH4-34. Cells expressing VH4-34 or a peptide / polypeptide or polypeptide complex containing VH4-34 may also be referred to herein as VH4-34-positive (i.e., VH4-34+) cells.

[0307]

[0337] The ability of antigen-binding molecules to bind to a given cell type can be analyzed by contacting cells with VH4-34 antigen-binding molecules and detecting antigen-binding molecules bound to cells after a washing step to remove unbound antigen-binding molecules, for example. The ability of antigen-binding molecules to bind to cells expressing immune cell surface molecules and / or cancer cell antigens can be analyzed by methods such as flow cytometry and immunofluorescence microscopy.

[0308]

[0338] In some embodiments, the antigen-binding molecule is one of the clones described herein: D011-1E11p, D011-6039, D011-6040, D011-7016, D011-1E11-5T2, D011-7016-LD1, D011-7016-LD2, D011-7016-LD2_H0L0, D011-7016-LD2_H1L1, D011-7016-LD2_H1L2, D011-7016-LD2_H1L3, D011-7016-LD2_H1L4, D011-7016-LD2_H3L1, D011-7016-LD2_H3L2, D011-7016-LD2_H The antigen-binding molecule binds to the same VH4-34 region or an overlapping VH4-34 region to an antigen-binding molecule containing one of the following VH and VL regions: 3L3, D011-7016-LD2_H3L4, D011-7016-LD2_H4L1, D011-7016-LD2_H4L2, D011-7016-LD2_H4L3, D011-7016-LD2_H4L4, D011-7016-LD2_H5L1, D011-7016-LD2_H5L2, D011-7016-LD2_H5L3, and D011-7016-LD2_H5L4.

[0309]

[0339] Whether a test antigen-binding molecule binds to a given target region that is identical to or overlaps with a reference antigen-binding molecule can be evaluated, for example, by analyzing (i) the interaction between the test antigen-binding molecule and the target in the absence of the reference antigen-binding molecule, and (ii) the interaction between the test antigen-binding molecule and the target in the presence of the reference antigen-binding molecule, or after incubation of the target with the reference antigen-binding molecule. A determination of a reduction in the level of interaction between the test antigen-binding molecule and the target after analysis according to (ii) compared to (i) may support the inference that the test and reference antigen-binding molecules bind to identical or overlapping regions of the target. Suitable assays for such analysis include, for example, competitive ELISA assays and epitope binning assays.

[0310]

[0340] In some embodiments, the antigen-binding molecule binds to VH4-34 in a region where it is bound by an interaction partner of VH4-34 (e.g., an I / i carbohydrate, a glycoprotein having N-acetyllactosamine (e.g., CD45 isoform B220), a lactosamine moiety, and / or antibody 9G4). In some embodiments, the VH4-34 antigen-binding molecule inhibits the interaction between VH4-34 and its interaction partner (e.g., an I / i carbohydrate, a glycoprotein having N-acetyllactosamine (e.g., CD45 isoform B220), a lactosamine moiety, and / or antibody 9G4). In some embodiments, the VH4-34 antigen-binding molecule is a competitive inhibitor of the binding of an interaction partner of VH4-34 (e.g., an I / i carbohydrate, a glycoprotein having N-acetyllactosamine (e.g., CD45 isoform B220), a lactosamine moiety, and / or antibody 9G4) to VH4-34. In some embodiments, the VH4-34 antigen-binding molecule is an allosteric inhibitor of the binding of VH4-34's interaction partner (e.g., an I / i carbohydrate, a glycoprotein having N-acetyllactosamine (e.g., CD45 isoform B220), a lactosamine moiety, and / or antibody 9G4) to VH4-34. In some embodiments, the VH4-34 antigen-binding molecule replaces VH4-34's interaction partner (e.g., an I / i carbohydrate, a glycoprotein having N-acetyllactosamine (e.g., CD45 isoform B220), a lactosamine moiety, and / or antibody 9G4) from a complex containing VH4-34 and its interaction partner. In some embodiments, the VH4-34 antigen-binding molecule does not bind to a complex containing VH4-34 and its interaction partner (e.g., an I / i carbohydrate, a glycoprotein having N-acetyllactosamine (e.g., CD45 isoform B220), a lactosamine moiety and / or antibody 9G4).

[0311]

[0341] The ability of a given antigen-binding molecule to inhibit the interaction between two species can be determined, for example, by analyzing the interaction in the presence of the given antigen-binding molecule or after incubation with one or both of its interaction partners. A suitable assay for determining whether a given antigen-binding molecule can inhibit the interaction between two interaction partners is a competitive ELISA assay. Antigen-binding molecules capable of inhibiting a given interaction (e.g., between VH4-34 and its interaction partner) are identified by observing a reduction / decrease in the level of interaction between the interaction partners in the presence of the VH4-34 antigen-binding molecule or after incubation with one or both of its interaction partners, compared to the level of interaction in the absence of the VH4-34 antigen-binding molecule (or in the presence of a suitable control antigen-binding molecule known not to inhibit the relevant interaction). Suitable analyses can be performed in vitro, for example, using recombinant interaction partners or cells expressing the interaction partners. Cells expressing interaction partners may do so endogenously or from nucleic acids introduced into the cells. For the purposes of such assays, one or both of the interaction partners and / or the VH4-34 antigen-binding molecule may be labeled or used together with a detectable entity for the purpose of detecting and / or measuring the level of interaction.

[0312]

[0342] In some embodiments, the antigen-binding molecule facilitates the interaction between a peptide / polypeptide / polypeptide complex containing VH4-34 and / or VH4-34 (e.g., the antigen-binding molecule) and an interaction partner of VH4-34 (e.g., an I / i carbohydrate, a glycoprotein having N-acetyllactosamine (e.g., CD45 isoform B220), a lactosamine moiety, and / or antibody 9G4) in a given assay, thereby binding the VH4-34 antigen. Inhibition to less than 1x the level of interaction observed in the absence of the compatible molecule or in the presence of an equal amount of a suitable control antigen-binding molecule, e.g., ≤0.99x, ≤0.95x, ≤0.9x, ≤0.85x, ≤0.8x, ≤0.75x, ≤0.7x, ≤0.65x, ≤0.6x, ≤0.55x, ≤0.5x, ≤0.45x, ≤0.4x, ≤0.35x, ≤0.3x, ≤0.25x, ≤0.2x, ≤0.15x, ≤0.1x, ≤0.05x, or ≤0.01x.

[0313]

[0343] In some embodiments, the antigen-binding molecule promotes (i.e., upregulates, enhances) cell death in cells encoding / containing / expressing VH4-34 and / or a peptide / polypeptide / polypeptide complex (e.g., the antigen-binding molecule) containing VH4-34.

[0314]

[0344] In some embodiments, antigen-binding molecules can reduce the number / percentage of cells encoding / containing / expressing VH4-34 and / or a peptide / polypeptide / polypeptide complex (e.g., antigen-binding molecule) containing VH4-34. In some embodiments, antigen-binding molecules can deplete / enhance such cells. Such cells may be cells of a B cell lineage.

[0315]

[0345] In some embodiments, the antigen-binding molecule reduces the number / percentage of cells encoding / containing / expressing a VH4-34 and / or VH4-34-containing peptide / polypeptide / polypeptide complex (e.g., antigen-binding molecule) to less than 1x of the number / percentage of such cells observed in a given assay in the absence of the VH4-34 antigen-binding molecule or in the presence of an equal amount of a suitable control antigen-binding molecule, for example, ≤0.99x, ≤0.95x, ≤0.9x, ≤0.85x, ≤0.8x, ≤0.75x, ≤0.7x, ≤0.65x, ≤0.6x, ≤0.55x, ≤0.5x, ≤0.45x, ≤0.4x, ≤0.35x, ≤0.3x, ≤0.25x, ≤0.2x, ≤0.15x, ≤0.1x, ≤0.05x, or ≤0.01x.

[0316]

[0346] The antigen-binding molecule may comprise one or more moieties that facilitate the reduction of the number / percentage of cells encoding / containing / expressing VH4-34 and / or a peptide / polypeptide / polypeptide complex (e.g., the antigen-binding molecule) containing VH4-34. For example, the antigen-binding molecule may comprise, for example, an Fc region and / or a drug moiety.

[0317]

[0347] The Fc region provides interactions with Fc receptors and other molecules of the immune system to produce functional effects. The IgG Fc-mediated effector function is outlined, for example, Jefferis et al., Immunol Rev 1998 163:59-76 (which is incorporated herein by reference in its entirety), and results from the interaction between the Fc region and Fc receptors expressed by immune cells, leading to Fc-mediated mobilization and activation of immune cells (e.g., macrophages, dendritic cells, neutrophils, basophils, eosinophils, platelets, mast cells, NK cells, and T cells), mobilization of complement pathway components by binding of the Fc region to the complement protein C1q, and consequent activation of the complement cascade. Fc-mediated functions include Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), complement-dependent cell-mediated cytotoxicity (CDC), membrane invasion complex (MAC) formation, cell degranulation, cytokine and / or chemokine production, and antigen processing and presentation.

[0318]

[0348] In some embodiments, the antigen-binding molecule includes an Fc region capable of promoting / directing one or more of ADCC, ADCP, and CDC to cells encoding / containing / expressing VH4-34 and / or a peptide / polypeptide / polypeptide complex (e.g., an antigen-binding molecule) (e.g., cells that encode / containing / express VH4-34 and / or a peptide / polypeptide / polypeptide complex (e.g., an antigen-binding molecule) on the cell surface), and / or promoting MAC formation or degranulation in cells encoding / containing / expressing VH4-34 and / or a peptide / polypeptide / polypeptide complex (e.g., an antigen-binding molecule) (e.g., cells that encode / containing / express VH4-34 and / or a peptide / polypeptide / polypeptide complex (e.g., an antigen-binding molecule) on the cell surface).

[0319]

[0349] In some embodiments, antigen-binding molecules can promote / direct ADCC in cells encoding / containing / expressing VH4-34 and / or VH4-34-containing peptide / polypeptide / polypeptide complexes (e.g., antigen-binding molecules).

[0320]

[0350] In some embodiments, antigen-binding molecules can promote / direct ADCP in cells encoding / containing / expressing VH4-34 and / or a peptide / polypeptide / polypeptide complex (e.g., antigen-binding molecule) containing VH4-34.

[0321]

[0351] The ability of antigen-binding molecules to promote / direct ADCC and / or ADCP can be evaluated, for example, by incubating target cells with the VH4-34 antigen-binding molecule and Jurkat-Lucia® NFAT cells (InvivoGen) or macrophages. Suitable methods are described herein.

[0322]

[0352] In some embodiments, the antigen-binding molecule includes a drug moiety. The VH4-34 antigen-binding molecule can be conjugated to the drug moiety. Antibody-drug conjugates are outlined, for example, in Parslow et al., Biomedicines. 2016 Sept; 4(3): pp. 14 (which is incorporated herein by reference in its entirety). In some embodiments, the drug moiety is or includes a cytotoxic agent, and as a result, the VH4-34 antigen-binding molecule exhibits cytotoxicity against cells encoding / containing / expressing VH4-34 and / or a peptide / polypeptide / polypeptide complex (e.g., the antigen-binding molecule) (e.g., cells encoding / containing / expressing VH4-34 and / or a peptide / polypeptide / polypeptide complex (e.g., the antigen-binding molecule) on the cell surface). In some embodiments, the drug moiety is or includes a chemotherapeutic agent.

[0323]

[0353] In some embodiments, the antigen-binding molecule includes a portion that involves immune cells. In some embodiments, the VH4-34 antigen-binding molecule includes a CD3 polypeptide-binding portion (e.g., an antigen-binding domain capable of binding to a CD3 polypeptide).

[0324]

[0354] In some embodiments, antigen-binding molecules can promote / direct T cell-mediated cytolytic activity in cells encoding / containing / expressing VH4-34 and / or VH4-34-containing peptide / polypeptide / polypeptide complexes (e.g., antigen-binding molecules).

[0325]

[0355] In some embodiments, the antigen-binding molecule reduces the number / percentage of cells encoding / containing / expressing a VH4-34 and / or VH4-34-containing peptide / polypeptide / polypeptide complex (e.g., antigen-binding molecule) to less than 1x the number / percentage of cells observed in a given assay in the absence of the VH4-34 antigen-binding molecule or in the presence of an equal amount of a suitable control antigen-binding molecule, for example, ≤0.99x, ≤0.95x, ≤0.9x, ≤0.85x, ≤0.8x, ≤0.75x, ≤0.7x, ≤0.65x, ≤0.6x, ≤0.55x, ≤0.5x, ≤0.45x, ≤0.4x, ≤0.35x, ≤0.3x, ≤0.25x, ≤0.2x, ≤0.15x, ≤0.1x, ≤0.05x, or ≤0.01x.

[0326]

[0356] In some embodiments, the antigen-binding molecule increases the level of cell death of cells encoding / containing / expressing VH4-34 and / or VH4-34-containing peptide / polypeptide / polypeptide complexes (e.g., the antigen-binding molecule) to more than 1x, e.g., ≥1.5x, ≥2x, ≥3x, ≥4x, ≥5x, ≥6x, ≥7x, ≥8x, ≥9x, ≥10x, ≥15x, ≥20x, ≥30x, ≥40x, or ≥50x the level of cell death observed in a given assay in the absence of the VH4-34 antigen-binding molecule or in the presence of an equal amount of a suitable control antigen-binding molecule.

[0327]

[0357] In some embodiments, antigen-binding molecules can reduce / decrease the levels of VH4-34 and / or VH4-34-containing peptide / polypeptide / polypeptide complexes (e.g., antigen-binding molecules) (or cells expressing such articles) in subjects to whom the VH4-34 antigen-binding molecule is administered. In some embodiments, antigen-binding molecules can reduce / decrease the levels of VH4-34 and / or VH4-34-containing peptide / polypeptide / polypeptide complexes (e.g., antigen-binding molecules) (or cells expressing such articles) in blood / plasma / serum, for example, in subjects to whom the VH4-34 antigen-binding molecule is administered.

[0328]

[0358] In some embodiments, the antigen-binding molecule reduces the serum half-life of VH4-34 and / or peptide / polypeptide / polypeptide complexes containing VH4-34 (e.g., the antigen-binding molecule) (or cells expressing such articles). In some embodiments, the antigen-binding molecule enhances / accelerates the clearance / efflux of VH4-34 and / or peptide / polypeptide / polypeptide complexes containing VH4-34 (e.g., the antigen-binding molecule) (or cells expressing such articles).

[0329]

[0359] In some embodiments, the antigen-binding molecule reduces the level of VH4-34 and / or VH4-34-containing peptide / polypeptide / polypeptide complex (e.g., the antigen-binding molecule) (or cells expressing such an article) in the target blood / plasma / serum to less than 1x the level observed in the absence of the VH4-34 antigen-binding molecule or after administration of an equal amount of a suitable control antigen-binding molecule, for example, ≤0.99x, ≤0.95x, ≤0.9x, ≤0.85x, ≤0.8x, ≤0.75x, ≤0.7x, ≤0.65x, ≤0.6x, ≤0.55x, ≤0.5x, ≤0.45x, ≤0.4x, ≤0.35x, ≤0.3x, ≤0.25x, ≤0.2x, ≤0.15x, ≤0.1x, ≤0.05x, or ≤0.01x. Chimeric antigen receptor (CAR)

[0360] This disclosure also provides a chimeric antigen receptor (CAR) comprising the antigen-binding polypeptide or polypeptide of this disclosure.

[0330]

[0361] CARs are recombinant receptors that provide both antigen-binding and T-cell activation functions. The structure and manipulation of CARs are outlined, for example, in Dotti et al., Immunol Rev (2014) 257(1), which is incorporated herein by reference in its entirety. CARs comprise an antigen-binding region linked to a cell membrane anchoring region and a signaling region. An optional hinge region can provide separation between the antigen-binding region and the cell membrane anchoring region and may act as a mobile linker.

[0331]

[0362] The CARs of this disclosure include an antigen-binding region comprising or consisting of the VH4-34 antigen-binding molecule of this disclosure, or a polypeptide in accordance with this disclosure.

[0332]

[0363] The cell membrane anchor region is provided between the antigen-binding region and the signaling region of the CAR, providing fixation of the CAR to the cell membrane of the cell expressing the CAR, placing the antigen-binding region in the extracellular space and the signaling region in the intracellular space. In some embodiments, the CAR includes a cell membrane anchor region comprising, or comprising an amino acid sequence comprising, one transmembrane amino acid sequence of CD3-ζ, CD4, CD8, or CD28. As used herein, the region "derived from" a reference amino acid sequence comprises an amino acid sequence having at least 60% sequence identity with respect to the reference sequence, for example, at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0333]

[0364] The signaling region of CAR enables T cell activation. The CAR signaling region may contain the amino acid sequence of the intracellular domain of CD3-ζ, which provides an immune receptor tyrosine-based activation motif (ITAM) for phosphorylation and activation of CAR-expressing T cells. Signaling regions containing sequences of other ITAM-containing proteins, such as FcγRI, have also been used in CARs (Haynes et al., 2001 J Immunol 166(1):182-187). The CAR signaling region may also contain a costimulatory sequence derived from the signaling region of a costimulatory molecule to facilitate activation of CAR-expressing T cells upon binding to a target protein. Suitable costimulatory molecules include CD28, OX40, 4-1BB, ICOS, and CD27. In some cases, CARs are engineered to provide costimulation of different intracellular signaling pathways. For example, CD28 costimulation-associated signaling preferentially activates the phosphatidylinositol 3-kinase (PI3K) pathway, while 4-1BB-mediated signaling is via the TNF receptor-associated factor (TRAF) adapter protein. Therefore, the signaling region of a CAR may contain costimulatory sequences derived from the signaling regions of more than one costimulatory molecule. In some embodiments, the CARs of this disclosure contain one or more costimulatory sequences comprising, or derived from, the amino acid sequences of one or more intracellular domains of CD28, OX40, 4-1BB, ICOS, and CD27.

[0334]

[0365] An optional hinge region may provide separation between the antigen-binding domain and the transmembrane domain and may act as a mobile linker. The hinge region may be derived from IgG1. In some embodiments, the CAR of this disclosure includes, comprises, or comprises an amino acid sequence derived from, an amino acid sequence of the hinge region of IgG1.

[0335]

[0366] Cells containing CARs according to this disclosure are also provided. CARs can be used to generate immune cells that express CARs, such as CAR-T or CAR-NK cells. Manipulating and introducing CARs into immune cells can be done during in vitro culture.

[0336]

[0367] The antigen-binding region of the CAR of this disclosure may be provided in any suitable format, such as scFv, scFab, etc. nucleic acids and vectors

[0368] This disclosure provides nucleic acids or multiple nucleic acids encoding antigen-binding molecules, polypeptides, or CARs according to this disclosure. In some embodiments, the nucleic acids include or consist of DNA and / or RNA.

[0337]

[0369] This disclosure also provides a vector or multiple vectors comprising nucleic acids or multiple nucleic acids in accordance with this disclosure.

[0370] Nucleic acids and vectors may be provided in purified or isolated forms, i.e., from other nucleic acids or naturally occurring biological materials.

[0338]

[0371] Nucleotide sequences may be contained in vectors, such as expression vectors. “Vector,” as used herein, is a nucleic acid molecule used as a medium for introducing exogenous nucleic acids into cells. A vector may be for the expression of nucleic acids in cells. Such a vector may include a promoter sequence operably ligated to a nucleotide sequence encoding the sequence to be expressed. A vector may also include termination codons and expression enhancers. Any suitable vector, promoter, enhancer, and termination codon known in the art may be used to express peptides or polypeptides from vectors according to this disclosure.

[0339]

[0372] The term "operably linked" may include situations in which a selected nucleic acid sequence and a regulatory nucleic acid sequence (e.g., a promoter and / or enhancer) are covalently linked in such a way that the expression of the nucleic acid sequence is under the influence or control of the regulatory sequence (thereby forming an expression cassette). Thus, a regulatory sequence is operably linked to a selected nucleic acid sequence if it is capable of influencing the transcription of the nucleic acid sequence. The resulting transcript(s) can then be translated into the desired peptide(s) / polypeptides(s).

[0340]

[0373] Suitable vectors include plasmids, binary vectors, DNA vectors, mRNA vectors, viral vectors (e.g., gamma retrovirus vectors (e.g., mouse leukemia virus (MLV)-derived vectors), lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, vaccinia virus vectors, and herpesvirus vectors), transposon-based vectors, and artificial chromosomes (e.g., yeast artificial chromosomes).

[0341]

[0374] In some embodiments, the vector is a eukaryotic vector, for example, a vector containing elements necessary for protein expression from the vector in eukaryotic cells. In some embodiments, the vector is a mammalian vector, for example, a cytomegalovirus (CMV) or SV40 promoter for driving protein expression.

[0342]

[0375] The polypeptide components of antigen-binding molecules may be encoded by different nucleic acids from multiple nucleic acids, or by different vectors from multiple vectors. Cells that encode / contain / express VH4-34 antigen-binding molecules and polypeptides.

[0376] This disclosure also provides cells containing or expressing antigen-binding molecules, polypeptides, or CARs in accordance with this disclosure. Furthermore, this disclosure also provides cells containing or expressing nucleic acids, multiple nucleic acids, vectors, or multiple vectors in accordance with this disclosure.

[0343]

[0377] The cells may be eukaryotic cells, for example, mammalian cells. Mammals may be primates (rhesus macaques, crab-eating macaques, non-human primates, or humans) or non-human mammals (for example, rabbits, guinea pigs, rats, mice or other rodents (including any animal in the order Rodentia), cats, dogs, pigs, sheep, goats, cattle (including cows, for example, dairy cows, or any animal in the order Artiodactyla), horses (including any animal in the order Perissodactyla), donkeys, and non-human primates).

[0344]

[0378] In some embodiments, the cells are or are derived from cell types commonly used for polypeptide expression for use in human therapy. Exemplary cells are described, for example, in Kunert and Reinhart, Appl Microbiol Biotechnol. (2016) 100:3451-3461 (which is incorporated herein by reference in its entirety), and include, for example, CHO, HEK 293, PER.C6, NS0, and BHK cells. In some embodiments, the cells are or are derived from CHO cells.

[0345]

[0379] The disclosure also provides a method for generating cells comprising nucleic acids or vectors, comprising the step of introducing nucleic acids, multiple nucleic acids, vectors, or multiple vectors into cells. In some embodiments, the step of introducing isolated nucleic acids or vectors into cells comprises transformation, transfection, electroporation, or transduction (e.g., retroviral transduction).

[0346]

[0380] This disclosure also provides a method for generating cells that express / contain antigen-binding molecules, polypeptides, or CARs according to this disclosure, the method comprising the step of introducing nucleic acids, multiple nucleic acids, vectors, or multiple vectors into cells. In some embodiments, the method further comprises the step of culturing the cells under conditions suitable for the expression of nucleic acids(s) or vectors(s) by the cells. In some embodiments, the method is carried out in vitro.

[0347]

[0381] This disclosure also provides cells obtained or that can be obtained by methods relating to this disclosure. Production of VH4-34 antigen-binding molecules and polypeptides

[0382] Antigen-binding molecules and polypeptides can be prepared according to methods for producing polypeptides known to those skilled in the art.

[0348]

[0383] Polypeptides can be prepared by chemical synthesis, for example, liquid-phase or solid-phase synthesis. For example, peptides / polypeptides can be synthesized using the methods described, for example, Chandrudu et al., Molecules (2013), 18:4373-4388, which are incorporated herein by reference in their entirety.

[0349]

[0384] Alternatively, antigen-binding molecules and polypeptides can be produced by recombinant expression. Molecular biology techniques suitable for recombinant polypeptide production are well known in the art, including those described in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th edition), Cold Spring Harbor Press, 2012, and Nat Methods. (2008); 5(2): pp. 135-146, both of which are incorporated herein by reference in their entirety. Methods for recombinant antigen-binding molecules are also described in Frenzel et al., Front Immunol. (2013); 4: 217, and Kunert and Reinhart, Appl Microbiol Biotechnol. (2016) 100: pp. 3451-3461, both of which are incorporated herein by reference in their entirety.

[0350]

[0385] In some cases, the VH4-34 antigen-binding molecules of this disclosure consist of more than one polypeptide chain. In such cases, the generation of the VH4-34 antigen-binding molecule may involve the transcription and translation of more than one polypeptide and subsequent association of the polypeptide chains to form the VH4-34 antigen-binding molecule.

[0351]

[0386] Any cell suitable for polypeptide expression may be used for recombinant generation according to this disclosure. The cell may be a prokaryote or a eukaryote. In some embodiments, the cell may be a prokaryotic cell, e.g., an archaeal or bacterial cell. In some embodiments, the bacterium may be a Gram-negative bacterium, e.g., a bacterium of the Enterobacteriaceae family, e.g., Escherichia coli. In some embodiments, the cell may be a eukaryotic cell, e.g., a yeast cell, plant cell, insect cell, or mammalian cell, e.g., the cells described above herein.

[0352]

[0387] In some cases, certain prokaryotic cells are not prokaryotic because they do not allow the same folding or post-translational modifications as eukaryotic cells. Furthermore, extremely high expression levels are possible in eukaryotes, and proteins can be more easily purified from eukaryotes using appropriate tags. Specific plasmids that enhance the secretion of proteins into culture media can also be utilized.

[0353]

[0388] In some embodiments, polypeptides are prepared by cell-free protein synthesis (CFPS) according to a system described, for example, Zemella et al. Chembiochem (2015) 16(17):2420-2431, which is incorporated herein by reference in its entirety.

[0354]

[0389] Production may involve culturing or fermenting eukaryotic cells modified to express the target polypeptide(s). Culturing or fermentation may be carried out in a bioreactor provided with adequate supply of nutrients, air / oxygen and / or growth factors. Secreted proteins may be collected by isolating the secreted polypeptide(s) by separating the culture medium / fermentation medium from the cells, extracting the protein content, and separating individual proteins. Culturing, fermentation, and isolation techniques are well known to those skilled in the art and are described, for example, in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th edition; incorporated herein by reference above).

[0355]

[0390] A bioreactor comprises one or more vessels in which cells can be cultured. Culture in a bioreactor may occur continuously with a continuous flow of reactants into the reactor and a continuous flow of cultured cells out of the reactor. Alternatively, culture may occur in batches. The bioreactor monitors and controls environmental conditions such as pH, oxygen, flow rates into and out of the vessels, and agitation within the vessels to provide optimal conditions for the cultured cells.

[0356]

[0391] After culturing cells expressing a VH4-34 antigen-binding molecule / polypeptide(s), the target polypeptide(s) may be isolated. Any suitable method known in the art for isolating proteins from cells may be used. It may be necessary to separate the cells from the nutrient medium to isolate the polypeptide(s). If the polypeptide(s) are secreted from the cells, the cells may be separated from the culture medium containing the secreted polypeptide(s) of interest by centrifugation. If the target polypeptide(s) accumulate within the cells, protein isolation may include centrifugation to separate the cells from the cell culture medium, treatment with lysis buffer for the cell pellet, and cell disruption by, for example, sonication, rapid freeze-thaw, or osmotic lysis.

[0357]

[0392] Next, it may be desirable to isolate the target polypeptide(s) from the supernatant or culture medium, which may contain other protein and non-protein components. A common approach to separating protein components from the supernatant or culture medium is by precipitation. Different soluble proteins precipitate at different concentrations of precipitating agents, such as ammonium sulfate. For example, water-soluble proteins are extracted at low concentrations of the precipitating agent. Thus, different soluble proteins can be identified by adding precipitating agents at different progressively increasing concentrations. Dialysis may then be used to remove ammonium sulfate from the separated proteins.

[0358]

[0393] Other methods for identifying different proteins are known in the art, and include, for example, ion-exchange chromatography and size chromatography. These may be used as an alternative to precipitation or performed following precipitation.

[0359]

[0394] Once the target polypeptide(s) are isolated from the culture, it may be desirable or necessary to concentrate them. Several methods for concentrating proteins, such as ultrafiltration or lyophilization, are known in the art. composition

[0395] This disclosure also provides compositions comprising VH4-34 antigen-binding molecules, polypeptides, CARs, nucleic acids, expression vectors, and cells as described herein.

[0360]

[0396] The VH4-34 antigen-binding molecules, polypeptides, CARs, nucleic acids, expression vectors, and cells described herein may be formulated as pharmaceutical compositions or pharmaceuticals for clinical use and may contain pharmaceutically acceptable carriers, diluents, excipients, or adjuvants. The compositions may be formulated for administration via topical, parenteral, systemic, intracavitary, intravenous, intra-arterial, intramuscular, intraarachnoid, intraocular, intraconjunctival, intratumoral, subcutaneous, intradermal, intraarachnoid, oral, or transdermal routes, which may include injection or infusion.

[0361]

[0397] Suitable formulations may contain VH4-34 antigen-binding molecules in sterile or isotonic media. Pharmaceuticals and pharmaceutical compositions may be formulated in the form of fluids, including gels. Fluid formulations may be formulated for administration by injection or infusion (e.g., via cannula) to selected areas of the human or animal body.

[0362]

[0398] In some embodiments, the composition is formulated for injection or infusion into, for example, a blood vessel, tissue / organ of interest.

[0399] This disclosure also provides methods for producing pharmaceutically useful compositions, such methods may comprise one or more steps selected from: producing antigen-binding molecules, polypeptides, CARs, nucleic acids (or more), expression vectors (or more) or cells described herein; isolating antigen-binding molecules, polypeptides, CARs, nucleic acids (or more), expression vectors (or more) or cells described herein; and / or mixing antigen-binding molecules, polypeptides, CARs, nucleic acids (or more), expression vectors (or more) or cells described herein with a pharmaceutically acceptable carrier, adjuvant, excipient or diluent.

[0363]

[0400] For example, further aspects of the present disclosure relate to methods for formulating or producing pharmaceuticals or pharmaceutical compositions for use in the treatment of a disease or condition (e.g., cancer), the methods comprising the step of formulating a pharmaceutical composition or pharmaceutical by mixing an antigen-binding molecule, polypeptide, CAR, nucleic acid (or more thereof), expression vector (or more thereof) or cell as described herein with a pharmaceutically acceptable carrier, adjuvant, excipient or diluent. Therapeutic and prophylactic applications

[0401] The VH4-34 antigen-binding molecules, polypeptides, CARs, nucleic acids, expression vectors, cells, and compositions described herein are used in therapeutic and prophylactic methods.

[0364]

[0402] This disclosure provides antigen-binding molecules, polypeptides, CARs, nucleic acids (or more), expression vectors (or more), cells, or compositions described herein for use in methods of medical treatment or prevention. It also provides the use of antigen-binding molecules, polypeptides, CARs, nucleic acids (or more), expression vectors (or more), cells, or compositions described herein in the manufacture of pharmaceuticals for the treatment or prevention of disease or condition. Furthermore, it provides methods for treating or preventing disease or condition, comprising the step of administering a therapeutically or prophylactically effective amount of antigen-binding molecules, polypeptides, CARs, nucleic acids (or more), expression vectors (or more), cells, or compositions described herein to a target.

[0365]

[0403] Therapeutic or preventive interventions relating to this disclosure may be effective in reducing the development or progression of a disease or condition, alleviating the symptoms of a disease or condition, or reducing the pathology of a disease or condition. Interventions may be effective in preventing the progression of a disease or condition, for example, preventing the worsening of a disease or condition, or slowing its rate of development. In some embodiments, interventions may lead to improvement of a disease or condition, for example, a reduction in the symptoms of a disease or condition, or a reduction in several other factors correlated with the severity / activity of a disease or condition. In some embodiments, interventions may prevent the development of a disease or condition into later stages (e.g., more severe or chronic stages).

[0366]

[0404] For example, the terms “develop,” “to develop,” and “development” in relation to a disorder, as used herein, refer to both the onset of the disease and the progression, exacerbation, or worsening of the disease state / related conditions.

[0367]

[0405] It will be acknowledged that the articles of this disclosure may be used to treat / prevent any disease or condition for which a therapeutic or preventive benefit would be obtained from reducing the levels of VH4-34 and / or VH4-34-containing peptide / polypeptide / polypeptide complexes (e.g., antigen-binding molecules), and / or reducing the number / percentage and / or activity of cells encoding / containing / expressing VH4-34 and / or VH4-34-containing peptide / polypeptide / polypeptide complexes (e.g., antigen-binding molecules).

[0368]

[0406] Therapies and preventive measures according to the embodiments and aspects disclosed herein primarily relate to diseases / conditions characterized by the expression of VH4-34, for example, in the form of a peptide / polypeptide / polypeptide complex (e.g., an antigen-binding molecule) containing VH4-34.

[0369]

[0407] Diseases or conditions characterized by VH4-34 expression may be associated, for example, with VH4-34 and / or VH4-34-containing peptide / polypeptide / polypeptide complexes (e.g., antigen-binding molecules) in blood / serum / plasma. Diseases or conditions characterized by VH4-34 expression may also be associated, for example, with VH4-34-containing IgG and / or IgM in blood / serum / plasma.

[0370]

[0408] Diseases or conditions characterized by VH4-34 expression may be associated with cells encoding / containing / expressing VH4-34 and / or VH4-34-containing peptide / polypeptide / polypeptide complexes (e.g., antigen-binding molecules). Diseases or conditions characterized by VH4-34 expression may be associated with cells encoding / containing / expressing VH4-34-containing IgG and / or IgM.

[0371]

[0409] In some embodiments, the disease or condition is a disease or condition in a subject that includes, for example, VH4-34 and / or a peptide / polypeptide / polypeptide complex (e.g., an antigen-binding molecule) containing VH4-34 in its blood / serum / plasma. In some embodiments, the disease or condition is a disease or condition in a subject that includes, for example, IgG and / or IgM containing VH4-34 in its blood / serum / plasma.

[0372]

[0410] In some embodiments, the disease or condition is a disease or condition in a subject that includes, for example, cells in its blood that encode / contain / express a peptide / polypeptide / polypeptide complex (e.g., an antigen-binding molecule) containing VH4-34. In some embodiments, the disease or condition is a disease or condition in a subject that includes, for example, cells in its blood that encode / contain / express IgG and / or IgM containing VH4-34.

[0373]

[0411] For example, a disease or condition may be a disease or condition in which cells encoding / containing / expressing VH4-34 and / or VH4-34-containing peptide / polypeptide / polypeptide complexes (e.g., antigen-binding molecules), and / or VH4-34 and / or VH4-34-containing peptide / polypeptide / polypeptide complexes (e.g., antigen-binding molecules) are pathologically involved.

[0374]

[0412] A disease or condition may be one in which VH4-34 and / or a peptide / polypeptide / polypeptide complex (e.g., an antigen-binding molecule) is positively associated with the onset, development, or progression of the disease or condition and / or the severity of one or more symptoms of the disease or condition, or in which VH4-34 and / or a peptide / polypeptide / polypeptide complex (e.g., an antigen-binding molecule) is a risk factor for the onset, development, or progression of the disease or condition.

[0375]

[0413] A disease or condition is one in which cells encoding / containing / expressing VH4-34 and / or cells encoding / containing / expressing peptide / polypeptide / polypeptide complexes (e.g., antigen-binding molecules) containing VH4-34 are positively associated with the onset, development, or progression of the disease or condition and / or the severity of one or more symptoms of the disease or condition, or such cells may be risk factors for the onset, development, or progression of the disease or condition.

[0376]

[0414] In some embodiments, the disease or condition being treated / prevented is characterized by an increase in the levels of VH4-34 and / or VH4-34-containing peptide / polypeptide / polypeptide complexes (e.g., antigen-binding molecules) in the subject or a sample obtained therefrom (e.g., a blood-derived sample) compared to the level in the absence of the disease or condition. In some embodiments, the disease or condition is characterized by an increase in the number / proportion of cells encoding / containing / expressing VH4-34 and / or cells encoding / containing / expressing VH4-34-containing peptide / polypeptide / polypeptide complexes (e.g., antigen-binding molecules) in the subject or a sample obtained therefrom (e.g., a blood-derived sample), compared to the number / proportion of such cells in the absence of the disease or condition.

[0377]

[0415] As per the preceding paragraph, an increase in the levels of VH4-34 and / or peptide / polypeptide / polypeptide complexes (e.g., antigen-binding molecules) containing VH4-34, and / or an increase in the number / percentage of cells encoding / containing / expressing VH4-34 and / or peptide / polypeptide / polypeptide complexes (e.g., antigen-binding molecules) containing VH4-34, may occur in tissues and / or organs where one or more symptoms of a disease or condition appear.

[0378]

[0416] Therapeutic or prophylactic interventions according to this disclosure may, for example, achieve a reduction in the levels of VH4-34 and / or VH4-34-containing peptide / polypeptide / polypeptide complexes (e.g., antigen-binding molecules) in the blood / plasma / serum of the treated subject. In some embodiments, the intervention may, for example, achieve a reduction in the levels of VH4-34-containing IgG and / or IgM in the blood / plasma / serum of the treated subject. In some embodiments, the intervention may, for example, achieve a reduction in the levels of VH4-34-containing IgG in the blood / plasma / serum of the treated subject. In some embodiments, the intervention may, for example, achieve a reduction in the levels of VH4-34-containing IgM in the blood / plasma / serum of the treated subject.

[0379]

[0417] Therapeutic or prophylactic interventions pursuant to this disclosure may achieve, for example, a reduction in the number / proportion of cells encoding / containing / expressing VH4-34 (e.g., B cells) and / or cells encoding / containing / expressing VH4-34-containing peptides / polypeptides / polypeptide complexes (e.g., antigen-binding molecules) in a treated subject (e.g., in the peripheral blood of the treated subject). In some embodiments, the intervention may achieve a reduction in the number / proportion of cells encoding / containing / expressing VH4-34-containing IgG and / or IgM (e.g., B cells) in a treated subject. In some embodiments, the intervention may achieve a reduction in the number / proportion of cells encoding / containing / expressing VH4-34-containing IgG (e.g., B cells) in a treated subject. In some embodiments, the intervention may achieve a reduction in the number / proportion of cells encoding / containing / expressing VH4-34-containing IgM (e.g., B cells) in a treated subject.

[0380]

[0418] In some embodiments, the disease or condition to be treated / prevented is characterized by a reduction in the number / percentage of cells expressing the CD45 B220 glycoform, for example, compared to the number / percentage of such cells in the absence of the disease or condition. In some embodiments, the disease or condition is characterized by B-cell lymphopenia. In some embodiments, the disease or condition is characterized by a reduction in red blood cell count and / or increased red blood cell aggregation, for example, compared to levels observed in the absence of the disease or condition. In some embodiments, the disease or condition is characterized by hemolysis and / or hemolytic anemia.

[0381]

[0419] Diseases / conditions characterized by the expression of VH4-34 (e.g., in the form of a peptide / polypeptide / polypeptide complex containing VH4-34, e.g., an antigen-binding molecule) include systemic lupus erythematosus (SLE), hairy cell leukemia (HCL), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), cold agglutinin disease, and Wiscott-Aldrich syndrome.

[0382]

[0420] In SLE, antibodies containing VH4-34 are thought to behave as anti-lymphocyte autoantibodies (see, for example, Cappione et al., J Immunol. (2004) 172(7): pp. 4298-307). VH4-34 binds to N-acetyllactosamine in the B220 glycoform of CD45 expressed by certain naive B cells, promoting its depletion. The proportion of IgG containing VH4-34 as part of total IgG in the serum of SLE patients has been shown to increase dramatically during SLE flares (Pugh-Bernard et al., J Clin Invest. (2001) 108(7): pp. 1061-70, Jenks et al., Arthritis Rheum. (2013) 65(12): pp. 3165-3175). In some embodiments, the VH4-34 antigen-binding molecules of this disclosure can alleviate symptoms of SLE, for example, by preventing / reducing B cell depletion. In some embodiments, the VH4-34 antigen-binding molecules of this disclosure can alleviate / prevent / reduce fatigue, skin rash, fever, pain, swelling, and / or inflammation associated with SLE.

[0383]

[0421] In cold agglutinin disease (CAD), VH4-34 binds to N-acetyllactosamine in I / i carbohydrates expressed by erythrocytes, causing their aggregation and destruction. VH4-34 antibodies are also involved in the pathogenesis of Wiscott-Aldrich syndrome (see, e.g., Kolhatkar et al., J Exp Med. (2015) 212(10): pp. 1663-1677). In some embodiments, the VH4-34 antigen-binding molecules of this disclosure can alleviate symptoms of CAD, for example, by reducing / preventing erythrocyte aggregation and / or destruction. In some embodiments, the VH4-34 antigen-binding molecules of this disclosure can alleviate / prevent / reduce fatigue, dizziness, headache, sweating, shortness of breath (dyspnea), rapid heart rate (tachycardia), and / or jaundice associated with CAD.

[0384]

[0422] VH4-34+ hairy cell leukemia (HCL) B cells have been shown to define a subtype of HCL known as hairy cell leukemia subtype (HCL-V). HCL-V is characterized by a particularly poor prognosis, with a median progression-free survival of only 5.1 months compared to 22.8 months for classical HCL (Arons et al., Blood (2009) 114(21): pp. 4687-95). See also Xi et al., Blood (2012) 119(14): pp. 3330-3332.

[0385]

[0423] IGHV4-34 is the most frequently used IGHV gene in chronic lymphocytic leukemia (CLL) cases expressing B-cell receptor immunoglobulin (BCR IG) with somatic high-frequency mutations in the IGHV gene (M-CLL; Xochelli et al., Clin Cancer Res. (2017) 23(17): pp. 5292-5301).

[0386]

[0424] Compared to approximately 10% of germinal center B-cell diffuse large B-cell lymphoma (GBC-DLBCL) and less than 4% of normal B cells, approximately 30% of activated B-cell diffuse large B-cell lymphoma (ABC-DLBCL) B cells encode VH4-34 BCR (Young et al., PNAS USA (2015) 112(44):134 pp. 47-54).

[0387]

[0425] In certain embodiments, a method for treating / preventing a disease or condition is described herein, comprising the step of administering a therapeutically effective amount of the VH4-34 antibody described herein. In some embodiments, a method for treating / preventing a disease or condition comprises the step of administering a therapeutically effective amount of a VH4-34 binding molecule that specifically binds to the VH4-34 epitope containing SEQ ID NO: 1 with one of the following amino acid residues: Q at position 32, W at position 33, A at position 49, V at position 50, and Y at position 51 of SEQ ID NO: 1. In some embodiments, a method for treating / preventing a disease or condition comprises the step of administering a therapeutically effective amount of a VH4-34 binding molecule that specifically binds to the VH4-34 epitope containing SEQ ID NO: 1 with one of the following amino acid residues: Q at position 32, W at position 33, A at position 49, V at position 50, and Y at position 51 of SEQ ID NO: 1.

[0388]

[0426] In some embodiments, the disease or condition to be treated / prevented is an autoimmune disease. In some embodiments, the autoimmune disease is selected from the group consisting of autoimmune hemolytic anemia (AIHA), unclassified immunodeficiency (CVID), rheumatoid arthritis, systemic lupus erythematosus (SLE), lupus nephritis, central nervous system (CNS) lupus, cold agglutinin disease (CAD), and Wiscott-Aldrich syndrome. In some embodiments, the autoimmune disease is cold agglutinin disease (CAD). In some embodiments, the autoimmune disease is systemic lupus erythematosus (SLE).

[0389]

[0427] In some embodiments, the disease or condition being treated / prevented is cancer. In some embodiments, cancer is a B-cell malignancy. In some embodiments, B-cell malignancies are selected from the group consisting of B-cell leukemia, Hairy cell leukemia (HCL), Hairy cell leukemia subtype (HCL-V), chronic lymphocytic leukemia (CLL), mutagenetic chronic lymphocytic leukemia (M-CLL), acute lymphoblastic leukemia (ALL), B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), activated B-cell diffuse large B-cell lymphoma (ABC-DLBCL), germinal center B-cell diffuse large B-cell lymphoma (GBC-DLBCL), primary central nervous system (CNS) lymphoma, and mantle cell lymphoma.

[0390]

[0428] In some embodiments, the diseases or conditions characterized by the expression of VH4-34 include B-cell leukemia, Hairy cell leukemia (HCL), Hairy cell leukemia subtype (HCL-V), chronic lymphocytic leukemia (CLL), mutagenetic chronic lymphocytic leukemia (M-CLL), acute lymphoblastic leukemia (ALL), B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), and activated B-cell diffuse large B-cell lymphoma (AB). The following conditions are selected: C-DLBCL), germinal center B-cell diffuse large B-cell lymphoma (GBC-DLBCL), primary central nervous system (CNS) lymphoma, mantle cell lymphoma, autoimmune hemolytic anemia (AIHA), unclassified immunodeficiency (CVID), rheumatoid arthritis, systemic lupus erythematosus (SLE), lupus nephritis, central nervous system (CNS) lupus, cold agglutinin disease (CAD), and Wiscott-Aldrich syndrome.

[0391]

[0429] In some embodiments, the diseases or conditions treated / prevented include B-cell malignancies, B-cell leukemia, Hairy cell leukemia (HCL), Hairy cell leukemia subtype (HCL-V), chronic lymphocytic leukemia (CLL), mutagenetic chronic lymphocytic leukemia (M-CLL), acute lymphoblastic leukemia (ALL), B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), activated B-cell diffuse large B-cell lymphoma (A The following are selected from BC-DLBCL), germinal center B-cell diffuse large B-cell lymphoma (GBC-DLBCL), primary central nervous system (CNS) lymphoma, mantle cell lymphoma, autoimmune hemolytic anemia (AIHA), unclassified immunodeficiency (CVID), rheumatoid arthritis, systemic lupus erythematosus (SLE), lupus nephritis, central nervous system (CNS) lupus, cold agglutinin disease, and Wiscott-Aldrich syndrome. In some embodiments, the disease or condition is systemic lupus erythematosus (SLE), Hairy cell leukemia (HCL), chronic lymphocytic leukemia (CLL), or diffuse large B-cell lymphoma (DLBCL).

[0392]

[0430] The administration of the articles of this disclosure is preferably in a “therapeutically effective” or “preventively effective” amount, which is sufficient to demonstrate a therapeutic or preventive benefit to the subject. The actual amount administered, as well as the rate and time course of administration, will vary depending on the nature and severity of the disease or condition and the specific article being administered. The prescription of treatment, such as the determination of dosage, is the responsibility of the general practitioner and other physicians, and usually takes into account the disease / disorder being treated, the condition of the individual subject, the site of delivery, the method of administration, and other factors known to the physician. Examples of the techniques and protocols describ...

Claims

1. (a) (i) HC-CDR1 having the amino acid sequence of SEQ ID NO: 172, HC-CDR2 having the amino acid sequence of SEQ ID NO: 55, HC-CDR3 having the amino acid sequence of SEQ ID NO: 56 A heavy chain variable (VH) region including, and (ii) LC-CDR1 having the amino acid sequence of SEQ ID NO: 144, LC-CDR2 having the amino acid sequence YAS, LC-CDR3 having the amino acid sequence of SEQ ID NO: 64 Light chain variable (VL) region including, or (b) (i) HC-CDR1 having the amino acid sequence of SEQ ID NO: 142, HC-CDR2 having the amino acid sequence of SEQ ID NO: 55, HC-CDR3 having the amino acid sequence of SEQ ID NO: 56 A heavy chain variable (VH) region including, and (ii) LC-CDR1 having the amino acid sequence of SEQ ID NO: 144, LC-CDR2 having the amino acid sequence YAS, LC-CDR3 having the amino acid sequence of SEQ ID NO: 64 Light chain variable (VL) region including, or (c) (i) HC-CDR1 having the amino acid sequence of SEQ ID NO: 38, HC-CDR2 having the amino acid sequence of SEQ ID NO: 39, HC-CDR3 having the amino acid sequence of SEQ ID NO: 40 A heavy chain variable (VH) region including, and (ii) LC-CDR1 having the amino acid sequence of SEQ ID NO: 46, LC-CDR2 having amino acid sequence AAS, LC-CDR3 having the amino acid sequence of SEQ ID NO: 48 Light chain variable (VL) region including, or (d) (i) HC-CDR1 having the amino acid sequence of SEQ ID NO: 54, HC-CDR2 having the amino acid sequence of SEQ ID NO: 55, HC-CDR3 having the amino acid sequence of SEQ ID NO: 56 A heavy chain variable (VH) region including, and (ii) LC-CDR1 having the amino acid sequence of SEQ ID NO: 62, LC-CDR2 having the amino acid sequence YAS, LC-CDR3 having the amino acid sequence of SEQ ID NO: 64 Light chain variable (VL) region including, or (e) (i) HC-CDR1 having the amino acid sequence of SEQ ID NO: 6, HC-CDR2 having the amino acid sequence of SEQ ID NO: 7, HC-CDR3 having the amino acid sequence of SEQ ID NO: 8 A heavy chain variable (VH) region including, and (ii) LC-CDR1 having the amino acid sequence of SEQ ID NO: 131, LC-CDR2 having amino acid sequence LVS, LC-CDR3 having the amino acid sequence of SEQ ID NO: 16 Light chain variable (VL) region including, or (f) (i) HC-CDR1 having the amino acid sequence of SEQ ID NO: 22, HC-CDR2 having the amino acid sequence of SEQ ID NO: 23, HC-CDR3 having the amino acid sequence of SEQ ID NO: 24 A heavy chain variable (VH) region including, and (ii) LC-CDR1 having the amino acid sequence of SEQ ID NO: 30, LC-CDR2 having amino acid sequence YTS, LC-CDR3 having the amino acid sequence of SEQ ID NO: 32 Light chain variable (VL) region including, or (g) (i) HC-CDR1 having the amino acid sequence of SEQ ID NO: 6, HC-CDR2 having the amino acid sequence of SEQ ID NO: 7, HC-CDR3 having the amino acid sequence of SEQ ID NO: 8 A heavy chain variable (VH) region including, and (ii) LC-CDR1 having the amino acid sequence of SEQ ID NO: 162, LC-CDR2 having amino acid sequence LVS, LC-CDR3 having the amino acid sequence of SEQ ID NO: 16 Light chain variable (VL) region including, or (h) (i) HC-CDR1 having the amino acid sequence of SEQ ID NO: 152, HC-CDR2 having the amino acid sequence of SEQ ID NO: 55, HC-CDR3 having the amino acid sequence of SEQ ID NO: 56 A heavy chain variable (VH) region including, and (ii) LC-CDR1 having the amino acid sequence of SEQ ID NO: 154, LC-CDR2 having the amino acid sequence YAS, LC-CDR3 having the amino acid sequence of SEQ ID NO: 64 Light chain variable (VL) region including, or (i) (i) HC-CDR1 having the amino acid sequence of SEQ ID NO: 185, HC-CDR2 having the amino acid sequence of SEQ ID NO: 55, HC-CDR3 having the amino acid sequence of SEQ ID NO: 56 A heavy chain variable (VH) region including, and (ii) LC-CDR1 having the amino acid sequence of SEQ ID NO: 144, LC-CDR2 having the amino acid sequence YAS, LC-CDR3 having the amino acid sequence of SEQ ID NO: 64 Light chain variable (VL) region including, or (j) (i) HC-CDR1 having the amino acid sequence of SEQ ID NO: 6, HC-CDR2 having the amino acid sequence of SEQ ID NO: 7, HC-CDR3 having the amino acid sequence of SEQ ID NO: 8 A heavy chain variable (VH) region including, and (ii) LC-CDR1 having the amino acid sequence of SEQ ID NO: 14, LC-CDR2 having amino acid sequence LVS, LC-CDR3 having the amino acid sequence of SEQ ID NO: 16 Light chain variable (VL) region including A VH4-34 antigen-binding molecule containing [the specified substance].

2. A VH region containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NOs. 5, 21, 37, 53, 125, 141, 151, 156, 167, 171, 176, 181, 182, or 184, and VL region containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NOs: 13, 29, 45, 61, 130, 143, 147, 153, 161, 189, 193, 198, 201, 205, or 209 A VH4-34 antigen-binding molecule according to claim 1, comprising:

3. (i) A VH region comprising an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 182, and A VL region containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 198, or (ii) A VH region comprising an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 182, and A VL region containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 193, or (iii) A VH region comprising an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 167, and A VL region containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 189, or (iv) A VH region containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 21, and A VL region containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 29, or (v) A VH region comprising an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 37, and A VL region containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 45, or (vi) A VH region comprising an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 53, and A VL region containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 61, or (vii) A VH region comprising an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 125, and A VL region containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 130, or (viiii) A VH region comprising an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 141, and A VL region containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 143, or (ix) A VH region containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 141, and A VL region containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 147, or (x) A VH region containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 156, and A VL region containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 161, or (xi) A VH region comprising an amino acid sequence having at least 80% sequence identity with respect to the amino acid sequence of SEQ ID NO: 151, and A VL region containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 153, or (xi) A VH region containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 184, and A VL region containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NOs: 189, 193, 198, 201, 205, or 209. or (xiiii) A VH region containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 167, and A VL region containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NOs: 189, 193, 198, 201, 205, or 209. or (xiv) A VH region containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 171, and A VL region containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NOs: 189, 193, 198, 201, 205, or 209. or (xv) A VH region containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 176, and A VL region containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NOs: 189, 193, 198, 201, 205, or 209. or (xvi) A VH region containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 181, and A VL region containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NOs: 189, 193, 198, 201, 205, or 209. or (xvii) A VH region containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 182, and A VL region containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NOs: 189, 193, 198, 201, 205, or 209. or (xviiii) A VH region containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 5, and A VL region containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:

13. A VH4-34 antigen-binding molecule according to any one of claims 1 or 2, comprising:

4. A VH4-34 antigen-binding molecule according to any one of claims 1 to 3, wherein the interaction between VH4-34 and a molecule containing an N-acetyllactosamine moiety is inhibited, and optionally the molecule containing the N-acetyllactosamine moiety is selected from I / I carbohydrates and CD45 isoform B220.

5. A VH4-34 antigen-binding molecule according to any one of claims 1 to 4, further comprising an Fc region.

6. A VH4-34 antigen-binding molecule according to any one of claims 1 to 5, which is a multispecific antigen-binding molecule and further comprises an antigen-binding domain that binds to antigens other than VH4-34.

7. A chimeric antigen receptor (CAR) comprising a VH4-34 antigen-binding molecule as described in any one of claims 1 to 6.

8. An optionally isolated nucleic acid or a plurality of nucleic acids encoding the VH4-34 antigen-binding molecule according to any one of claims 1 to 6 or the CAR according to claim 7.

9. An expression vector comprising the nucleic acid or a plurality of nucleic acids as described in claim 8, or a plurality of expression vectors.

10. A cell comprising a VH4-34 antigen-binding molecule according to any one of claims 1 to 6, a CAR according to claim 7, a nucleic acid or a plurality of nucleic acids according to claim 8, or an expression vector or a plurality of expression vectors according to claim 9.

11. A method comprising the step of culturing the cells described in claim 10 under conditions suitable for the expression of antigen-binding molecules or CARs by the cells.

12. A composition comprising a VH4-34 antigen-binding molecule according to any one of claims 1 to 6, a CAR according to claim 7, a nucleic acid or a plurality of nucleic acids according to claim 8, an expression vector or a plurality of expression vectors according to claim 9 or a cell according to claim 10, and a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.

13. A VH4-34 antigen-binding molecule according to any one of claims 1 to 6, a CAR according to claim 7, a nucleic acid or a plurality of nucleic acids according to claim 8, an expression vector or a plurality of expression vectors according to claim 9, a cell according to claim 10, or a composition according to claim 12, for use in a method of medical treatment or prevention.

14. A VH4-34 antigen-binding molecule according to any one of claims 1 to 6, a CAR according to claim 7, a nucleic acid or a plurality of nucleic acids according to claim 8, an expression vector or a plurality of expression vectors according to claim 9, a cell according to claim 10, or a composition according to claim 12, for use in a method of treating or preventing a disease or condition characterized by the expression of VH4-34.

15. Use of a VH4-34 antigen-binding molecule according to any one of claims 1 to 6, a CAR according to claim 7, a nucleic acid or a plurality of nucleic acids according to claim 8, an expression vector or a plurality of expression vectors according to claim 9, a cell according to claim 10, or a composition according to claim 12 in the manufacture of a pharmaceutical product for treating or preventing a disease or condition characterized by the expression of VH4-34.

16. The use according to claim 14 or 15, wherein the disease or condition characterized by the expression of VH4-34 is an autoimmune disease.

17. The use according to claim 16, wherein the autoimmune disease is selected from the group consisting of autoimmune hemolytic anemia (AIHA), unclassified immunodeficiency (CVID), rheumatoid arthritis, systemic lupus erythematosus (SLE), lupus nephritis, central nervous system (CNS) lupus, cold agglutinin disease (CAD), and Wiscott-Aldrich syndrome.

18. The use according to claim 17, wherein the autoimmune disease is cold agglutinin disease (CAD).

19. The use according to claim 17, wherein the autoimmune disease is systemic lupus erythematosus (SLE).

20. The use according to claim 14 or 15, wherein the disease or condition characterized by the expression of VH4-34 is cancer.

21. The use according to claim 20, wherein the cancer is a B-cell malignant tumor.

22. The use according to claim 21, wherein the B-cell malignancy is selected from the group consisting of B-cell leukemia, hairy cell leukemia (HCL), hairy cell leukemia subtype (HCL-V), chronic lymphocytic leukemia (CLL), mutant chronic lymphocytic leukemia (M-CLL), acute lymphoblastic leukemia (ALL), B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), activated B-cell diffuse large B-cell lymphoma (ABC-DLBCL), germinal center B-cell diffuse large B-cell lymphoma (GBC-DLBCL), primary central nervous system (CNS) lymphoma, and mantle cell lymphoma.

23. The use according to claim 22, wherein the B-cell malignant tumor is chronic lymphocytic leukemia (CLL).

24. Diseases or conditions characterized by VH4-34 expression include B-cell leukemia, Hairy cell leukemia (HCL), Hairy cell leukemia subtype (HCL-V), chronic lymphocytic leukemia (CLL), mutant chronic lymphocytic leukemia (M-CLL), acute lymphoblastic leukemia (ALL), B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), activated B-cell diffuse large B-cell lymphoma (ABC-DLBCL), and germinal center B Use according to claim 14 or 15, selected from diffuse large B-cell lymphoma (GBC-DLBCL), primary central nervous system (CNS) lymphoma, mantle cell lymphoma, autoimmune hemolytic anemia (AIHA), unclassified immunodeficiency (CVID), rheumatoid arthritis, systemic lupus erythematosus (SLE), lupus nephritis, central nervous system (CNS) lupus, cold agglutinin disease (CAD), and Wiscott-Aldrich syndrome.

25. A method for treating or preventing a disease or condition characterized by the expression of VH4-34 in a subject, comprising the step of administering to the subject a VH4-34 antigen-binding molecule according to any one of claims 1 to 6, a CAR according to claim 8, a nucleic acid or a plurality of nucleic acids according to claim 8, an expression vector or a plurality of expression vectors according to claim 9, a cell according to claim 10, or a composition according to claim 12.

26. The method according to claim 25, wherein the disease or condition characterized by the expression of VH4-34 is an autoimmune disease.

27. The method according to claim 26, wherein the autoimmune disease is selected from the group consisting of autoimmune hemolytic anemia (AIHA), unclassified immunodeficiency (CVID), rheumatoid arthritis, systemic lupus erythematosus (SLE), lupus nephritis, central nervous system (CNS) lupus, cold agglutinin disease (CAD), and Wiscott-Aldrich syndrome.

28. The method according to claim 27, wherein the autoimmune disease is cold agglutinin disease (CAD).

29. The method according to claim 25, wherein the disease or condition characterized by the expression of VH4-34 is cancer.

30. The method according to claim 29, wherein the cancer is a B-cell malignant tumor.

31. The method according to claim 30, wherein the B-cell malignancy is selected from the group consisting of B-cell leukemia, hairy cell leukemia (HCL), hairy cell leukemia subtype (HCL-V), chronic lymphocytic leukemia (CLL), mutant chronic lymphocytic leukemia (M-CLL), acute lymphoblastic leukemia (ALL), B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), activated B-cell diffuse large B-cell lymphoma (ABC-DLBCL), germinal center B-cell diffuse large B-cell lymphoma (GBC-DLBCL), primary central nervous system (CNS) lymphoma, and mantle cell lymphoma.

32. The method according to claim 31, wherein the B-cell malignant tumor is chronic lymphocytic leukemia (CLL).

33. Diseases or conditions characterized by VH4-34 expression include B-cell leukemia, Hairy cell leukemia (HCL), Hairy cell leukemia subtype (HCL-V), chronic lymphocytic leukemia (CLL), mutant chronic lymphocytic leukemia (M-CLL), acute lymphoblastic leukemia (ALL), B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), activated B-cell diffuse large B-cell lymphoma (ABC-DLBCL), and embryonic The method according to claim 25, selected from cardiac B-cell diffuse large B-cell lymphoma (GBC-DLBCL), primary central nervous system (CNS) lymphoma, mantle cell lymphoma, autoimmune hemolytic anemia (AIHA), unclassified immunodeficiency (CVID), rheumatoid arthritis, systemic lupus erythematosus (SLE), lupus nephritis, central nervous system (CNS) lupus, cold agglutinin disease (CAD), and Wiscott-Aldrich syndrome.

34. Use of a VH4-34 antigen-binding molecule according to any one of claims 1 to 6 to promote the death of cells expressing VH4-34.

35. An optionally isolated in vitro complex comprising a VH4-34 antigen-binding molecule according to any one of claims 1 to 6, conjugated to VH4-34.

36. A method for detecting VH4-34 in a sample, comprising the steps of contacting a sample containing or suspected to contain VH4-34 with a VH4-34 antigen-binding molecule described in any one of claims 1 to 6, and detecting the formation of a complex of the VH4-34 antigen-binding molecule with VH4-34.

37. A method for selecting or stratifying subjects for treatment using a drug targeting VH4-34, comprising the steps of: contacting a sample derived from the subject in vitro with a VH4-34 antigen-binding molecule described in any one of claims 1 to 6; and detecting the formation of a complex of the VH4-34 antigen-binding molecule with VH4-34.

38. Use of the VH4-34 antigen-binding molecule according to any one of claims 1 to 6 as an in vitro or in vivo diagnostic or prognostic agent.

39. A method for detecting, locating, or imaging a disease or condition characterized by the expression of VH4-34, using a VH4-34 antigen-binding molecule according to any one of claims 1 to 6, wherein the disease or condition characterized by the expression of VH4-34 is optionally B-cell leukemia, hairy cell leukemia (HCL), hairy cell leukemia subtype (HCL-V), chronic lymphocytic leukemia (CLL), mutant chronic lymphocytic leukemia (M-CLL), acute lymphoblastic leukemia (ALL), B-cell lymphoma, Use is selected from diffuse large B-cell lymphoma (DLBCL), activated B-cell diffuse large B-cell lymphoma (ABC-DLBCL), germinal center B-cell diffuse large B-cell lymphoma (GBC-DLBCL), primary central nervous system (CNS) lymphoma, mantle cell lymphoma, autoimmune hemolytic anemia (AIHA), unclassified immunodeficiency (CVID), rheumatoid arthritis, systemic lupus erythematosus (SLE), lupus nephritis, central nervous system (CNS) lupus, cold agglutinin disease (CAD), and Wiscott-Aldrich syndrome.

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