BAFF-R antibody and its use

Abstract

This invention provides antibodies, compositions, and methods for producing and using them for use in antibody therapy. [Solution] The present invention provides a BAFF-R antibody comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises CDR L1, CDR L2, and CDR L3 as specified by a specific sequence; and the heavy chain variable region comprises CDR H1, CDR H2, and CDR H3 as specified by a specific sequence.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 346,324, filed on June 6, 2016, which is incorporated herein by reference in its entirety. [Background technology]

[0002] Antibody therapy is one of the most successful immunotherapies available in clinics treating hematological malignancies. An exemplary example is rituximab, which targets CD20 and induces cytotoxic effects against B-cell lymphoma. However, a major concern with rituximab is the emergence of rituximab resistance, which is thought to be due to the downregulation of CD20 and thus the prevention of the antibody from binding to target cells. [Overview of the Initiative]

[0003] A B-cell activator receptor (BAFF-R) antibody comprising a light chain variable region and a heavy chain variable region is provided herein. The light chain variable region comprises CDR L1 as specified in SEQ ID NO: 1, CDR L2 as specified in SEQ ID NO: 2, and CDR L3 as specified in SEQ ID NO: 3. The heavy chain variable region comprises CDR H1 as specified in SEQ ID NO: 4, CDR H2 as specified in SEQ ID NO: 5, and CDR H3 as specified in SEQ ID NO: 6. In another embodiment, the light chain variable region comprises CDR L1 as specified in SEQ ID NO: 7, CDR L2 as specified in SEQ ID NO: 8, and CDR L3 as specified in SEQ ID NO: 9. The heavy chain variable region comprises CDR H1 as specified in SEQ ID NO: 10, CDR H2 as specified in SEQ ID NO: 11, and CDR H3 as specified in SEQ ID NO: 12. Optionally, the antibody is a humanized antibody. Functional fragments of the disclosed antibodies are also provided.

[0004] BAFF-R and K below approximately 4nM DA humanized B-cell activator receptor (BAFF-R) antibody or a functional fragment thereof that can be bound to is provided.

[0005] BAFF-R and K below approximately 4nM D Humanized B-cell activator receptor (BAFF-R) antibodies that bind to it are also provided.

[0006] Chimeric antigen receptors (CARs) containing antibodies or functional fragments thereof, as provided herein, are also provided.

[0007] Isolated nucleic acids encoding BAFF-R antibodies or functional fragments of antibodies are provided herein.

[0008] Pharmaceutical compositions comprising a therapeutically effective amount of a BAFF-R antibody or a functional fragment thereof as disclosed herein and a pharmaceutically acceptable excipient are also provided.

[0009] Mouse fibroblasts expressing the human BAFF-R protein or a functional fragment thereof are provided, with the human BAFF-R protein or its functional fragment expressed on the cell surface.

[0010] A method is provided for treating cancer in a subject that requires it. The method comprises administering a therapeutically effective amount of the chimeric antigen receptor provided herein to a subject, thereby treating cancer in the subject.

[0011] A method for treating cancer in a subject requiring such treatment is further provided, comprising administering a therapeutically effective amount of the antibody or a functional fragment thereof disclosed herein to the subject, thereby treating the cancer in the subject.

[0012] A method is provided for treating autoimmune diseases in subjects in need. The method comprises administering a therapeutically effective dose of an antibody or a functional fragment thereof, as disclosed herein, to a subject, thereby treating the autoimmune disease in the subject.

[0013] Methods for inhibiting the proliferation of cells are also provided. The methods include contacting a cell with a BAFF-R antibody or a functional fragment thereof as disclosed herein, thereby forming the contacted cell. The BAFF-R antibody or a functional fragment thereof binds to the BAFF-R protein on the contacted cell, thereby inhibiting the proliferation of the cell. Optionally, the cell is a lymphocyte-like cell.

[0014] Methods for producing an anti-human BAFF-R antibody or a functional fragment thereof are provided. The methods include administering mouse fibroblasts expressing a BAFF-R protein or a fragment thereof as provided herein to a mouse, thereby forming an immunized BAFF-R mouse. Splenocytes from the immunized BAFF-R mouse are fused with human myeloma cells, thereby forming BAFF-R hybridoma cells. The BAFF-R hybridoma cells are then made to express a BAFF-R antibody, thereby producing an anti-BAFF-R antibody.

Brief Description of the Drawings

[0015] [Figure 1-1]Figures 1A, 1B, 1C, 1D and 1E are FACS images showing the generation and specificity of novel monoclonal antibodies against human BAFF-R. Figure 1A is a FACS analysis of cell surface expression of hBAFF-R-GFP fusion protein in mouse fibroblasts (L cells). Gating on GFP-positive cells, engineered L cell clones (right plot) are compared to parental L cells (left plot). Clone D2C was selected for further study. Figures 1B, 1C, 1D and 1E are FACS traces of fluorescence counts of anti-BAFF-R antibodies binding to cell lines and patient samples. Figure 1B shows affinity-purified hybridoma mAbs (C90, C67, C55 and C53) that bind to BAFF-R-positive human MCL lines (including Mino, JeKo-1, REC-1, JVM-13 and Z-138) at a concentration of 0.05 μg mAb / 106 cells. The BAFF-R-negative 293T embryonic kidney cell line was used as a control. Figure 1C shows chimeric antibodies C55 and C90 that bind to hBAFF-R-expressing L cells at high and low concentrations. Only parental L cells and secondary anti-hIgG-APC antibody were used as controls. Figure 1D shows a panel of alexa fluor 488-conjugated chimeric antibody-binding NHL cell lines. Figure 1E shows chimeric antibodies that bind to three types of NHL primary patient samples. The data are representative of three independent experiments. For all of Figures 1B - 1E, the top-to-bottom trace as shown in the figure correlates top-to-bottom with the variables used (e.g., antibody type or cell type) shown below or next to the figure. [Figure 1-2]Figures 1A, 1B, 1C, 1D, and 1E are FACS images showing the generation and specificity of novel monoclonal antibodies against human BAFF-R. Figure 1A is a FACS analysis of cell surface expression of hBAFF-R-GFP fusion protein in mouse fibroblasts (L cells). Manipulated L cell clones (right plot) are compared to parental L cells (left plot) after gating for GFP-positive cells. Clone D2C was selected for further study. Figures 1B, 1C, 1D, and 1E are FACS traces of fluorescence counts of anti-BAFF-R antibodies binding to cell lines and patient samples. Figure 1B shows affinity-purified hybridoma mAbs (C90, C67, C55, and C53) binding to BAFF-R-positive human MCL lines (including Mino, JeKo-1, REC-1, JVM-13, and Z-138) at a concentration of 0.05 μg mAb / 10⁶ cells. A BAFF-R negative 293T embryonic kidney cell line was used as a control. Figure 1C shows chimeric antibodies C55 and C90 binding to hBAFF-R expressing L cells at high and low concentrations. Only parental L cells and secondary anti-hIgG-APC antibodies were used as controls. Figure 1D shows a panel of alexa fluor 488 conjugate chimeric antibody-conjugated NHL cell lines. Figure 1E shows chimeric antibodies binding to three types of primary NHL patient samples. Data are representative of three independent experiments. For all of Figures 1B–1E, the top-to-bottom traces shown in the figures correlate with the top-to-bottom variables used (e.g., antibody type or cell type) shown below or next to the figures. [Figure 1-3]Figures 1A, 1B, 1C, 1D, and 1E are FACS images showing the generation and specificity of novel monoclonal antibodies against human BAFF-R. Figure 1A is a FACS analysis of cell surface expression of hBAFF-R-GFP fusion protein in mouse fibroblasts (L cells). Manipulated L cell clones (right plot) are compared to parental L cells (left plot) after gating for GFP-positive cells. Clone D2C was selected for further study. Figures 1B, 1C, 1D, and 1E are FACS traces of fluorescence counts of anti-BAFF-R antibodies binding to cell lines and patient samples. Figure 1B shows affinity-purified hybridoma mAbs (C90, C67, C55, and C53) binding to BAFF-R-positive human MCL lines (including Mino, JeKo-1, REC-1, JVM-13, and Z-138) at a concentration of 0.05 μg mAb / 10⁶ cells. A BAFF-R negative 293T embryonic kidney cell line was used as a control. Figure 1C shows chimeric antibodies C55 and C90 binding to hBAFF-R expressing L cells at high and low concentrations. Only parental L cells and secondary anti-hIgG-APC antibodies were used as controls. Figure 1D shows a panel of alexa fluor 488 conjugate chimeric antibody-conjugated NHL cell lines. Figure 1E shows chimeric antibodies binding to three types of primary NHL patient samples. Data are representative of three independent experiments. For all of Figures 1B–1E, the top-to-bottom traces shown in the figures correlate with the top-to-bottom variables used (e.g., antibody type or cell type) shown below or next to the figures. [Figure 1-4]Figures 1A, 1B, 1C, 1D, and 1E are FACS images showing the generation and specificity of novel monoclonal antibodies against human BAFF-R. Figure 1A is a FACS analysis of cell surface expression of hBAFF-R-GFP fusion protein in mouse fibroblasts (L cells). Manipulated L cell clones (right plot) are compared to parental L cells (left plot) after gating for GFP-positive cells. Clone D2C was selected for further study. Figures 1B, 1C, 1D, and 1E are FACS traces of fluorescence counts of anti-BAFF-R antibodies binding to cell lines and patient samples. Figure 1B shows affinity-purified hybridoma mAbs (C90, C67, C55, and C53) binding to BAFF-R-positive human MCL lines (including Mino, JeKo-1, REC-1, JVM-13, and Z-138) at a concentration of 0.05 μg mAb / 10⁶ cells. A BAFF-R negative 293T embryonic kidney cell line was used as a control. Figure 1C shows chimeric antibodies C55 and C90 binding to hBAFF-R expressing L cells at high and low concentrations. Only parental L cells and secondary anti-hIgG-APC antibodies were used as controls. Figure 1D shows a panel of alexa fluor 488 conjugate chimeric antibody-conjugated NHL cell lines. Figure 1E shows chimeric antibodies binding to three types of primary NHL patient samples. Data are representative of three independent experiments. For all of Figures 1B–1E, the top-to-bottom traces shown in the figures correlate with the top-to-bottom variables used (e.g., antibody type or cell type) shown below or next to the figures. [Figure 1-5]Figures 1A, 1B, 1C, 1D, and 1E are FACS images showing the generation and specificity of novel monoclonal antibodies against human BAFF-R. Figure 1A is a FACS analysis of cell surface expression of hBAFF-R-GFP fusion protein in mouse fibroblasts (L cells). Manipulated L cell clones (right plot) are compared to parental L cells (left plot) after gating for GFP-positive cells. Clone D2C was selected for further study. Figures 1B, 1C, 1D, and 1E are FACS traces of fluorescence counts of anti-BAFF-R antibodies binding to cell lines and patient samples. Figure 1B shows affinity-purified hybridoma mAbs (C90, C67, C55, and C53) binding to BAFF-R-positive human MCL lines (including Mino, JeKo-1, REC-1, JVM-13, and Z-138) at a concentration of 0.05 μg mAb / 10⁶ cells. A BAFF-R negative 293T embryonic kidney cell line was used as a control. Figure 1C shows chimeric antibodies C55 and C90 binding to hBAFF-R expressing L cells at high and low concentrations. Only parental L cells and secondary anti-hIgG-APC antibodies were used as controls. Figure 1D shows a panel of alexa fluor 488 conjugate chimeric antibody-conjugated NHL cell lines. Figure 1E shows chimeric antibodies binding to three types of primary NHL patient samples. Data are representative of three independent experiments. For all of Figures 1B–1E, the top-to-bottom traces shown in the figures correlate with the top-to-bottom variables used (e.g., antibody type or cell type) shown below or next to the figures. [Figure 2-1]Figures 2A, 2B, and 2C are graphs showing the specific in vitro cytotoxicity of BAFF-R monoclonal antibodies against B-cell tumor lines. Antibody-induced cytotoxicity was measured by chromium-51 release after incubation with C55, C90, or rituximab and an effector (NK cells or complement-containing serum). The ratio of NK effector cells to target cells (E:T) was 20:1. The percentage of cell-specific lysis of target cells is as follows: The first panel shows BAFF-R-expressing L cells or control parental L cells; the second and third panels show BAFF-R-positive JeKo-1 MCL or BAFF-R-negative U266 multiple myeloma cells, shown as dose-response curves with varying antibody concentrations. Figure 2B shows the specific lysis of antibody (1:3 dilution) mixed with active complement-containing human serum against CDC-sensitive (Raji) and CDC-resistant (Raji-2P) cells. Figure 2C shows the ADCC effect of BAFF-R chimeric antibody on NHL strains JeKo-1, SU-DHL-6, Raji, and RL with or without NK effector cells (E:T=20:1). Data are shown as mean ± standard deviation of a triple sample. *P<0.05 compared to NK cells by two-sided Student's t-test. [Figure 2-2]Figures 2A, 2B, and 2C are graphs showing the specific in vitro cytotoxicity of BAFF-R monoclonal antibodies against B-cell tumor lines. Antibody-induced cytotoxicity was measured by chromium-51 release after incubation with C55, C90, or rituximab and an effector (NK cells or complement-containing serum). The ratio of NK effector cells to target cells (E:T) was 20:1. The percentage of cell-specific lysis of target cells is as follows: The first panel shows BAFF-R-expressing L cells or control parental L cells; the second and third panels show BAFF-R-positive JeKo-1 MCL or BAFF-R-negative U266 multiple myeloma cells, shown as dose-response curves with varying antibody concentrations. Figure 2B shows the specific lysis of antibody (1:3 dilution) mixed with active complement-containing human serum against CDC-sensitive (Raji) and CDC-resistant (Raji-2P) cells. Figure 2C shows the ADCC effect of BAFF-R chimeric antibody on NHL strains JeKo-1, SU-DHL-6, Raji, and RL with or without NK effector cells (E:T=20:1). Data are shown as mean ± standard deviation of a triple sample. *P<0.05 compared to NK cells by two-sided Student's t-test. [Figure 2-3]Figures 2A, 2B, and 2C are graphs showing the specific in vitro cytotoxicity of BAFF-R monoclonal antibodies against B-cell tumor lines. Antibody-induced cytotoxicity was measured by chromium-51 release after incubation with C55, C90, or rituximab and an effector (NK cells or complement-containing serum). The ratio of NK effector cells to target cells (E:T) was 20:1. The percentage of cell-specific lysis of target cells is as follows: The first panel shows BAFF-R-expressing L cells or control parental L cells; the second and third panels show BAFF-R-positive JeKo-1 MCL or BAFF-R-negative U266 multiple myeloma cells, shown as dose-response curves with varying antibody concentrations. Figure 2B shows the specific lysis of antibody (1:3 dilution) mixed with active complement-containing human serum against CDC-sensitive (Raji) and CDC-resistant (Raji-2P) cells. Figure 2C shows the ADCC effect of BAFF-R chimeric antibody on NHL strains JeKo-1, SU-DHL-6, Raji, and RL with or without NK effector cells (E:T=20:1). Data are shown as mean ± standard deviation of a triple sample. *P<0.05 compared to NK cells by two-sided Student's t-test. [Figure 3-1] Figures 3A and 3B are graphs showing that the BAFF-R monoclonal antibody induces in vitro antibody-dependent cell-mediated cytotoxicity (ADCC) against primary B-cell tumors. The ADCC effect was measured by chromium-51 release after incubation with C55, C90, or rituximab and effector (NK cells). The percentage of cell-specific lysis of target cells is as follows. Figure 3A shows NHL patient samples (E:T=20:1 or 10:1); Figure 3B shows primary MCL and CLL samples (E:T=20:1) from rituximab-refractory patients. Data are shown as mean ± standard deviation of triple samples. *P<0.05 compared to NK cells by two-sided Student's t-test. [Figure 3-2]Figures 3A and 3B are graphs showing that the BAFF-R monoclonal antibody induces in vitro antibody-dependent cell-mediated cytotoxicity (ADCC) against primary B-cell tumors. The ADCC effect was measured by chromium-51 release after incubation with C55, C90, or rituximab and effector (NK cells). The percentage of cell-specific lysis of target cells is as follows. Figure 3A shows NHL patient samples (E:T=20:1 or 10:1); Figure 3B shows primary MCL and CLL samples (E:T=20:1) from rituximab-refractory patients. Data are shown as mean ± standard deviation of triple samples. *P<0.05 compared to NK cells by two-sided Student's t-test. [Figure 4-1] Figure 4A is a schematic diagram showing the treatment schedule following attack administration of the tumor on day 0 with the minimum lethal dose. Treatment was administered via IV tail vein injection. Figures 4B and 4C are images showing the efficacy of human BAFF-R-targeted chimeric antibody-induced in vivo therapy against B-cell tumors. Luciferase-expressing tumors: Bioluminescence images of mice attacked with JeKo-1 (MCL) (Figure 4B) or RS4;11 (ALL) (Figure 4C). The experimental group was treated with a chimeric BAFF-R mAb (C55 or C90 as shown). Control mice were treated with PBS, NK cells alone, or rituximab on the same schedule. Data are representative of three independent experiments. [Figure 4-2]Figure 4A is a schematic diagram showing the treatment schedule following attack administration of the tumor on day 0 with the minimum lethal dose. Treatment was administered via IV tail vein injection. Figures 4B and 4C are images showing the efficacy of human BAFF-R-targeted chimeric antibody-induced in vivo therapy against B-cell tumors. Luciferase-expressing tumors: Bioluminescence images of mice attacked with JeKo-1 (MCL) (Figure 4B) or RS4;11 (ALL) (Figure 4C). The experimental group was treated with a chimeric BAFF-R mAb (C55 or C90 as shown). Control mice were treated with PBS, NK cells alone, or rituximab on the same schedule. Data are representative of three independent experiments. [Figure 4-3] Figure 4A is a schematic diagram showing the treatment schedule following attack administration of the tumor on day 0 with the minimum lethal dose. Treatment was administered via IV tail vein injection. Figures 4B and 4C are images showing the efficacy of human BAFF-R-targeted chimeric antibody-induced in vivo therapy against B-cell tumors. Luciferase-expressing tumors: Bioluminescence images of mice attacked with JeKo-1 (MCL) (Figure 4B) or RS4;11 (ALL) (Figure 4C). The experimental group was treated with a chimeric BAFF-R mAb (C55 or C90 as shown). Control mice were treated with PBS, NK cells alone, or rituximab on the same schedule. Data are representative of three independent experiments. [Figure 5-1]Figures 5A, 5B, and 5C are images or graphs showing that the chimeric BAFF-R antibody induces ADCC in vitro in a drug-resistant lymphoma model. Figure 5A is a scatter plot of FACS analysis showing CD20 binding on JeKo-1 cells following CRISPR / HDR knockout of the CD20 gene. CD20 expression for selected CD20- / - clone number 25 was compared to wild-type JeKo-1. The ADCC effect was measured by chromium-51 release after incubation with C55, C90, or rituximab and effector NK cells (E:T=20:1). Percentages of cell-specific lysis of target cells are as follows: Rituximab-resistant JeKo-1-CD20-KO (Figure 5B) and ibrutinib-resistant Z-138 and SP49-IR (Figure 5C). All data are representative of two or more identical experiments. Data are presented as the mean ± standard deviation of a triple sample. *P<0.05 compared to NK cells by two-sided Student's t-test. [Figure 5-2] Figures 5A, 5B, and 5C are images or graphs showing that the chimeric BAFF-R antibody induces ADCC in vitro in a drug-resistant lymphoma model. Figure 5A is a scatter plot of FACS analysis showing CD20 binding on JeKo-1 cells following CRISPR / HDR knockout of the CD20 gene. CD20 expression for selected CD20- / - clone number 25 was compared to wild-type JeKo-1. The ADCC effect was measured by chromium-51 release after incubation with C55, C90, or rituximab and effector NK cells (E:T=20:1). Percentages of cell-specific lysis of target cells are as follows: Rituximab-resistant JeKo-1-CD20-KO (Figure 5B) and ibrutinib-resistant Z-138 and SP49-IR (Figure 5C). All data are representative of two or more identical experiments. Data are presented as the mean ± standard deviation of a triple sample. *P<0.05 compared to NK cells by two-sided Student's t-test. [Figure 5-3]Figures 5A, 5B, and 5C are images or graphs showing that the chimeric BAFF-R antibody induces ADCC in vitro in a drug-resistant lymphoma model. Figure 5A is a scatter plot of FACS analysis showing CD20 binding on JeKo-1 cells following CRISPR / HDR knockout of the CD20 gene. CD20 expression for selected CD20- / - clone number 25 was compared to wild-type JeKo-1. The ADCC effect was measured by chromium-51 release after incubation with C55, C90, or rituximab and effector NK cells (E:T=20:1). Percentages of cell-specific lysis of target cells are as follows: Rituximab-resistant JeKo-1-CD20-KO (Figure 5B) and ibrutinib-resistant Z-138 and SP49-IR (Figure 5C). All data are representative of two or more identical experiments. Data are presented as the mean ± standard deviation of a triple sample. *P<0.05 compared to NK cells by two-sided Student's t-test. [Figure 6-1] Images (Figures 6A and 6B) and graphs (Figure 6C) demonstrate the efficacy of human BAFF-R-targeted chimeric antibody-induced in vivo therapy for drug-resistant B-cell tumors. Bioluminescence images of mice attacked with luciferase-expressing tumors using JeKo-1-CD20-KO cells (Figure 6A) or ibrutinib-resistant Z-138 cells (Figure 6B), followed by antibody therapy as shown in Figure 4. Control mice were administered PBS, NK cells alone, or rituximab on the same schedule. Figure 6C shows the 80-day tumor-free and overall survival curves for the mice shown in (A) and (B), respectively. Differences in tumor-free rates and survival between the experimental group and all control groups were analyzed by log-rank test (**P<0.001). Data are representative of three independent experiments. [Figure 6-2]Images (Figures 6A and 6B) and graphs (Figure 6C) demonstrate the efficacy of human BAFF-R-targeted chimeric antibody-induced in vivo therapy for drug-resistant B-cell tumors. Bioluminescence images of mice attacked with luciferase-expressing tumors using JeKo-1-CD20-KO cells (Figure 6A) or ibrutinib-resistant Z-138 cells (Figure 6B), followed by antibody therapy as shown in Figure 4. Control mice were administered PBS, NK cells alone, or rituximab on the same schedule. Figure 6C shows the 80-day tumor-free and overall survival curves for the mice shown in (A) and (B), respectively. Differences in tumor-free rates and survival between the experimental group and all control groups were analyzed by log-rank test (**P<0.001). Data are representative of three independent experiments. [Figure 6-3] Images (Figures 6A and 6B) and graphs (Figure 6C) demonstrate the efficacy of human BAFF-R-targeted chimeric antibody-induced in vivo therapy for drug-resistant B-cell tumors. Bioluminescence images of mice attacked with luciferase-expressing tumors using JeKo-1-CD20-KO cells (Figure 6A) or ibrutinib-resistant Z-138 cells (Figure 6B), followed by antibody therapy as shown in Figure 4. Control mice were administered PBS, NK cells alone, or rituximab on the same schedule. Figure 6C shows the 80-day tumor-free and overall survival curves for the mice shown in (A) and (B), respectively. Differences in tumor-free rates and survival between the experimental group and all control groups were analyzed by log-rank test (**P<0.001). Data are representative of three independent experiments. [Figure 7-1]Figures 7A and 7B show the generation of anti-human BAFF-R monoclonal antibodies and clonal selection. Figure 7A is a schematic diagram showing that BALB / c mice were immunized according to the schedule shown using L cell clone D2C (which stably expresses human hBAFF-R with a C-terminal GFP tag on its intracellular domain). Spleen tissue was harvested on day 20 to establish B cell hybridoma clones. Figure 7B is a table showing ELISA results from five hybridoma supernatants using anti-mouse IgG-HRP. Clones 53, 55, 67, and 90 produced BAFF-R specific mAbs, while clone 37 did not (representative of other negative clones). [Figure 7-2] Figures 7A and 7B show the generation of anti-human BAFF-R monoclonal antibodies and clonal selection. Figure 7A is a schematic diagram showing that BALB / c mice were immunized according to the schedule shown using L cell clone D2C (which stably expresses human hBAFF-R with a C-terminal GFP tag on its intracellular domain). Spleen tissue was harvested on day 20 to establish B cell hybridoma clones. Figure 7B is a table showing ELISA results from five hybridoma supernatants using anti-mouse IgG-HRP. Clones 53, 55, 67, and 90 produced BAFF-R specific mAbs, while clone 37 did not (representative of other negative clones). [Figure 8] These are flow cytometry results confirming that the selected hybridoma clones bind to MCL cells. The binding of the supernatants (1 / 10, 1 / 50, and 1 / 200 dilutions) of hybridoma clones 53, 55, 67, and 90 to Mino (mantle cell lymphoma) cell lines and 293T (negative control) cell lines was assessed by flow cytometry performed with anti-mouse IgG-APC. [Figure 9]This graph shows the dose-dependent binding of purified mAbs to human BAFF-R. Mouse mAbs from hybridoma clones 53, 55, 67, and 90 were purified by protein A affinity chromatography. Binding of sequentially diluted (1 μg / 10⁶ cells to 1.6 ng / 10⁶ cells) purified mouse mAbs to Mino cells was assessed by flow cytometry using an anti-mouse IgG-APC secondary antibody. [Figure 10] This is the result of a FACS screening analysis showing that the hBAFF-R mAb recognized non-Hodgkin lymphoma cell lines in vitro. Mouse mAb clones 55 and 90 were conjugated to additional cell lines: JeKo-1 (mantle cell lymphoma), SU-DHL-6 (diffuse large B-cell lymphoma), Raji (Burkitt lymphoma), and RL (follicular lymphoma) at high doses (2 μg mAb / 10⁶ cells) and low doses (0.05 μg mAb / 10⁶ cells). Flow cytometry analysis was performed with anti-mouse IgG-APC. The top-to-bottom trace shown in the figure correlates with the top-to-bottom trace of the variable used (e.g., antibody type or cell type) shown next to the figure. [Figure 11] This graph shows that the hBAFF-R mAb recognized lymphoma patient samples. Patient samples of mantle cell lymphoma, diffuse large B-cell lymphoma, and follicular lymphoma were stained with mouse mAbs C55 and C90 at high doses (2 μg / 10⁶ cells) and low doses (0.05 μg / 10⁶ cells). Flow cytometry analysis was performed using anti-mouse IgG-APC. The top-to-bottom trace shown in the figure correlates from left to right with the variables used (e.g., antibody type or cell type) shown below the figure. [Figure 12] This graph shows that a chimeric antibody induced ADCC in BAFF-R-expressing L cells. BAFF-R-expressing D2C L cells (target) were labeled with chromium-51 and subsequently incubated overnight with chimeric mAb+NK cells (effector-to-target ratio, 20:1). The culture supernatant was analyzed for released chromium. [Figure 13]This graph shows the chimeric antibodies required by NK cells for cytotoxicity against tumor cells. JeKo-1 cells (target) were labeled with chromium-51. The cells were incubated with or without NK cells (effector) using chimeric mAbs (C55, C90, or rituximab) and in an effector-to-target ratio of 20:1. Chimeric antibodies were added at concentrations of 50–0.005 μg / mL. The culture supernatant was analyzed for released chromium. [Figure 14] FACS results demonstrating that the hBAFF-R mAb blocked the BAFF / BAFF-R interaction. BAFF-R expressing D2C L cell clones were incubated with C90 (0-1000 ng / 10⁶ cells) at 4°C for 45 minutes, followed by incubation with recombinant BAFF ligand (0.5 μg / 10⁶ cells) at 4°C for 90 minutes. Flow cytometry was performed and gated for anti-BAFF-PE. The signal plots show the BAFF / BAFF-R binding signals in the presence of each mAb concentration. The concentrations shown in the signal plots are indicated above each of the FACS results. [Figure 15] FACS results show limited internal migration observed with BAFF-R mAb. Mino cells were incubated with mAb C90 (0.05 μg / 10⁶ cells) at 4°C for 20 minutes, followed by incubation at 37°C for 1 hour. Flow cytometry analysis was performed using anti-mouse IgG-APC. Cells were gated for surface localization antibody (out) and loss of cell surface staining (in). [Figure 16-1]FACS results (Figure 16A) and gel image (16B) show that CD20 knockout clones were generated by CRISPR. JeKo-1 CD20 knockout clones were generated using a commercial CRISPR / HDR system that substituted RFP at the CD20 locus. In Figure 16A, clones were screened and selected by flow cytometry for CD20- / RFP+ expression. In Figure 16B, Western blotting with anti-CD20 antibody was performed on whole cell lysates from CD20- / RFP+ clones. β-actin was used as a loading control for blotting. Figure 16C shows FACS results for the same clones as in Figure 16A, confirming that BAFF-R expression was not affected by the CD20 CRISPR / HDR manipulation after screening for BAFF-R / RFP+ expression. [Figure 16-2] FACS results (Figure 16A) and gel image (16B) show that CD20 knockout clones were generated by CRISPR. JeKo-1 CD20 knockout clones were generated using a commercial CRISPR / HDR system that substituted RFP at the CD20 locus. In Figure 16A, clones were screened and selected by flow cytometry for CD20- / RFP+ expression. In Figure 16B, Western blotting with anti-CD20 antibody was performed on whole cell lysates from CD20- / RFP+ clones. β-actin was used as a loading control for blotting. Figure 16C shows FACS results for the same clones as in Figure 16A, confirming that BAFF-R expression was not affected by the CD20 CRISPR / HDR manipulation after screening for BAFF-R / RFP+ expression. [Figure 16-3]FACS results (Figure 16A) and gel image (16B) show that CD20 knockout clones were generated by CRISPR. JeKo-1 CD20 knockout clones were generated using a commercial CRISPR / HDR system that substituted RFP at the CD20 locus. In Figure 16A, clones were screened and selected by flow cytometry for CD20- / RFP+ expression. In Figure 16B, Western blotting with anti-CD20 antibody was performed on whole cell lysates from CD20- / RFP+ clones. β-actin was used as a loading control for blotting. Figure 16C shows FACS results for the same clones as in Figure 16A, confirming that BAFF-R expression was not affected by the CD20 CRISPR / HDR manipulation after screening for BAFF-R / RFP+ expression. [Figure 17-1] This is a FACS result illustrating the characteristic binding of BAFF-R to normal B cells. PBMCs from healthy donors were co-stained with an APC-conjugated C90 chimeric antibody and either (A) a lymphocyte marker panel (anti-CD20-PE, anti-CD3-PacificBlue, and anti-CD56-FITC) or (B) a myeloid cell marker panel (anti-CD45-PE, anti-CD15-PerCP-Cy5.5, and anti-CD14-PacificBlue). Each specific immune cell subpopulation was analyzed after being gated for binding to the BAFF-R antibody. [Figure 17-2] This is a FACS result illustrating the characteristic binding of BAFF-R to normal B cells. PBMCs from healthy donors were co-stained with an APC-conjugated C90 chimeric antibody and either (A) a lymphocyte marker panel (anti-CD20-PE, anti-CD3-PacificBlue, and anti-CD56-FITC) or (B) a myeloid cell marker panel (anti-CD45-PE, anti-CD15-PerCP-Cy5.5, and anti-CD14-PacificBlue). Each specific immune cell subpopulation was analyzed after being gated for binding to the BAFF-R antibody. [Figure 18]Figures 18A and 18B show FACS results illustrating the characterization of hBAFF-R mAbs in normal immune cells from peripheral blood. Mouse mAb clones 55 and 90 were tested for binding to isolated human immune cell subpopulations. B cells, T cells, and NK cells were isolated using commercial specific cell type isolation kits and stained with C55 and C90 (0.05 μg mAb / 10⁶ cells). Flow cytometry analysis was performed with anti-mouse IgG. Myeloid cells from PBMCs were gated for CD66b+ and stained for mAbC55 and C90. The top-to-bottom traces shown in the figures correlate with the top-to-bottom traces of the variables used (e.g., antibody type or cell type) shown next to the figures. [Figure 19-1] Figures 19A and 19B are immunohistochemical images. In Figure 19A, immunohistochemistry was performed to identify the tissue specificity of the anti-BAFF-R antibody. Tissue samples were stained using a 1:150 dilution of 1 mg / mL antibody. Tissue specificity of C55 mAb against human BAFF-R (20× objective lens): 1:150 dilution of 1 mg / mL stock. In Figure 19B, immunohistochemistry was additionally performed on tonsil and breast tissue to identify the tissue specificity of the anti-BAFF-R antibody. Tissue samples were stained using a 1:150 dilution of 1 mg / mL antibody. Tissue specificity of mAb against human BAFF-R (upper panel: tonsil tissue; lower panel: breast tissue; 20× objective lens). [Figure 19-2] Figures 19A and 19B are immunohistochemical images. In Figure 19A, immunohistochemistry was performed to identify the tissue specificity of the anti-BAFF-R antibody. Tissue samples were stained using a 1:150 dilution of 1 mg / mL antibody. Tissue specificity of C55 mAb against human BAFF-R (20× objective lens): 1:150 dilution of 1 mg / mL stock. In Figure 19B, immunohistochemistry was additionally performed on tonsil and breast tissue to identify the tissue specificity of the anti-BAFF-R antibody. Tissue samples were stained using a 1:150 dilution of 1 mg / mL antibody. Tissue specificity of mAb against human BAFF-R (upper panel: tonsil tissue; lower panel: breast tissue; 20× objective lens). [Figure 20-1]Figures 20A and 20B are graphs showing functional in vitro assays performed against humanized variants. In Figure 20A, ELISA assays were performed against nine humanized variants of C90. Recombinant extracellular domains of human BAFF-R were used as antigens. Antibodies were administered at variable concentrations from 0.78 to 100 ng / mL, and their absorbances were collected at 450 nm. In Figure 20B, humanized variants were tested against JeKo-1 cells in a chromium release assay. Cells were allowed to take up chromium and subsequently treated with humanized C90 variants and effector NK cells. Cells were incubated for 6 hours, and the supernatant was sampled for chromium levels. [Figure 20-2] Figures 20A and 20B are graphs showing functional in vitro assays performed against humanized variants. In Figure 20A, ELISA assays were performed against nine humanized variants of C90. Recombinant extracellular domains of human BAFF-R were used as antigens. Antibodies were administered at variable concentrations from 0.78 to 100 ng / mL, and their absorbances were collected at 450 nm. In Figure 20B, humanized variants were tested against JeKo-1 cells in a chromium release assay. Cells were allowed to take up chromium and subsequently treated with humanized C90 variants and effector NK cells. Cells were incubated for 6 hours, and the supernatant was sampled for chromium levels. [Figure 21-1] Figures 21A and 21B are graphs showing the analysis of the specific cytotoxicity of humanized antibodies C90-4 and C90-5 in various lymphoma strains. In Figure 21A, chromium release assays were performed on JeKo-1, Z138, and RS4 cells using humanized antibodies C90-4 and C90-5. Antibodies were administered to the cell lines at concentrations between 0 and 5 μg / mL and incubated with NK cells at an E:T ratio of 20:1 for 6 hours. The cell supernatant was analyzed for chromium content. In Figure 21B, chromium release assays were performed on LY-10, MEC-2, RL, and Raji lymphoma strains using humanized antibodies C90-4 and C90-5. Antibodies were administered to the cell lines at a concentration of 5 μg / mL and incubated with NK cells at an E:T ratio of 20:1 for 6 hours. The cell supernatant was analyzed for chromium content. [Figure 21-2] Figures 21A and 21B are graphs showing the analysis of the specific cytotoxicity of humanized antibodies C90-4 and C90-5 in various lymphoma strains. In Figure 21A, chromium release assays were performed on JeKo-1, Z138, and RS4 cells using humanized antibodies C90-4 and C90-5. Antibodies were administered to the cell lines at concentrations between 0 and 5 μg / mL and incubated with NK cells at an E:T ratio of 20:1 for 6 hours. The cell supernatant was analyzed for chromium content. In Figure 21B, chromium release assays were performed on LY-10, MEC-2, RL, and Raji lymphoma strains using humanized antibodies C90-4 and C90-5. Antibodies were administered to the cell lines at a concentration of 5 μg / mL and incubated with NK cells at an E:T ratio of 20:1 for 6 hours. The cell supernatant was analyzed for chromium content. [Figure 22-1] Figures 22A and 22B show FACS results and graphs indicating the leading candidate humanized C90 antibody tested for binding and cytotoxicity to primary MCL samples. In Figure 22A, three primary MCL tumor samples were co-stained with CD20-APC and biotinylated humanized C90, and the signal was subsequently detected using PE-conjugated streptavidin. In Figure 22B, the cytotoxicity of humanized C90 to primary tumor samples was evaluated by a chromium release assay. Cells were incubated with chromium-51 and subsequently treated with the antibody and effector NK cells. Following an overnight incubation, the supernatant was sampled and the chromium content was determined. [Figure 22-2]Figures 22A and 22B show FACS results and graphs indicating the leading candidate humanized C90 antibody tested for binding and cytotoxicity to primary MCL samples. In Figure 22A, three primary MCL tumor samples were co-stained with CD20-APC and biotinylated humanized C90, and the signal was subsequently detected using PE-conjugated streptavidin. In Figure 22B, the cytotoxicity of humanized C90 to primary tumor samples was evaluated by a chromium release assay. Cells were incubated with chromium-51 and subsequently treated with the antibody and effector NK cells. Following an overnight incubation, the supernatant was sampled and the chromium content was determined. [Figure 23] This is a FACS result showing flow cytometry analysis of biotinylated humanized C90-4 and C90-5. PBMCs were stained using antibodies and subsequently detected with a fluorescent PE streptavidin probe. PBMCs were also labeled with granulocyte marker CD66b-PerCP-Cy5.5, monocyte marker CD14-PE-Cy7, B cell marker CD20-APC, T cell marker CD3-PE-Cy5, and NK cell marker CD56-FITC. PBMCs were analyzed by flow cytometry. [Figure 24] This image illustrates in vivo tumor therapy with BAFF-R chimeric antigen receptor (CAR) T cells. Donor T cells were engineered to express a chimeric C55 anti-BAFF-R single-chain (sFv) on a T cell receptor signaling domain containing a 4-1BB motif. NSG mice were attacked with a minimum lethal dose (1 × 10⁶ cells) of NHL JeKo-1-Luci cells. Tumor cells were allowed to engraft until the tumor was detectable by bioluminescence imaging (day 9). Mice were administered either T cell therapy (5 × 10⁶ CAR-T cells) or a control 9 and 15 days after tumor attack administration. Tumor development was tracked by closely monitoring and imaging mice every 3 days. [Modes for carrying out the invention]

[0016] In particular, BAFF-R antibodies containing light chain variable regions and heavy chain variable regions are provided herein. Functional fragments of antibodies are also provided. The BAFF-R antibodies and their functional fragments provided herein can bind to the human BAFF-R protein and induce antibody-dependent cell-mediated cytotoxicity (ADCC) in BAFF-R expressing cells (e.g., B cells). Optionally, the light chain variable regions and heavy chain variable regions of the antibodies provided herein can form part of a chimeric antigen receptor (CAR). Therefore, the compositions and methods provided herein may be used, in particular, for the treatment of cancer (e.g., B-cell malignancies) or autoimmune diseases.

[0017] When used herein, BAFF-R, BAFF receptor, or BAFF-R protein refers to any recombinant or naturally occurring form of B cell activator receptor (BAFF-R), which maintains BAFF-R activity (e.g., at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of activity compared to BAFF-R), and is also known as tumor necrosis factor receptor superfamily member 13C (TNFRSF13C) or its variants or homologs. Optionally, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a continuous portion of 50, 100, 150, or 200 amino acids) compared to naturally occurring BAFF-R. Optionally, BAFF-R is substantially identical to the protein identified by UniProt reference number Q96RJ3 or a variant or homolog having substantial identity to it. Optionally, BAFF-R is substantially identical to the protein identified by UniProt reference number Q9D8D0 or a variant or homolog having substantial identity to it. Optionally, BAFF-R is substantially identical to the protein identified by NCBI reference number GI:16445027 or a variant or homolog having substantial identity to it. Optionally, BAFF-R is substantially identical to the protein identified by NCBI reference number GI:16306481 or a variant or homolog having substantial identity to it.

[0018] A B-cell activator receptor (BAFF-R) antibody is provided, comprising a light chain variable region and a heavy chain variable region. The light chain variable region includes CDR L1 as specified in SEQ ID NO: 1, CDR L2 as specified in SEQ ID NO: 2, and CDR L3 as specified in SEQ ID NO: 3. The heavy chain variable region includes CDR H1 as specified in SEQ ID NO: 4, CDR H2 as specified in SEQ ID NO: 5, and CDR H3 as specified in SEQ ID NO: 6. Optionally, the light chain variable region may also include CDR L1 as specified in SEQ ID NO: 7, CDR L2 as specified in SEQ ID NO: 8, and CDR L3 as specified in SEQ ID NO: 9. The heavy chain variable region may also include CDR H1 as specified in SEQ ID NO: 10, CDR H2 as specified in SEQ ID NO: 11, and CDR H3 as specified in SEQ ID NO: 12. Optionally, the antibody is a humanized antibody. Functional fragments of the disclosed antibodies are also provided.

[0019] Humanized antibodies, such as those provided herein, can bind to the BAFF-R protein and include at least one mouse CDR or a functional fragment or variant of the BAFF-R antibody provided herein (e.g., CDR L1 of SEQ ID NO: 1 or 7, CDR L2 of SEQ ID NO: 2 or 8, CDR L3 of SEQ ID NO: 3 or 9, CDR H1 of SEQ ID NO: 4 or 10, CDR H2 of SEQ ID NO: 5 or 11, CDR H3 of SEQ ID NO: 7 or 13). A functional fragment of a CDR is a portion of the complete CDR amino acid sequence, but an antibody or fragment containing a functional fragment can still bind to the antigen (e.g., BAFF-R). A functional variant of a CDR is a CDR with one or more changes to the CDR sequence, but an antibody or functional fragment containing a functional variant can still bind to the antigen (e.g., BAFF-R). For example, a functional variant of the nucleic acid sequence encoding a CDR may contain one or more changes, but still encode the same amino acid sequence of the CDR. Furthermore, a functional variant of the polypeptide sequence of a CDR can contain one or more amino acid changes, insofar as the antibody or its functional fragment binds to the antigen. Therefore, a functional fragment or variant of a CDR typically contains the amino acid residue required for antibody binding to the antigen (e.g., BAFF-R). If a humanized antibody contains at least one CDR, then at least one CDR or its functional fragment is derived from a donor antibody. Optionally, the donor antibody is a mouse antibody. Those skilled in the art will immediately recognize that a humanized antibody containing at least one mouse CDR is a humanized antibody with at least one mouse CDR derived from a donor antibody, and that the additional CDRs are derived from an acceptor antibody (e.g., if the light chain contains a total of three CDRs and the heavy chain contains a total of three CDRs).

[0020] When the BAFF-R antibody provided herein is a humanized antibody, the antibody may include a humanized heavy chain variable region and / or a humanized light chain variable region. Optionally, the humanized light chain variable region and the humanized heavy chain variable region may include a combined mouse CDR or a functional fragment or variant of a mouse CDR. Thus, the humanized light chain variable region and the humanized heavy chain variable region may include six combined CDRs, where at least one of the six CDRs is a mouse CDR. When the humanized light chain variable region and the humanized heavy chain variable region include a combined mouse CDR, the humanized light chain variable region or the humanized heavy chain variable region includes one mouse CDR. For example, the humanized antibody may include CDR L3 (e.g., mouse, also referred to herein as mouse CDR L3) derived from a donor antibody, and CDR L1, CDR L2, CDR H1, CDR H2, and CDR H3 (i.e., human) derived from an acceptor antibody.

[0021] Optionally, the humanized light chain variable region and the humanized heavy chain variable region include two combined mouse CDRs. When the humanized light chain variable region and the humanized heavy chain variable region include two combined mouse CDRs, the humanized light chain variable region and the humanized heavy chain variable region each include one mouse CDR (i), the humanized light chain variable region includes two mouse CDRs (ii), or the humanized heavy chain variable region includes two mouse CDRs (iii). For example, the humanized antibody may include CDR L3 and CDR H3 derived from a donor antibody (e.g., mouse, also referred to herein as mouse CDR L3 and mouse CDR H3, respectively), and CDR L1, CDR L2, CDR H1, and CDR H2 derived from an acceptor antibody (i.e., human).

[0022] Optionally, the humanized light chain variable region and the humanized heavy chain variable region include three combined mouse CDRs. When the humanized light chain variable region and the humanized heavy chain variable region include three combined mouse CDRs, the humanized light chain variable region may include one mouse CDR and the humanized heavy chain variable region may include two mouse CDRs (i), the humanized light chain variable region may include two mouse CDRs and the humanized heavy chain variable region may include one mouse CDR (ii), the humanized light chain variable region may include three mouse CDRs (iii), or the humanized heavy chain variable region may include three mouse CDRs (iv). For example, the humanized antibody may include CDR L3, CDR H3, and CDR L2 derived from a donor antibody (e.g., mouse, also referred to herein as mouse CDR L3, mouse CDR H3, and mouse CDR L2, respectively), and CDR L1, CDR H1, and CDR H2 derived from an acceptor antibody (i.e., human).

[0023] The humanized light chain variable region and the humanized heavy chain variable region may contain four combined mouse CDRs. When the humanized light chain variable region and the humanized heavy chain variable region contain four combined mouse CDRs, the humanized light chain variable region may contain one mouse CDR and the humanized heavy chain variable region may contain three mouse CDRs (i), the humanized light chain variable region may contain three mouse CDRs and the humanized heavy chain variable region may contain one mouse CDR (ii), or the humanized light chain variable region may contain two mouse CDRs and the humanized heavy chain variable region may contain two mouse CDRs (iii). For example, the humanized antibody may contain CDR L3, CDR H3, CDR L2 and CDR L1 derived from a donor antibody (e.g., mouse, also referred to herein as mouse CDR L3, mouse CDR H3, mouse CDR L2 and mouse CDR L1, respectively), and CDR H1 and CDR H2 (i.e., human) derived from an acceptor antibody.

[0024] Each of the humanized light chain variable region and the humanized heavy chain variable region may contain at least one mouse CDR. When each of the humanized light chain variable region and the humanized heavy chain variable region contains at least one mouse CDR, the humanized light chain variable region contains at least one mouse CDR, and the humanized heavy chain variable region contains at least one mouse CDR. Therefore, the humanized light chain variable region may contain mouse CDR L1, and the humanized heavy chain may contain mouse CDR H1. Optionally, mouse CDR L1 contains the amino acid sequence of SEQ ID NO: 1, and mouse CDR H1 contains the amino acid sequence of SEQ ID NO: 4. Optionally, mouse CDR L1 is the amino acid sequence of SEQ ID NO: 1, and mouse CDR H1 is the amino acid sequence of SEQ ID NO: 4. Optionally, the humanized light chain variable region may contain mouse CDR L2, and the humanized heavy chain variable region may contain mouse CDR H2. Optionally, mouse CDR L2 contains the amino acid sequence of SEQ ID NO: 2, and mouse CDR H2 contains the amino acid sequence of SEQ ID NO: 5. Optionally, mouse CDR L2 has the amino acid sequence of SEQ ID NO: 2, and mouse CDR H2 has the amino acid sequence of SEQ ID NO: 5. Optionally, the humanized light chain variable region includes mouse CDR L3, and the humanized heavy chain variable region includes mouse CDR H3. Optionally, mouse CDR L3 has the amino acid sequence of SEQ ID NO: 3, and mouse CDR H3 has the amino acid sequence of SEQ ID NO: 6. Optionally, CDR L3 has the amino acid sequence of SEQ ID NO: 3, and mouse CDR H3 has the amino acid sequence of SEQ ID NO: 6.

[0025] Optionally, mouse CDR L1 contains the amino acid sequence of SEQ ID NO: 7, and mouse CDR H1 contains the amino acid sequence of SEQ ID NO: 10. Optionally, mouse CDR L1 is the amino acid sequence of SEQ ID NO: 7, and mouse CDR H1 is the amino acid sequence of SEQ ID NO: 10. Optionally, the humanized light chain variable region contains mouse CDR L2, and the humanized heavy chain variable region contains mouse CDR H2. Optionally, mouse CDR L2 contains the amino acid sequence of SEQ ID NO: 8, and mouse CDR H2 contains the amino acid sequence of SEQ ID NO: 11. Optionally, mouse CDR L2 is the amino acid sequence of SEQ ID NO: 8, and mouse CDR H2 is the amino acid sequence of SEQ ID NO: 11. Optionally, the humanized light chain variable region contains mouse CDR L3, and the humanized heavy chain variable region contains mouse CDR H3. Optionally, mouse CDR L3 contains the amino acid sequence of SEQ ID NO: 9, and mouse CDR H3 contains the amino acid sequence of SEQ ID NO: 12. For the purposes of this discussion, CDR L3 is the amino acid sequence of SEQ ID NO: 9, and mouse CDR H3 is the amino acid sequence of SEQ ID NO: 12.

[0026] The presence of mouse CDR L3 and mouse CDR H3 may be sufficient for the binding of the humanized antibody to BAFF-R. Therefore, the humanized antibody does not need to contain mouse CDR L1, mouse CDR L2, CDR H1, or mouse CDR H2. If the humanized antibody does not contain mouse CDR L1, mouse CDR L2, mouse CDR H1, or mouse CDR H2, then the humanized antibody contains CDR L1, CDR L2, CDR H1, or CDR H2 derived from the acceptor antibody (i.e., human). Therefore, a humanized antibody that does not contain mouse CDR L1, mouse CDR L2, mouse CDR H1, or mouse CDR H2 does not contain CDR L1, CDR L2, CDR H1, or CDR H2 from the donor antibody (e.g., mouse, rat, rabbit), but contains CDR L1, CDR L2, CDR H1, or CDR H2 from the acceptor antibody (i.e., human). Therefore, the humanized light chain variable region does not need to contain mouse CDR L1 or mouse CDR L2, and the humanized heavy chain variable region does not contain mouse CDR H1 or mouse CDR H2. Optionally, the humanized light chain variable region does not contain mouse CDR L1 and mouse CDR L2, and the humanized heavy chain variable region does not contain mouse CDR H1 and mouse CDR H2.

[0027] Optionally, the humanized light chain variable region includes mouse CDR L2 and mouse CDR L3, and the humanized heavy chain variable region includes mouse CDR H2 and mouse CDR H3. Optionally, the humanized light chain variable region includes mouse CDR L1, mouse CDR L2 and mouse CDR L3, and the humanized heavy chain variable region includes mouse CDR H1, mouse CDR H2 and mouse CDR H3. Optionally, the humanized light chain variable region includes mouse CDR L1 as specified in SEQ ID NO: 1, mouse CDR L2 as specified in SEQ ID NO: 2, and mouse CDR L3 as specified in SEQ ID NO: 3, and the humanized heavy chain variable region includes mouse CDR H1 as specified in SEQ ID NO: 4, mouse CDR H2 as specified in SEQ ID NO: 5, and mouse CDR H3 as specified in SEQ ID NO: 6. Optionally, the humanized light chain variable region includes mouse CDR L1 as specified in SEQ ID NO: 7, mouse CDR L2 as specified in SEQ ID NO: 8, and mouse CDR L3 as specified in SEQ ID NO: 9, and the humanized heavy chain variable region includes mouse CDR H1 as specified in SEQ ID NO: 10, mouse CDR H2 as specified in SEQ ID NO: 11, and mouse CDR H3 as specified in SEQ ID NO: 12.

[0028] The positions of the CDR and FR can be defined by the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, USD Department of Health and Human Services, US Government Printing Office (1991)). Similarly, the positions occupied by individual residues within the light or heavy chain of an antibody can be defined by the Kabat numbering system. Therefore, the locations of residues required for binding within the humanized light and heavy chains of a humanized antibody can be defined by the positions of the residues according to the Kabat numbering system, as is well known in the art. As described above, a humanized antibody may be an antibody having a CDR from a donor antibody (e.g., mouse) and a variable region framework (FR) from a human antibody. The framework region (FR) is said to hold the CDR in the correct location within the humanized antibody. Proceeding from the amino terminus, these regions are denoted as FR L1, FR L2, FR L3, and FR L4 for the light chain and FR H1, FR H2, FR H3, and FR H4 for the heavy chain, respectively. Humanized antibodies containing one or more residues within the framework region are provided herein. Optionally, these residues are important for epitope binding of the humanized antibody. Framework region residues that are involved in (or important for) epitope binding (e.g., BAFF-R binding) are referred herein to as binding framework region residues. Binding framework region residues may be located within the framework region of the humanized light chain variable region (i.e., FR L1, FR L2, FR L3, FR L4) or they may be located within the framework of the humanized heavy chain variable region (i.e., FR H1, FR H2, FR H3, FR H4). Binding framework residues located within the FR L3 region of the humanized light chain are referred herein to as FR L3 binding framework region residues. Therefore, binding framework region residues present in the FR H3 region of the humanized heavy chain are referred to herein as FR H3 binding framework region residues.

[0029] Optionally, the humanized antibody contains at least one binding framework region residue. Optionally, the humanized light chain variable region contains at least one binding framework region residue. Optionally, the humanized light chain variable region contains one or more FR L1, FR L2, FR L3, or FR L4 binding framework region residues. Optionally, the humanized light chain variable region contains one or more FR L1 binding framework region residues. Optionally, the humanized light chain variable region contains one or more FR L2 binding framework region residues. Optionally, the humanized light chain variable region contains one or more FR L3 binding framework region residues. Optionally, the humanized light chain variable region contains one or more FR L4 binding framework region residues. Optionally, the humanized heavy chain variable region contains one or more FR H1, FR H2, FR H3, or FR H4 binding framework region residues. Optionally, the humanized heavy chain variable region contains one or more FR H1 binding framework region residues. Optionally, the humanized heavy chain variable region includes one or more FR H2 binding framework region residues. Optionally, the humanized heavy chain variable region includes one or more FR H3 binding framework region residues. Optionally, the humanized heavy chain variable region includes one or more FR H4 binding framework region residues.

[0030] The humanized light chain variable region contains at least one binding framework region residue (e.g., residues 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more), and The t-modified heavy chain variable region may contain at least one binding framework region residue (e.g., residues 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50 or more). The location of the binding framework region residue in the humanized antibody may be defined by a Kabat numbering system similar to the location of CDR residues.

[0031] Optionally, the light chain variable region contains serine at the position corresponding to Kabat position 7. Optionally, the light chain variable region contains proline at the position corresponding to Kabat position 8. Optionally, the light chain variable region contains valine at the position corresponding to Kabat position 15. Optionally, the light chain variable region contains threonine at the position corresponding to Kabat position 22. Optionally, the light chain variable region contains glutamine at the position corresponding to Kabat position 24. Optionally, the light chain variable region contains glycine at the position corresponding to Kabat position 41. Optionally, the light chain variable region contains lysine at the position corresponding to Kabat position 42. Optionally, the light chain variable region contains alanine at the position corresponding to Kabat position 43. Optionally, the light chain variable region contains proline at the position corresponding to Kabat position 44. Optionally, the light chain variable region contains threonine at the position corresponding to Kabat position 56. Optionally, the light chain variable region contains threonine at the position corresponding to Kabat position 72. Optionally, the light chain variable region contains phenylalanine at the position corresponding to Kabat position 73. Optionally, the light chain variable region contains glutamine at the position corresponding to Kabat position 79. Optionally, the light chain variable region contains valine at the position corresponding to Kabat position 104.

[0032] Optionally, the light chain variable region may contain serine at the position corresponding to Kabat position 7, proline at the position corresponding to Kabat position 8, valine at the position corresponding to Kabat position 15, threonine at the position corresponding to Kabat position 22, glutamine or serine at the position corresponding to Kabat position 24, glycine at the position corresponding to Kabat position 41, lysine at the position corresponding to Kabat position 42, alanine or threonine at the position corresponding to Kabat position 43, proline at the position corresponding to Kabat position 44, threonine at the position corresponding to Kabat position 56, threonine at the position corresponding to Kabat position 72, phenylalanine or lysine at the position corresponding to Kabat position 73, glutamine at the position corresponding to Kabat position 79, or valine at the position corresponding to Kabat position 104.

[0033] Optionally, the light chain variable region may include serine at the position corresponding to Kabat position 7, proline at the position corresponding to Kabat position 8, valine at the position corresponding to Kabat position 15, threonine at the position corresponding to Kabat position 22, glutamine or serine at the position corresponding to Kabat position 24, glycine at the position corresponding to Kabat position 41, lysine at the position corresponding to Kabat position 42, alanine or threonine at the position corresponding to Kabat position 43, proline at the position corresponding to Kabat position 44, threonine at the position corresponding to Kabat position 56, threonine at the position corresponding to Kabat position 72, phenylalanine or lysine at the position corresponding to Kabat position 73, glutamine at the position corresponding to Kabat position 79, and valine at the position corresponding to Kabat position 104.

[0034] Optionally, the light chain variable region includes binding framework residues such as serine at Kabat position 7, proline at Kabat position 8, valine at Kabat position 15, threonine at Kabat position 22, glutamine or serine at Kabat position 24, glycine at Kabat position 41, lysine at Kabat position 42, alanine or threonine at Kabat position 43, proline at Kabat position 44, threonine at Kabat position 56, threonine at Kabat position 72, phenylalanine or lysine at Kabat position 73, glutamine at Kabat position 79, or valine at Kabat position 104.

[0035] Optionally, the heavy chain variable region contains threonine or alanine at the position corresponding to Kabat position 10. Optionally, the heavy chain variable region contains lysine at the position corresponding to Kabat position 11. Optionally, the heavy chain variable region contains valine at the position corresponding to Kabat position 12. Optionally, the heavy chain variable region contains threonine at the position corresponding to Kabat position 15. Optionally, the heavy chain variable region contains threonine at the position corresponding to Kabat position 19. Optionally, the heavy chain variable region contains threonine at the position corresponding to Kabat position 23. Optionally, the heavy chain variable region contains proline at the position corresponding to Kabat position 41. Optionally, the heavy chain variable region contains alanine at the position corresponding to Kabat position 44. Optionally, the heavy chain variable region contains proline or threonine at the position corresponding to Kabat position 61. Optionally, the heavy chain variable region contains arginine at the position corresponding to Kabat position 66. Optionally, the heavy chain variable region contains threonine at the position corresponding to Kabat position 70. Optionally, the heavy chain variable region contains lysine at the position corresponding to Kabat position 75. Optionally, the heavy chain variable region contains valine at the position corresponding to Kabat position 79. Optionally, the heavy chain variable region contains threonine at the position corresponding to Kabat position 81. Optionally, the heavy chain variable region contains methionine at the position corresponding to Kabat position 82. Optionally, the heavy chain variable region contains asparagine at the position corresponding to Kabat position 82B. Optionally, the heavy chain variable region contains methionine at the position corresponding to Kabat position 82C. Optionally, the heavy chain variable region contains proline at the position corresponding to Kabat position 84. Optionally, the heavy chain variable region contains valine at the position corresponding to Kabat position 85. Optionally, the heavy chain variable region contains lysine at the position corresponding to Kabat position 108. Optionally, the heavy chain variable region includes valine at the position corresponding to Kabat position 109.

[0036] Optionally, the heavy chain variable region is threonine or alanine at the position corresponding to Kabat position 10, lysine at the position corresponding to Kabat position 11, valine at the position corresponding to Kabat position 12, threonine at the position corresponding to Kabat position 15, threonine at the position corresponding to Kabat position 19, threonine at the position corresponding to Kabat position 23, proline at the position corresponding to Kabat position 41, alanine or proline at the position corresponding to Kabat position 44, serine or threonine at the position corresponding to Kabat position 61, arginine at the position corresponding to Kabat position 66, Ka It contains threonine at the position corresponding to bat position 70, lysine at the position corresponding to Kabat position 75, valine at the position corresponding to Kabat position 79, threonine or lysine at the position corresponding to Kabat position 81, methionine at the position corresponding to Kabat position 82, asparagine at the position corresponding to Kabat position 82B, methionine at the position corresponding to Kabat position 82C, proline at the position corresponding to Kabat position 84, valine at the position corresponding to Kabat position 85, lysine at the position corresponding to Kabat position 108, or valine at the position corresponding to Kabat position 109.

[0037] Optionally, the heavy chain variable region can be threonine or alanine at the position corresponding to Kabat position 10, lysine at the position corresponding to Kabat position 11, valine at the position corresponding to Kabat position 12, threonine at the position corresponding to Kabat position 15, threonine at the position corresponding to Kabat position 19, threonine at the position corresponding to Kabat position 23, proline at the position corresponding to Kabat position 41, alanine or proline at the position corresponding to Kabat position 44, serine or threonine at the position corresponding to Kabat position 61, and arginine or K at the position corresponding to Kabat position 66. It contains threonine at the position corresponding to Kabat position 70, lysine at the position corresponding to Kabat position 75, valine at the position corresponding to Kabat position 79, threonine or lysine at the position corresponding to Kabat position 81, methionine at the position corresponding to Kabat position 82, asparagine at the position corresponding to Kabat position 82B, methionine at the position corresponding to Kabat position 82C, proline at the position corresponding to Kabat position 84, valine at the position corresponding to Kabat position 85, lysine at the position corresponding to Kabat position 108, and valine at the position corresponding to Kabat position 109.

[0038] Optionally, the heavy chain variable region is located at the position corresponding to Kabat position 10 (threonine or alanine), the position corresponding to Kabat position 11 (lysine), the position corresponding to Kabat position 12 (valine), the position corresponding to Kabat position 15 (threonine), the position corresponding to Kabat position 19 (threonine), the position corresponding to Kabat position 23 (threonine), the position corresponding to Kabat position 41 (proline), the position corresponding to Kabat position 44 (alanine or proline), the position corresponding to Kabat position 61 (serine or threonine), the position corresponding to Kabat position 66 (arginine), and at Kabat position 70. It contains binding framework region residues that are threonine at the corresponding position, lysine at the position corresponding to Kabat position 75, valine at the position corresponding to Kabat position 79, threonine or lysine at the position corresponding to Kabat position 81, methionine at the position corresponding to Kabat position 82, asparagine at the position corresponding to Kabat position 82B, methionine at the position corresponding to Kabat position 82C, proline at the position corresponding to Kabat position 84, valine at the position corresponding to Kabat position 85, lysine at the position corresponding to Kabat position 108, or valine at the position corresponding to Kabat position 109.

[0039] A humanized BAFF-R antibody is provided, comprising a humanized light chain variable region containing mouse CDR L1, mouse CDR L2, or mouse CDR L3, and a humanized heavy chain variable region containing mouse CDR H1, mouse CDR H2, or mouse CDR H3. The humanized light chain variable region may contain mouse CDR L1 as specified in SEQ ID NO: 1, mouse CDR L2 as specified in SEQ ID NO: 2, or mouse CDR L3 as specified in SEQ ID NO: 3. The humanized light chain variable region may contain mouse CDR L1 as specified in SEQ ID NO: 1, mouse CDR L2 as specified in SEQ ID NO: 2, and mouse CDR L3 as specified in SEQ ID NO: 3. The humanized heavy chain variable region may contain mouse CDR H1 as specified in SEQ ID NO: 4, mouse CDR H2 as specified in SEQ ID NO: 5, or mouse CDR H3 as specified in SEQ ID NO: 6. The humanized heavy chain variable region may include mouse CDR H1 as specified in SEQ ID NO: 4, mouse CDR H2 as specified in SEQ ID NO: 5, and mouse CDR H3 as specified in SEQ ID NO: 6. Optionally, the humanized light chain variable region may include mouse CDR L1 as specified in SEQ ID NO: 1. Optionally, the humanized light chain variable region may include mouse CDR L2 as specified in SEQ ID NO: 2. Optionally, the humanized light chain variable region may include mouse CDR L3 as specified in SEQ ID NO: 3. Optionally, the humanized heavy chain variable region may include mouse CDR H1 as specified in SEQ ID NO: 4. Optionally, the humanized heavy chain variable region may include mouse CDR H2 as specified in SEQ ID NO: 5. Optionally, the humanized light chain variable region may include mouse CDR H3 as specified in SEQ ID NO: 6. In further embodiments, the humanized light chain variable region may include at least one binding framework region residue. In other further embodiments, the humanized heavy chain variable region includes at least one binding framework region residue.

[0040] A humanized BAFF-R antibody is provided, comprising a humanized light chain variable region containing mouse CDR L1, mouse CDR L2, or mouse CDR L3, and a humanized heavy chain variable region containing mouse CDR H1, mouse CDR H2, or mouse CDR H3. The humanized light chain variable region may contain mouse CDR L1 as specified in SEQ ID NO: 7, mouse CDR L2 as specified in SEQ ID NO: 8, or mouse CDR L3 as specified in SEQ ID NO: 9. The humanized light chain variable region may contain mouse CDR L1 as specified in SEQ ID NO: 7, mouse CDR L2 as specified in SEQ ID NO: 8, and mouse CDR L3 as specified in SEQ ID NO: 9. The humanized heavy chain variable region may contain mouse CDR H1 as specified in SEQ ID NO: 10, mouse CDR H2 as specified in SEQ ID NO: 11, or mouse CDR H3 as specified in SEQ ID NO: 12. The humanized heavy chain variable region may include mouse CDR H1 as specified in SEQ ID NO: 10, mouse CDR H2 as specified in SEQ ID NO: 11, and mouse CDR H3 as specified in SEQ ID NO: 12. Optionally, the humanized light chain variable region may include mouse CDR L1 as specified in SEQ ID NO: 7. Optionally, the humanized light chain variable region may include mouse CDR L2 as specified in SEQ ID NO: 8. Optionally, the humanized light chain variable region may include mouse CDR L3 as specified in SEQ ID NO: 9. Optionally, the humanized heavy chain variable region may include mouse CDR H1 as specified in SEQ ID NO: 10. Optionally, the humanized heavy chain variable region may include mouse CDR H2 as specified in SEQ ID NO: 11. Optionally, the humanized light chain variable region may include mouse CDR H3 as specified in SEQ ID NO: 12. In further embodiments, the humanized light chain variable region may include at least one binding framework region residue. In other further embodiments, the humanized heavy chain variable region includes at least one binding framework region residue.

[0041] Optionally, the light chain variable region may contain the sequence of sequence number 18, sequence number 20, or sequence number 22. Optionally, the light chain variable region may contain the sequence of sequence number 18. Optionally, the light chain variable region may contain the sequence of sequence number 20. Optionally, the light chain variable region may contain the sequence of sequence number 22. Optionally, the light chain variable region is the sequence of sequence number 18. Optionally, the light chain variable region is the sequence of sequence number 20. Optionally, the light chain variable region is the sequence of sequence number 22. Optionally, the heavy chain variable region may contain the sequence of sequence number 24, sequence number 26, or sequence number 28. Optionally, the heavy chain variable region may contain the sequence of sequence number 24. Optionally, the heavy chain variable region may contain the sequence of sequence number 26. Optionally, the heavy chain variable region may contain the sequence of sequence number 28. Optionally, the heavy chain variable region is the sequence of sequence number 24. Optionally, the heavy chain variable region is the sequence of SEQ ID NO: 26. Optionally, the heavy chain variable region is the sequence of SEQ ID NO: 28. Therefore, in another embodiment, a humanized BAFF-R antibody is provided that includes a humanized light chain variable region and a humanized heavy chain variable region, where the humanized light chain variable region includes the sequence of SEQ ID NO: 18 and the heavy chain variable region includes the sequence of SEQ ID NO: 24. In another embodiment, a humanized BAFF-R antibody is provided that includes a humanized light chain variable region and a humanized heavy chain variable region, where the humanized light chain variable region includes the sequence of SEQ ID NO: 20 and the heavy chain variable region includes the sequence of SEQ ID NO: 26. In another embodiment, a humanized BAFF-R antibody is provided that includes a humanized light chain variable region and a humanized heavy chain variable region, where the humanized light chain variable region includes the sequence of SEQ ID NO: 22 and the heavy chain variable region includes the sequence of SEQ ID NO: 28.

[0042] Optionally, the antibody is a chimeric antibody. Optionally, the light chain variable region contains the sequence of SEQ ID NO: 14. Optionally, the heavy chain variable region contains the sequence of SEQ ID NO: 16. Optionally, the light chain variable region is the sequence of SEQ ID NO: 14. Optionally, the heavy chain variable region is the sequence of SEQ ID NO: 16. Therefore, in another embodiment, a chimeric BAFF-R antibody is provided comprising a light chain variable region and a heavy chain variable region, where the light chain variable region contains the sequence of SEQ ID NO: 14 and the heavy chain variable region contains the sequence of SEQ ID NO: 16.

[0043] Optionally, the light chain variable region includes the sequence of SEQ ID NO: 30. Optionally, the heavy chain variable region includes the sequence of SEQ ID NO: 32. Optionally, the light chain variable region is the sequence of SEQ ID NO: 30. Optionally, the heavy chain variable region is the sequence of SEQ ID NO: 32. Therefore, in another embodiment, a chimeric BAFF-R antibody is provided that includes a light chain variable region and a heavy chain variable region, where the light chain variable region includes the sequence of SEQ ID NO: 30 and the heavy chain variable region includes the sequence of SEQ ID NO: 32.

[0044] Functional fragments may be used in each of the cases enumerated herein where an antibody is present. Thus, a Fab' fragment is provided which may include, for example, a heavy chain (including, for example, a constant region and a variable region) and a light chain (including, for example, a constant region and a variable region). Optionally, the Fab' fragment may include a humanized heavy chain (including, for example, a constant region and a variable region) and a humanized light chain (including, for example, a constant region and a variable region).

[0045] Optionally, the BAFF-R antibody or its fragment contains a human constant region. Optionally, the BAFF-R antibody or its fragment is IgG. Optionally, the BAFF-R antibody or its fragment is IgG1. Optionally, the BAFF-R antibody or its fragment is IgG2. Optionally, the BAFF-R antibody or its fragment is IgG3. Optionally, the BAFF-R antibody or its fragment is IgG4. Optionally, the BAFF-R antibody or its fragment is IgA. Optionally, the BAFF-R antibody or its fragment is IgM.

[0046] Optionally, BAFF-R antibodies or fragments thereof are single-chain antibodies. Single-chain antibodies contain a variable light chain and a variable heavy chain. Those skilled in the art will immediately recognize that, in contrast to immunoglobulin antibodies (which contain two identical pairs of polypeptide chains, each pair having one light chain and one heavy chain), single-chain antibodies contain a single light chain and a single heavy chain. Each light chain and heavy chain consists of two regions: a variable ("V") region (i.e., the variable light chain and variable heavy chain) involved in binding to the target antigen, and a constant ("C") region that interacts with other components of the immune system. The variable light chain and variable heavy chain in a single-chain antibody can be linked via a linker peptide. An example of a linker peptide for a single-chain antibody is described in Bird, RE, et al., Science. 242(4877):423-6 (1988). A method for producing scFv antibodies is described. See Huse et al., Science 246:1275-1281 (1989); Ward et al., Nature 341:544-546 (1989); and Vaughan et al., Nature Biotech. 14:309-314 (1996). Briefly, mRNA is isolated from B cells from immunized animals and cDNA is prepared. The cDNA is amplified using specific primers for the variable regions of the heavy and light chains of immunoglobulin. The PCR product is purified and the nucleic acid sequences are ligated. If a linker peptide is desired, the nucleic acid sequence encoding the peptide is inserted between the heavy and light chain nucleic acid sequences. The nucleic acid encoding scFv is inserted into the vector and expressed in a suitable host cell.

[0047] The ability of an antibody or its functional fragment to bind to a specific epitope (e.g., BAFF-R) is determined by the equilibrium dissociation constant (K). D It may be described by the equilibrium dissociation constant (K) as defined herein. D ) is the ratio of the dissociation rate (K-off) and association rate (K-on) of the BAFF-R antibody to the BAFF-R protein. It is given by the following formula: K D Described as =K-off / K-on. Optionally, BAFF-R antibodies have an equilibrium dissociation constant (K) lower than approximately 5 nM.D ) can bind to the BAFF-R protein. Optionally, the BAFF-R antibody has an equilibrium dissociation constant (K D ) that can bind to the BAFF-R protein. Optionally, the BAFF-R antibody has an equilibrium dissociation constant (K D ) that can bind to the BAFF-R protein. Optionally, the BAFF-R antibody has an equilibrium dissociation constant (K D ) that can bind to the BAFF-R protein. Optionally, the BAFF-R antibody has an equilibrium dissociation constant (K D ) that can bind to the BAFF-R protein. Optionally, the BAFF-R antibody has an equilibrium dissociation constant (K D ) that can bind to the BAFF-R protein. Optionally, the BAFF-R antibody has an equilibrium dissociation constant (K D ) that can bind to the BAFF-R protein. Optionally, the BAFF-R antibody has an equilibrium dissociation constant (K D ) that can bind to the BAFF-R protein. Optionally, the BAFF-R antibody has an equilibrium dissociation constant (K D ) that can bind to the BAFF-R protein. Optionally, the BAFF-R antibody has an equilibrium dissociation constant (K D ) that can bind to the BAFF-R protein.

[0048] Optionally, the BAFF-R antibody or a functional fragment thereof has an equilibrium dissociation constant (K D ) that can bind to the BAFF-R protein. Optionally, the BAFF-R antibody or a functional fragment thereof has an equilibrium dissociation constant (K D ) that can bind to the BAFF-R protein. Optionally, the BAFF-R antibody or a functional fragment thereof has an equilibrium dissociation constant (K D ) that can bind to the BAFF-R protein. Optionally, the BAFF-R antibody or a functional fragment thereof has an equilibrium dissociation constant (K DIt can bind to the BAFF-R protein at ).Optionally, the BAFF-R antibody or its functional fragment can bind to an equilibrium dissociation constant (K) of approximately 2.5 nM. D It can bind to the BAFF-R protein at ).Optionally, the BAFF-R antibody or its functional fragment has an equilibrium dissociation constant of approximately 3 nM (K). D It can bind to the BAFF-R protein at a rate of approximately 3.5 nM. Optionally, the BAFF-R antibody or its functional fragment can bind to the BAFF-R protein at a rate of approximately 3.5 nM. D It can bind to the BAFF-R protein at ).Optionally, the BAFF-R antibody or its functional fragment has an equilibrium dissociation constant of approximately 4 nM (K). D It can bind to the BAFF-R protein at ).Optionally, the BAFF-R antibody or its functional fragment can bind to an equilibrium dissociation constant (K) of approximately 4.5 nM. D It can bind to the BAFF-R protein at ).Optionally, the BAFF-R antibody or its functional fragment has an equilibrium dissociation constant of approximately 5 nM (K). D It can bind to the BAFF-R protein at ) Optionally, the BAFF-R antibody or its functional fragment can bind to an equilibrium dissociation constant (K) of approximately 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 or 5 nM. D It can bind to the BAFF-R protein.

[0049] Optionally, BAFF-R and K below approximately 4nM D A humanized B-cell activator receptor (BAFF-R) antibody is provided that can be bound to BAFF-R. Optionally, a K2 antibody with a K2 M lower than approximately 4 nM is also provided. D A humanized B-cell activator receptor (BAFF-R) antibody that binds to this antibody is provided. Optionally, the antibody does not induce BAFF-R activity.

[0050] Optionally, the BAFF-R antibody binds to the BAFF-R protein. Optionally, the BAFF-R protein is the human BAFF-R protein. Optionally, the BAFF-R protein is encoded by a nucleic acid sequence identified by NCBI gene identification number 115650. Optionally, the BAFF-R protein forms part of a cell. Optionally, the BAFF-R protein is expressed on the surface of the cell. Optionally, the cell is a lymphocyte-like cell. Optionally, the cell is a B cell. Optionally, the cell is a cancer cell. Optionally, the cancer cell is a lymphoma cell.

[0051] Various diagnostic and therapeutic portions, and combinations thereof, may be conjugated to the BAFF-R antibody or its functional fragment (including embodiments thereof) provided herein, thereby providing highly stable and / or versatile drug delivery and / or diagnostic compositions. Optionally, the BAFF-R antibody or its functional fragment includes a therapeutic portion or a diagnostic portion. Optionally, the therapeutic portion or diagnostic portion is conjugated to the BAFF-R antibody or its functional fragment via a chemical linker. Optionally, the chemical linker is a covalent or non-covalent linker. Techniques for conjugating therapeutic portions into antibodies are well known (e.g., Arnon et al., Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy (Monoclonal Antibodies And Cancer Therapy, edited by Reisfeld et al., pp. 243-56 (Alan R. Liss, Inc. 1985)); Hellstrom et al., Antibodies For Drug Delivery in Controlled Drug Delivery (2nd edition), edited by Robinson et al., pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review in Monoclonal Antibodies '84: Biological And Clinical Applications, edited by Pinchera et al., pp. 475-506 (1985); and Thorpe et al., The Preparation And Cytotoxic Properties Of Antibody-Toxin) See Conjugates, Immunol. Rev., 62:119-58 (1982). As used herein, the terms antibody drug conjugate or ADC refer to a therapeutic moiety conjugated to an antibody or a functional fragment thereof, or otherwise covalently bound to a therapeutic moiety.

[0052] The term "therapeutic portion" as provided herein is used in its plain, ordinary sense and refers to a monovalent compound that, when administered to a target requiring it, provides a therapeutic benefit (e.g., prevention, eradication, or improvement of the underlying disorder being treated). Examples of therapeutic portions provided herein include, but are not limited to, peptides, proteins, nucleic acids, nucleic acid analogs, small molecules, antibodies, enzymes, prodrugs, and cytotoxic agents (e.g., toxins), including, but not limited to, lysine, doxorubicin, daunorubicin, taxol, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxyanthracin dione, actinomycin D, diphtheria toxin, Pseudomonas aeruginosa exotoxin (PE) A, PE40, abrin, and glucocorticoids. Optionally, a therapeutic portion may be an anticancer agent or chemotherapeutic agent as described herein. Optionally, the therapeutic portion is a nucleic acid portion, a peptide portion, or a small molecule drug portion. Optionally, the therapeutic portion is a nucleic acid portion. Optionally, the therapeutic portion is an antibody portion. Optionally, the therapeutic portion is a peptide portion. Optionally, the therapeutic portion is a small molecule drug portion. Optionally, the therapeutic portion is a nuclease. Optionally, the therapeutic portion is an immunostimulant. Optionally, the therapeutic portion is a toxin. Optionally, the therapeutic portion is a nuclease.

[0053] Chimeric antigen receptors (CARs) containing antibodies or functional fragments thereof, as provided herein, are also provided herein.

[0054] Provided herein are isolated nucleic acids encoding BAFF-R antibodies or functional fragments thereof (including embodiments thereof). The BAFF-R antibodies or functional fragments encoded by the isolated nucleic acids are described in detail throughout this application (including the above description and example sections). For example, the nucleic acid may encode at least one CDR, a specific residue involved in epitope binding, or a binding framework residue. For example, the nucleic acid may encode a light chain containing the sequence of SEQ ID NO: 1.

[0055] Optionally, the isolated nucleic acid may contain the sequences of SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 29, or SEQ ID NO: 31. Optionally, the isolated nucleic acid may contain the sequences of SEQ ID NO: 13 and SEQ ID NO: 15. Optionally, the isolated nucleic acid may contain the sequences of SEQ ID NO: 29 and SEQ ID NO: 31.

[0056] Optionally, the isolated nucleic acid may contain the sequences of SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, or SEQ ID NO: 27. Optionally, the isolated nucleic acid may contain the sequences of SEQ ID NO: 17 and SEQ ID NO: 23. Optionally, the isolated nucleic acid may contain the sequences of SEQ ID NO: 19 and SEQ ID NO: 25. Optionally, the isolated nucleic acid may contain the sequences of SEQ ID NO: 21 and SEQ ID NO: 27.

[0057] A pharmaceutical composition is provided comprising a therapeutically effective amount of the BAFF-R antibody or a functional fragment thereof and a pharmaceutically acceptable excipient.

[0058] The therapeutically effective doses provided herein refer to amounts effective in achieving their intended purpose. The actual effective dose for a particular application will depend, among other things, on the condition being treated. When administered in a manner that treats a disease, the pharmaceutical compositions described herein will contain an amount of humanized antibody with sufficient activity to achieve the desired outcome (e.g., modifying the activity of a target molecule (e.g., BAFF-R), and / or reducing, eliminating, or slowing the progression of disease symptoms (e.g., cancer, autoimmune disease)). Determining the therapeutically effective dose of the BAFF-R antibody provided herein is well within the capabilities of those skilled in the art, particularly in light of the detailed disclosure herein.

[0059] Acceptable carriers, excipients, or stabilizers are nontoxic to the recipient at the dosage and concentration used and typically include buffers (phosphates, citrates, or acetates, etc.) at a pH of 5.0–8.0 (optionally 6.0–7.0); salts for isotonicity (sodium chloride, potassium chloride, and similar substances, etc.); antioxidants; preservatives; low molecular weight polypeptides; proteins; hydrophilic polymers (polysorbate 80, etc.); amino acids (glycine, etc.); carbohydrates; chelating agents; sugars; and other standard components known to those skilled in the art (Remington: The Science and Practice of Pharmacy, 22nd edition, edited by Loyd V. Allen et al., Pharmaceutical Press (2012)). mAbs may be present at concentrations of 0.1–100 mg / ml (e.g., 1–10 mg / ml or 10–50 mg / ml, e.g., 5, 10, 20, 30, 40, 50, or 60 mg / ml).

[0060] Pharmaceutical compositions comprising antibodies (e.g., humanized antibodies) or functional fragments thereof as described herein may be administered by a variety of methods known in the art. The route and / or mode of administration will vary depending on the desired outcome. Optionally, administration may be intravenous, intramuscular, intraperitoneal, subcutaneous, or proximal to the target site. Pharmacochemically acceptable excipients may be suitable for intravenous, intramuscular, subcutaneous, parenteral, vertebral, or epidermal administration (e.g., by injection or infusion).

[0061] Pharmaceutical compositions of antibodies or functional fragments thereof may be prepared according to methods practiced in the art in a well-known routine. See, for example, Remington: The Science and Practice of Pharmacy, 22nd edition, edited by Loyd V. Allen et al., Pharmaceutical Press (2012); and Sustained and Controlled Release Drug Delivery Systems, edited by JR Robinson, Marcel Dekker, Inc., New York, 1978. The pharmaceutical compositions are preferably manufactured under GMP conditions. Typically, a therapeutically effective dose or effective dose of a humanized antibody is used in the pharmaceutical composition. The provided humanized antibody may be formulated into a pharmaceutically acceptable dosage form by conventional methods known to those skilled in the art. The dosage regimen is adjusted to provide the optimal desired response (e.g., therapeutic response). For example, a single bolus may be administered, multiple divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the requirements of the treatment situation. Formulating the humanized antibody in combination with other therapies or drugs may be advantageous. For ease of administration and uniformity of dosage, it may be advantageous to formulate parenteral compositions in dose units. When used herein, dose unit refers to a physically separated unit suitable as a single dose for the subject being treated; each unit contains a predetermined amount of humanized antibody intended to produce the desired therapeutic effect in conjunction with the required pharmaceutical excipients.

[0062] The actual dosage level of the active ingredient in a pharmaceutical composition may be varied to obtain an amount of the active ingredient effective in achieving the desired therapeutic response for a particular patient, composition, and mode of administration without toxicity to the patient. The selected dosage level depends on various pharmacokinetic factors, including the activity of the particular composition used, the route of administration, the time of administration, the rate of excretion of the particular antibody used, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular composition used, the age, sex, weight, condition, overall health, and medical history of the patient being treated, as well as homogeneous factors.

[0063] A physician or veterinarian may start with a dose of the antibody or functional fragment used in a pharmaceutical composition at a level lower than required to achieve the desired therapeutic effect, and gradually increase the dose until the desired effect is achieved. In general, the effective dose of a composition may vary depending on different factors (such as the specific disease or condition being treated, the means of administration, the target site, the patient's physiological state, whether the patient is human or animal, other pharmaceuticals being administered, and whether the treatment is preventive or therapeutic). Therapeutic doses should be titrated to optimize safety and efficacy. For antibody administration, the dose is in the range of approximately 0.0001 to 100 mg / kg (usually 0.01 to 5 mg / kg) relative to host body weight. For example, the dose may be 1 mg / kg body weight or 10 mg / kg body weight, or within the range of 1 to 10 mg / kg. Exemplary treatment regimens involve administration once every two or three weeks, once a month, or once every three to six months.

[0064] The BAFF-R antibody or its functional fragments provided herein may be administered on multiple occasions. The interval between single doses may be once a week, once a month, or once a year. The interval may also be irregular, as indicated by measuring the blood levels of humanized antibodies in the patient. In some methods, the dose is adjusted to achieve plasma antibody concentrations of 1–1000 μg / ml, and in some methods, 25–300 μg / ml. Alternatively, the antibody may be administered as a sustained-release formulation, in which case less frequent administration is required. The dose and frequency vary depending on the half-life of the antibody in the patient. Generally, humanized antibodies exhibit longer half-lives than chimeric antibodies and non-human antibodies. The dose and frequency of administration may vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, relatively low doses are administered over a long period at relatively infrequent intervals. Some patients continue treatment for the rest of their lives. In therapeutic applications, sometimes relatively high doses are required at relatively short intervals until the progression of the disease is reduced or terminated, and preferably until the patient shows partial or complete improvement in the symptoms of the disease. Thereafter, the patient may be administered a prophylactic regimen.

[0065] Mouse fibroblasts expressing the human BAFF-R protein or a fragment thereof are provided, and the human BAFF-R protein or fragment thereof is expressed on the cell surface of the cells. Optionally, the human BAFF-R protein or fragment thereof includes a detectable portion. Optionally, the detectable portion is a fluorescent portion. Optionally, the detectable portion is enhanced green fluorescent protein (eGFP).

[0066] A method is provided for treating cancer in a subject that requires it. The method comprises administering a therapeutically effective amount of the chimeric antigen receptor provided herein to a subject, thereby treating cancer in the subject.

[0067] In another embodiment, a method for treating cancer in a subject requiring it is provided, comprising administering a therapeutically effective amount of an antibody or functional fragment thereof provided herein to the subject, thereby treating the cancer in the subject. Optionally, cancer is lymphoma, leukemia, or myeloma. Optionally, cancer is lymphoma. Optionally, lymphoma is mantle cell lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, marginal zone lymphoma, or Burkitt lymphoma. Optionally, lymphoma is mantle cell lymphoma. Optionally, lymphoma is follicular lymphoma. Optionally, lymphoma is diffuse large B-cell lymphoma. Optionally, lymphoma is marginal zone lymphoma. Optionally, lymphoma is Burkitt lymphoma.

[0068] Optionally, cancer is leukemia. Optionally, leukemia is lymphoblastic leukemia, chronic lymphocytic leukemia, or hairy cell leukemia. Optionally, leukemia is lymphoblastic leukemia. Optionally, leukemia is chronic lymphocytic leukemia. Optionally, leukemia is hairy cell leukemia.

[0069] At will, cancer is myeloma. At will, myeloma is multiple myeloma.

[0070] Optionally, the method further comprises administering a second therapeutic agent to the subject. Optionally, the therapeutic agent is a chimeric monoclonal antibody capable of binding to the CD20 antigen. Optionally, the therapeutic agent is rituximab. The term "rituximab" refers in the usual sense to a monoclonal antibody against the protein CD20, identified by the ATC code L01XC02.

[0071] Methods for treating autoimmune diseases in subjects requiring such treatment are also provided. The method comprises administering a therapeutically effective dose of an antibody or a functional fragment thereof, as provided herein, to a subject, thereby treating the autoimmune disease in the subject. Optionally, the autoimmune disease is rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, glomerulonephritis, Sjögren's syndrome, or autoimmune hemolytic anemia. Optionally, the autoimmune disease is rheumatoid arthritis. Optionally, the autoimmune disease is systemic lupus erythematosus. Optionally, the autoimmune disease is multiple sclerosis. Optionally, the autoimmune disease is glomerulonephritis. Optionally, the autoimmune disease is Sjögren's syndrome. Optionally, the autoimmune disease is autoimmune hemolytic anemia. Optionally, the method further comprises administering a second therapeutic agent to the subject.

[0072] In another embodiment, a method for inhibiting cell division and proliferation is provided. The method involves contacting cells with a BAFF-R antibody or a functional fragment thereof (including embodiments thereof) as provided herein, thereby forming contacted cells. The BAFF-R antibody or functional fragment thereof binds to the BAFF-R protein on the contacted cells, thereby inhibiting cell division and proliferation. Optionally, the cells are lymphocyte-like cells. Optionally, the cells are B cells. Optionally, the cells are cancer cells. Optionally, the cells are lymphoma cells.

[0073] In another embodiment, a method for producing an anti-human BAFF-R antibody is provided. The method comprises administering mouse fibroblasts, as provided herein, to a mouse to form an immunized BAFF-R mouse. Splenocytes from the immunized BAFF-R mouse are fused with human myeloma cells to form BAFF-R hybridoma cells. The BAFF-R hybridoma cells are then made to express BAFF-R antibodies, thereby producing an anti-BAFF-R antibody. Optionally, the anti-BAFF-R antibody is an antibody, as provided herein.

[0074] Various embodiments and aspects are shown and described herein, but it will be apparent to those skilled in the art that such embodiments and aspects are provided merely as examples. Numerous variations, changes, and substitutions will come to mind for those skilled in the art. It should be understood that various alternatives to the embodiments described herein may be used.

[0075] Antibodies are large complex molecules with a complex internal structure (molecular weight of approximately 150,000 Da or approximately 1320 amino acids). Natural antibody molecules contain two identical pairs of polypeptide chains, each pair having one light chain and one heavy chain. Each light and heavy chain consists of two regions: a variable ("V") region involved in binding to the target antigen, and a constant ("C") region that interacts with other components of the immune system. The light chain variable region and the heavy chain variable region come together in three-dimensional space to form the variable region that binds to the antigen (e.g., a receptor on the surface of a cell). Within each light chain variable region or heavy chain variable region are three short segments (averaging 10 amino acids in length) called complementarity-determining regions ("CDRs"). The six CDRs in the antibody variable domain (three from the light chain and three from the heavy chain) fold together in three-dimensional space to form the actual antibody binding site (paratope), which docks onto the target antigen (epitope). The location and length of the CDR were precisely defined by Kabat, E. et al., Sequences of Proteins of Immunological Interest, USD Department of Health and Human Services, 1983, 1987. Parts of the variable region not contained within the CDR are called the framework ("FR"), which forms the environment for the CDR.

[0076] The term antibody is used according to its common known meaning in the art. Antibodies exist, for example, as intact immunoglobulins. However, whenever the term antibody(s) is enumerated herein, functional antibody fragment(s) may be used. For example, many well-characterized functional antibody fragments can be produced by digestion with various peptidases. Thus, for example, pepsin digests the antibody under a disulfide bond in the hinge region, and F(ab)'2(Fab(light chain is disulfide bonded V H -C H1 F(ab)'2 produces a dimer of the F(ab)'2 itself (when linked to F(ab)'2). F(ab)'2 can be reduced under mild conditions that break the disulfide bond in the hinge region, thereby converting the F(ab)'2 dimer to a Fab' monomer. The Fab' monomer is essentially a Fab with a portion of the hinge region (see Fundamental Immunology (Paul, ed., 3rd edition (1993))). Various antibody fragments are defined in terms of the digestion of intact antibodies, but those skilled in the art will recognize that such fragments can be synthesized de novo, either chemically or by recombinant DNA methodologies. Thus, the term antibody is illustrative when used herein, and antibody fragments produced by modification of whole antibodies, or those synthesized de novo using recombinant DNA methodologies (e.g., single-stranded Fv) or identified using phage display libraries (e.g., McCafferty et al., Nature 348:552-554 (1990)) may be used as descriptions of antibodies.

[0077] Any technique known in the art may be used to prepare monoclonal or polyclonal antibodies (see, for example, Kohler & Milstein, Nature 256:495-497 (1975); Kozbor et al., Immunology Today 4:72 (1983); Cole et al. (Monoclonal Antibodies and Cancer Therapy (1985), pp. 77-96)). A monoclonal antibody (mAb) refers to an antibody derived from a single clone. Techniques for producing single-chain antibodies (U.S. Patent No. 4,946,778) may be adapted to produce antibodies against the polypeptides described herein. Humanized antibodies can also be expressed using transgenic mice or other organisms (such as other mammals). Alternatively, phage display technology can be used to identify Fab fragments of antibodies and heteromers that specifically bind to selected antigens (see, for example, McCafferty et al., Nature 348:552-554 (1990); Marks et al., Biotechnology 10:779-783 (1992)).

[0078] An mAb epitope is the region of the antigen to which the mAb binds. If each antibody competitively inhibits (blocks) the binding of the other to the antigen, then the two antibodies bind to the same or overlapping epitopes. That is, as measured in competitive binding assays, a 1×, 5×, 10×, 20×, or 100× excess of one antibody inhibits the binding of the other by at least 30%, but preferably 50%, 75%, 90%, or even up to 99% (see, e.g., Junghans et al., Cancer Res. 50:1495, 1990). Alternatively, if essentially all amino acid mutations in the antigen that reduce or eliminate the binding of one antibody also reduce or eliminate the binding of the other, then the two antibodies have the same epitope. If several amino acid mutations that reduce or eliminate the binding of one antibody also reduce or eliminate the binding of the other, then the two antibodies have overlapping epitopes.

[0079] A ligand refers to a drug (e.g., a polypeptide or other molecule) that can bind to a receptor molecule (e.g., an antibody).

[0080] The labeled or detectable portion is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. For example, useful labels include: 32 Examples include phosphate (P), fluorescent dyes, high electron-density reagents, enzymes (such as those typically used in ELISA), biotin, digoxigenin, or haptens and proteins or other entities that can be detected by using radioisotope labeling in peptides or antibodies that are specifically reactive with the target peptide. Any suitable method known in the art may be used for conjugating the antibody to the label (e.g., using the method described in Hermanson, Bioconjugate Techniques 1996, Academic Press, Inc., San Diego).

[0081] Contact, used in its simple and ordinary sense, refers to a process that allows at least two different kinds of things (e.g., chemical compounds, including biomolecules or cells) to react, interact, or come close enough to physically touch. However, it should be recognized that the resulting reaction products can be produced directly from reactions between added reagents or from intermediates from one or more of the added reagents that can be produced in the reaction mixture.

[0082] The term "contact" includes enabling two species to react, interact, or come into physical contact, where the two species could be, for example, an antibody and a BAFF-R protein, as described herein. Contact includes enabling a humanized antibody, for example, as described herein, to interact with BAFF-R.

[0083] As used herein, treatment of a condition, disease, or disorder, or symptoms associated with a condition, disease, or disorder, or treatment thereof, refers to an approach to obtain a beneficial or desired outcome (including clinical outcomes). Beneficial or desired clinical outcomes may include, but are not limited to, the alleviation or improvement of one or more symptoms or conditions, reduction of the severity of a condition, disorder, or disorder, stabilization of the condition, disorder, or disorder, prevention of the onset of a condition, disorder, or disorder, prevention of the transmission of a condition, disorder, or disorder, delay or slowing of the progression of a condition, disorder, or disorder, delay or slowing of the onset of a condition, disorder, or disorder, remission or alleviation of the condition, disorder, or disorder, and remission (whether partial or total). Treatment may also mean long-term survival of the subject beyond what would be expected in the absence of treatment. In some cases, treatment includes permanently halting the progression of a condition, disorder, or disorder, but may also mean inhibiting the progression of a condition, disorder, or disorder, or temporarily slowing the progression of a condition, disorder, or disorder. When used herein, the terms treatment, cure, or cure mean the effect of one or more symptoms of a disease or condition characterized by protease expression, or a method of reducing the symptoms of a disease or condition characterized by protease expression. Accordingly, in the disclosed methods, treatment may mean a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of an established disease, condition, or symptoms of a disease or condition. For example, a method of treating a disease is determined to be a treatment if there is a 10% reduction in one or more symptoms of the disease in the subject compared to a control. Accordingly, the reduction may be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percentage reduction between 10% and 100% compared to the native level or the control level. It is understood that treatment does not necessarily mean the cure or complete elimination of a disease, condition, or the symptoms of a disease or condition.Furthermore, as used herein, references to reduction, decrease, or inhibition include changes of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, and 90% or more compared to a control level, and such terms may, but may not, include complete disappearance.

[0084] The terms polypeptide, peptide, and protein are used interchangeably herein to refer to polymers of amino acid residues, where the polymer may be conjugated to a non-amino acid portion. This term applies to naturally occurring and non-naturally occurring amino acid polymers, as well as amino acid polymers in which one or more amino acid residues are artificial chemical mimics of corresponding naturally occurring amino acids. A fusion protein refers to a chimeric protein encoding two or more separate protein sequences expressed by recombination as a single portion. The terms peptidyl and peptidyl moiety refer to monovalent peptides.

[0085] The term "amino acid," as used herein, refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those that are later modified (e.g., hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine). Amino acid analogs refer to compounds (e.g., homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium) that have the same basic chemical structure as naturally occurring amino acids (i.e., an α-carbon bonded to hydrogen, a carboxyl group, an amino group, and an R group). Such analogs may have a modified R group (e.g., norleucine) or a modified peptide skeleton, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimics refer to chemical compounds that have a structure different from the general chemical structure of amino acids, but that function in a manner similar to naturally occurring amino acids. The terms "naturally occurring amino acids" and "non-natural amino acids" refer to amino acid analogs, synthetic amino acids, and amino acid mimics that are not found in nature.

[0086] Amino acids may be referred herein by either their commonly known three-letter or one-letter symbols, as recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides may similarly be referred by their commonly recognized one-letter codes.

[0087] The term "conservatively modified variant" applies to both amino acid sequences and nucleic acid sequences. For a given nucleic acid sequence, a conservatively modified variant refers to a nucleic acid that codes for the same or essentially the same amino acid sequence. Due to the degeneracy of the genetic code, many nucleic acid sequences will code for any given protein. For example, the codons GCA, GCC, GCG, and GCU all code for the amino acid alanine. Therefore, at every position where alanine is defined by a codon, the codon can be changed to any of the corresponding codons listed without altering the coded polypeptide. Such nucleic acid variations are silent variations, and they are a type of conservatively modified variation. All nucleic acid sequences in this specification that code for polypeptides also describe all possible silent variations of the nucleic acid. Those skilled in the art will recognize that each codon in a nucleic acid (except AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan) can be modified to result in a functionally identical molecule. Thus, each silent variation of a polypeptide-coding nucleic acid is implicit in each described sequence.

[0088] With respect to amino acid sequences, those skilled in the art will recognize that individual substitutions, deletions, or additions to nucleic acid, peptide, polypeptide, or protein sequences (which alter, add, or delete a single amino acid or a small percentage of amino acids in the encoded sequence) are conserved variants if the modification results in the substitution of an amino acid with a chemically similar amino acid. A catalog of conserved substitutions that provide functionally similar amino acids is well known in the art. Such conserved variants are added to, and not excluded from, polymorphic variants, interspecies homologs, and alleles.

[0089] The following 8 groups: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), glutamic acid (E); 3) Asparagine (N), glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), leucine (L), methionine (M), valine (V); 6) Phenylalanine (F), tyrosine (Y), tryptophan (W); 7) Serine (S), threonine (T); and 8) Cysteine ​​(C), Methionine (M) Each of these contains an amino acid that is a conserved substitution with respect to the other (see, for example, Creighton, Proteins (1984)).

[0090] The percentage of sequence identity is determined by comparing two optimally aligned sequences across a comparison window, where, for the optimal alignment of the two sequences, the portion of the polynucleotide or polypeptide sequence in the comparison window may contain additions or deletions (i.e., gaps) compared to a reference sequence (which contains no additions or deletions). The percentage is calculated by determining the number of positions in which identical nucleic acid bases or amino acid residues occur in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity.

[0091] In the context of two or more nucleic acid or polypeptide sequences, the terms identical or percent identical refer to two or more sequences or subsequences that, when compared and aligned for maximum correspondence over a specified region, such as using a comparison window or one of the following sequence comparison algorithms or by manual alignment and visual inspection, are the same or have a specified percentage of identical amino acid residues or nucleotides (i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity over a specified region of the entire polypeptide sequence or individual domains, for example). Such sequences are then said to be substantially identical. This definition also refers to complements of test sequences. Optionally, identity exists over a region of at least about 50 nucleotides in length, more preferably over a region of 100 to 500 or 1000 or more nucleotides in length. This description includes polypeptides substantially identical to any of sequence numbers 30 to 51.

[0092] For sequence comparison, typically one sequence acts as a reference sequence, against which the test sequence is compared. When using a sequence comparison algorithm, the test sequence and reference sequence are input into the computer, sub-sequence coordinates are specified if necessary, and sequence algorithm program parameters are specified. Default program parameters may be used, or alternative parameters may be specified. The sequence comparison algorithm then calculates the percentage sequence identity of the test sequence compared to the reference sequence based on the program parameters.

[0093] When used herein, the comparison window includes, for example, a full-length sequence or a reference to any one segment of consecutive positions selected from the group consisting of 20 to 600, about 50 to about 200, or about 100 to about 150 amino acids or nucleotides, where the sequences can be compared to the same number of consecutive reference sequences after the two sequences have been optimally aligned. Methods for aligning sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith and Waterman (1970) Adv. Appl. Math. 2:482c, by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search method for similarity of Pearson and Lipman (1988) Proc. Nat'l. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package (Genetics Computer Group, 575 Science Dr., Madison, WI)), or by manual alignment and visual inspection (see, for example, Ausubel et al., Current Protocols in Molecular Biology (1995 supplement)).

[0094] Examples of suitable algorithms for determining percent sequence identity and sequence similarity include the BLAST algorithm and the BLAST 2.0 algorithm, which are described in Altschul et al. (1977) Nuc. Acids Res. 25:3389-3402 and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively. Software for performing BLAST real-time analysis is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ). This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of the same length in the query sequence (either matching or satisfying some positive threshold score T) when aligned with a word of length W in the database sequence. T is referred to as the neighbor word score threshold (Altschul et al., ibid.). These initial neighbor word hits act as seeds to initiate the search, finding longer HSPs containing the seeds. Word hits are extended in both directions along each sequence as long as the cumulative alignment score can increase. For nucleotide sequences, the cumulative score is calculated using parameters M (reward score for matching residue pairs; always > 0) and N (penalty score for residue mismatches; always < 0). For amino acid sequences, the cumulative score is calculated using a scoring matrix. The extension of word hits in each direction stops if the cumulative alignment score falls by an amount X from its maximum achieved value; if the cumulative score becomes 0 or less due to the accumulation of one or more negative-scoring residue alignments; or if it reaches the end of either sequence. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program uses a word length of 11 (W), an expected value of 10 (E), M=5, N=-4, and a comparison of both strands as defaults for nucleotide sequences.Regarding amino acid sequences, the BLASTP program uses, by default, a word length of 3, an expected value of 10 (E), and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915), alignment of 50 (B), expected value of 10 (E), M=5, N=-4, and comparison of both strands.

[0095] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the minimum sum probability (P(N)), which provides an indicator of the probability that the match between two nucleotide or amino acid sequences occurs by chance. For example, in the comparison of a test nucleic acid to a reference nucleic acid, if the minimum sum probability is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001, the nucleic acid is considered similar to the reference sequence.

[0096] An indicator that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross-reactive with an antibody made against the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to the second polypeptide if, for example, the two peptides differ only in conservative substitutions. Another indicator that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize with each other under stringent conditions, as described below. Yet another indicator that two nucleic acid sequences are substantially identical is that the sequences can be amplified using the same primers.

[0097] An amino acid residue in an antibody corresponds to a given residue if it occupies the same essential structural position within the antibody as the given residue. For example, a selected residue in a comparative antibody corresponds to position 48 in the antibody provided herein (according to the Kabat numbering system described herein) if the selected residue occupies the same essential spatial or structural relationship to Kabat position 48, as assessed using methods applicable in the art. For example, if the comparative antibody is aligned with the antibody provided herein with maximum sequence homology, the position in the alignment comparative antibody that aligns with Kabat position 48 can be determined to correspond to it. Alternatively, instead of (or in addition to) the primary sequence alignment described above, three-dimensional structural alignment can also be used, for example, if the structure of the comparative antibody is aligned with the antibody provided herein with maximum correspondence and the overall structures are compared. In this case, amino acids that occupy the same essential position as Kabat position 48 in the structural model can be said to correspond.

[0098] The term "isolated," when applied to proteins, means that the protein is essentially free from other cellular components to which it would naturally be bound. It can be either dry or aqueous solution, but is preferably homogeneous. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high-performance liquid chromatography. The dominant protein species present in the preparation is substantially purified. The term "isolated" means that the protein essentially produces a single band on the electrophoretic gel. Specifically, it means that the protein is at least 85% pure, more preferably at least 95% pure, and most preferably at least 99% pure.

[0099] The phrases "specifically (or selectively) bind to an antibody" or "specifically (or selectively) immunoreactive with" refer to a binding reaction that, when referring to a protein or peptide, is a determinant of the presence of a protein in a heterogeneous population of proteins and other biologics. Therefore, under specified immunoassay conditions, a given antibody will bind to a specific protein at least twice the background level and substantially not to other proteins present in the sample in significant amounts. Typically, a specific or selective reaction will be at least twice the background signal or noise, and more typically 10 to 100 times or more the background level.

[0100] When used herein, "cell" refers to a cell that performs metabolic or other functions sufficient to maintain or replicate genomic DNA. Cells can be identified by methods well known in the art (e.g., the presence of an intact membrane, staining with a specific dye, the ability to produce offspring, or, in the case of gametes, the ability to combine with a second gamete to produce viable offspring). Examples of cells include prokaryotes and eukaryotic cells. Examples of prokaryotes include, but are not limited to, bacteria. Examples of eukaryotic cells include, but are not limited to, yeast cells and cells derived from plants and animals (e.g., mammalian cells, insect (e.g., Spodoptera) cells, and human cells).

[0101] When defined herein, the terms inhibitor, inhibit, and similar terms mean, with respect to a protein inhibitor (e.g., a BAFF-R antibody provided herein), negatively affecting (e.g., reducing) the activity or function of a protein compared to the activity or function of the protein in the absence of the inhibitor (e.g., a BAFF-R antibody). Inhibition includes reducing a disease or the symptoms of a disease (e.g., cancer or autoimmune disease). Therefore, inhibition includes blocking a stimulus, reducing signaling or enzyme activity or the amount of a protein, preventing, delaying activation, inactivating, desensitizing, or downregulating, at least partially, partially or completely. Similarly, an inhibitor is a compound or protein that inhibits BAFF-R activity, for example, by binding, partially or completely blocking, reducing, preventing, delaying, inactivating, desensitizing, or downregulating activity (e.g., BAFF-R signaling activity).

[0102] The agents provided herein (e.g., antibodies) are often administered as pharmaceutical compositions comprising active therapeutic agents and a variety of other pharmaceutically acceptable components. See Remington: The Science and Practice of Pharmacy, 22nd edition, edited by Loyd V. Allen et al., Pharmaceutical Press (2012). The preferred form depends on the intended mode of administration and therapeutic application. Depending on the desired formulation, the composition may also include pharmaceutically acceptable non-toxic carriers or diluents (usually defined as vehicles used in formulating pharmaceutical compositions for animal or human administration). The diluents are selected so as not to affect the biological activity of the combination. Examples of such diluents include distilled water, physiological phosphate-buffered saline, Ringer's solution, dextrose solution, and Hanks' solution. In addition, pharmaceutical compositions or formulations may also include other carriers, adjuvants, or non-toxic, non-therapeutic, non-immunogenic stabilizers and similar substances.

[0103] The composition may be administered for therapeutic or prophylactic purposes. In therapeutic applications, the composition is administered to patients suffering from a disease (e.g., cancer) in a therapeutically effective dose. The effective dose for this use will depend on the severity of the disease and the patient's general health condition. Single or multiple doses of the composition may be administered as needed, depending on the dosage and frequency, and may be tolerable to the patient. Patients or subjects may include both humans and other animals (especially mammals). Therefore, the method is applicable to both human treatment and animal applications. Optionally, the patient may be a mammal, a primate, or a human.

[0104] Formulations suitable for oral administration may consist of (a) solutions (an effective amount of the antibody provided herein, etc., suspended in water or saline or a diluent such as PEG 400); (b) capsules, sachets or tablets (each containing a predetermined amount of the active ingredient as a liquid, solid, granule or gelatin); (c) suspensions in a suitable liquid; and (d) suitable emulsions. Tablet forms may contain one or more of the following: lactose, sucrose, mannitol, sorbitol, calcium phosphate, corn starch, potato starch, microcrystalline cellulose, gelatin, colloidal silicon dioxide, talc, magnesium stearate, stearic acid and other excipients, colorants, fillers, binders, diluents, buffers, wetting agents, preservatives, flavoring agents, dyes, disintegrants, and pharmaceutically compatible carriers. Lozenge forms contain the active ingredient in a fragrance (e.g., sucrose), and similarly, lozenges contain the active ingredient in an inert base (gelatin and glycerin or sucrose and acacia emulsion, gel, etc.), and these may also contain carriers known in the art in addition to the active ingredient.

[0105] The pharmaceutical composition may also include large, slowly metabolized macromolecules, such as proteins, polysaccharides (e.g., chitosan), polylactic acid, polyglycolic acid and copolymers (e.g., latex, functionalized Sepharose™, agarose, cellulose, and similar materials), polymeric amino acids, amino acid copolymers, and lipid aggregates (e.g., oil droplets or liposomes). In addition, these carriers may function as immunostimulants (i.e., adjuvants).

[0106] Suitable formulations for rectal administration include, for example, suppositories (consisting of nucleic acids packaged in a suppository base). Suitable suppository bases include natural or synthetic triglycerides or paraffinic hydrocarbons. In addition, gelatin rectal capsules consisting of a combination of a base (e.g., liquid triglycerides, polyethylene glycol, and paraffinic hydrocarbons) and a selected compound can also be used.

[0107] Suitable formulations for parenteral administration (e.g., via intra-articular, intravenous, intramuscular, intratumor, intradermal, intraperitoneal, and subcutaneous routes) include aqueous and non-aqueous isotonic sterile injection solutions (which may contain antioxidants, buffers, bacteriostatic agents, and solutes to make the formulation isotonic with the blood of the intended recipient), and aqueous and non-aqueous sterile suspensions (which may contain suspending agents, solubilizers, thickeners, stabilizers, and preservatives). The composition may be administered orally, topically, intraperitoneally, intravesically, or intrasacrally, for example, by intravenous infusion. Parenteral, oral, and intravenous administration are preferred methods of administration. Formulations of the compound may be provided in sealed containers (ampoules and vials, etc.) in unit or multiple doses.

[0108] Injectable solutions and suspensions may be prepared from the types of sterile powders, granules, and tablets described herein. Cells transduced with nucleic acids for ex vivo therapies may also be administered intravenously or parenterally as described above.

[0109] Pharmaceutical preparations may be in unit dosage form. In such form, the preparation is subdivided into unit doses containing appropriate amounts of active components. A unit dosage form may be a packaged preparation, and the package contains discrete amounts of the preparation (tablets, capsules, powders, etc., divided into vials or ampoules). Furthermore, a unit dosage form may be a capsule, tablet, cachet, or lozenge itself, or it may be an appropriate number of any of these in packaged form. The composition may also contain other suitable therapeutic agents, if desired.

[0110] Combination administration involves co-administration using separate formulations or single pharmaceutical formulations, and a sequence of administrations in either order, where preferably, there is a period during which both (or all) active agents exert their biological activity simultaneously.

[0111] The effective dose of the compositions provided herein may vary depending on different factors (including the means of administration, the target site, the patient's physiological state, whether the patient is human or animal, other medicinal substances administered, and whether the treatment is preventive or therapeutic). However, those skilled in the art will immediately recognize appropriate and / or equivalent doses by considering the dosages of compositions approved for the treatment and prevention of cancer, as provided for guidance.

[0112] The term "disease" or "condition" refers to a condition being treated by the compounds, pharmaceutical compositions, or methods provided herein, or to a health condition of a patient or subject that can be treated in this manner. Optionally, a disease is cancer (e.g., lung cancer, ovarian cancer, osteosarcoma, bladder cancer, cervical cancer, liver cancer, kidney cancer, skin cancer (e.g., Merkel cell carcinoma), testicular cancer, leukemia, lymphoma, head and neck cancer, colorectal cancer, prostate cancer, pancreatic cancer, melanoma, breast cancer, neuroblastoma). A disease may be an autoimmune disease, an inflammatory disease, a cancerous disease, an infectious disease, a metabolic disease, a developmental disease, a cardiovascular disease, a liver disease, a gastrointestinal disease, an endocrine disease, a nervous system disease, or any other disease.

[0113] As used herein, the term cancer refers to all types of cancer, neoplasms, or malignant tumors found in mammals, including leukemia, lymphoma, melanoma, neuroendocrine tumors, carcinomas, and sarcomas. Exemplary cancers that can be treated by the compounds, pharmaceutical compositions, or methods provided herein include lymphoma, sarcoma, bladder cancer, bone cancer, brain tumor, cervical cancer, colon cancer, esophageal cancer, gastric cancer, head and neck cancer, kidney cancer, myeloma, thyroid cancer, leukemia, prostate cancer, breast cancer (e.g., triple-negative, ER-positive, ER-negative, chemotherapy-resistant, Herceptin-resistant, HER2-positive, doxorubicin-resistant, tamoxifen-resistant, tubal carcinoma, lobular carcinoma, primary, metastatic), and ovarian cancer. These include pancreatic cancer, liver cancer (e.g., hepatocellular carcinoma), lung cancer (e.g., non-small cell lung cancer, squamous cell lung cancer, adenocarcinoma, large cell lung cancer, small cell lung cancer, carcinoid, sarcoma), glioblastoma pleomorphonosum, glioma, melanoma, prostate cancer, castration-resistant prostate cancer, breast cancer, triple-negative breast cancer, glioblastoma, ovarian cancer, lung cancer, squamous cell carcinoma (e.g., head, neck, or esophagus), colorectal cancer, leukemia, acute myeloid leukemia, lymphoma, B-cell lymphoma, or multiple myeloma. Additional examples include thyroid cancer, endocrine cancer, brain tumors, breast cancer, cervical cancer, colorectal cancer, head and neck cancer, esophageal cancer, liver cancer, kidney cancer, lung cancer, non-small cell lung cancer, melanoma, mesothelioma, ovarian cancer, sarcoma, gastric cancer, uterine cancer or medulloblastoma, Hodgkin's disease, non-Hodgkin lymphoma, multiple myeloma, neuroblastoma, glioma, glioblastoma multiforme, ovarian cancer, rhabdomyosarcoma, primary thrombocythemia, primary macroglobulinemia, primary brain tumors, cancer, malignant pancreatic insulin These include rinoma, malignant carcinoid, bladder cancer, pre-malignant skin lesions, testicular cancer, lymphoma, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary cancer, malignant hypercalcemia, endometrial cancer, adrenocortical carcinoma, neoplasms of the pancreatic endocrine or exocrine glands, medullary thyroid carcinoma, medullary thyroid carcinoma, melanoma, colorectal cancer, papillary thyroid carcinoma, hepatocellular carcinoma, Paget's disease of the breast, phyllodes tumor, lobular carcinoma, tubular carcinoma, stellate cell carcinoma of the pancreas, stellate cell carcinoma of the hepatic gland, or prostate cancer.

[0114] The term leukemia broadly refers to a progressive malignant disease of the hematopoietic organs, generally characterized by the distorted division, proliferation, and development of white blood cells and their precursors in the blood and bone marrow. Leukemia is generally clinically classified based on (1) the duration and characteristics of the disease (acute or chronic); (2) the type of cells involved; myeloid (myelogenic), lymphogenic (lymphogenic), or monocytic; and (3) an increase or non-increase in the number of abnormal cells in the blood (leukemic or non-leukemic (subleukemic)). Exemplary leukemias that can be treated by the compounds, pharmaceutical compositions, or methods provided herein include, for example, acute non-lymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, non-leukemic leukemia, and leukocystic leukemia. Leukemia, basophilic leukemia, blastocyte leukemia, bovine leukemia, chronic myeloid leukemia, cutaneous leukemia, stem cell leukemia, eosinophilic leukemia, Gross leukemia, hairy cell leukemia, hemoblastic leukemia, hemocytoblastic leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenic leukemia, lymphocytic leukemia, lymphoblastic leukemia, lymphoid leukemia, lymphangiogenic leukemia, lymphocytic leukemia, Lymphosarcoma leukemia, mast cell leukemia, megakaryocytic leukemia, micromyeloblastic leukemia, monocytic leukemia, myeloblastic leukemia, myeloid leukemia, myeloid granulocytic leukemia, myelomonocytic leukemia, Naegeli's leukemia, plasma cell leukemia, multiple myeloma, plasma cell leukemia, promyelocytic leukemia, Rieder cell leukemia, Schilling's leukemia, stem cell leukemia, subleukemia, or anaplastic cell leukemia.

[0115] As used herein, the terms metastasis and metastatic cancer are used interchangeably and may refer to the propagation of a mitotic disease or disorder (e.g., cancer) from one organ or another non-adjacent organ or part of the body. Cancer originates in a site of origin (e.g., the breast), which is referred to as the primary tumor (e.g., primary breast cancer). Some cancer cells in the primary tumor or site of origin acquire the ability to penetrate and invade the surrounding normal tissue in the local area, and / or to penetrate the walls of the circulatory lymphatic or vascular system through the system to other parts and tissues throughout the body. A second clinically detectable tumor formed from cancer cells of the primary tumor is referred to as a metastatic tumor or secondary tumor. When cancer cells metastasize, it is presumed that the metastatic tumor and its cells are similar to those of the original tumor. Therefore, if lung cancer metastasizes to the breast, the secondary tumor in the breast site will consist of abnormal lung cells rather than abnormal mammary gland cells. The secondary tumor in the breast is referred to as metastatic lung cancer. Therefore, the term metastatic cancer refers to a disease in which the subject has or has had a primary tumor and has one or more secondary tumors. The term non-metastatic cancer, or a subject with non-metastatic cancer, refers to a disease in which the subject has a primary tumor but does not have one or more secondary tumors. For example, metastatic lung cancer refers to a disease in a subject in which the subject has or has a history of a primary lung tumor and has one or more secondary tumors at a second or multiple locations (e.g., in the breast).

[0116] The terms "associated with" or "associated with" mean, in the context of a substance or the activity or function of a substance associated with a disease (e.g., cancer (e.g., leukemia, lymphoma, B-cell lymphoma, or multiple myeloma)), that the substance or the activity or function of a substance causes (in whole or in part) the disease (e.g., cancer (e.g., leukemia, lymphoma, B-cell lymphoma, or multiple myeloma)) or the symptoms of the disease (in whole or in part).

[0117] As used herein, autoimmune disease refers to a disease or disorder resulting from a modification of the immune response of the subject's immune system to substances, tissues and / or cells that are normally present in the subject's body. Examples of autoimmune diseases include, but are not limited to, arthritis, rheumatoid arthritis, psoriatic arthritis, juvenile idiopathic arthritis, scleroderma, systemic scleroderma, multiple sclerosis, systemic lupus erythematosus (SLE), myasthenia gravis, juvenile diabetes mellitus, type 1 diabetes mellitus, Guillain-Barré syndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis, ankylosing spondylitis, psoriasis, Sjögren's syndrome, vasculitis, glomerulonephritis, autoimmune thyroiditis, Behçet's disease, Crohn's disease, ulcerative colitis, bullous pemphigoid, sarcoidosis, psoriasis, ichthyosis, Graves' ophthalmopathy, inflammatory bowel disease, Addison's disease, vitiligo, asthma, and allergic asthma.

[0118] As used herein, inflammatory disease refers to a disease or disorder associated with an abnormality or alteration of inflammation. Inflammation is a biological response initiated by the immune system as part of a healing process in response to pathogens, damaged cells or tissues, or irritants. Chronic inflammation can lead to a variety of diseases. Inflammatory diseases include, but are not limited to, atherosclerosis, allergies, asthma, rheumatoid arthritis, transplant rejection, celiac disease, chronic prostatitis, inflammatory bowel disease, pelvic inflammatory disease, and inflammatory muscle disease.

[0119] Humanized antibodies are genetically modified antibodies in which at least one CDR (or a functional fragment or variant thereof) from a mouse antibody ("donor antibody," which may be from a rat, hamster, or other non-human species) is grafted onto a human antibody framework ("acceptor antibody"). Optionally, two or more mouse CDRs are grafted (e.g., all six mouse CDRs are grafted). The sequence of the acceptor antibody may be, for example, a mature human antibody sequence (or a fragment thereof), a consensus sequence of a human antibody sequence (or a fragment thereof), or a germline region sequence (or a fragment thereof). Thus, a humanized antibody may be an antibody having one or more CDRs from a donor antibody and a variable region framework (FR). The FR may form part of the constant region and / or variable region within the human antibody. In addition, to maintain high binding affinity, amino acids in the human acceptor sequence may be replaced by corresponding amino acids from the donor sequence, for example, (1) the amino acid is located in the CDR, or (2) the amino acid is located in the human framework region (e.g., the amino acid is directly adjacent to one of the CDRs). See U.S. Patents 5,530,101 and 5,585,089 (incorporated herein by reference) for detailed instructions for constructing humanized antibodies. Humanized antibodies often incorporate all six CDRs (including the hypervariable loop H1, as defined by Kabat, e.g., but often by Chothia) from mouse antibodies, but they can also be constructed from fewer mouse CDRs and / or smaller than the complete mouse CDR sequence (e.g., functional fragments of CDRs) (e.g., Pascalis et al., J.Immunol.169:3076,2002; Vajdos et al., Journal of Molecular Biology, 320:415-428,2002; Iwahashi et al., Mol.Immunol.36:1079-1091,1999; Tamura et al, Journal of Immunology, 164:1432-1441,2000).

[0120] Typically, a humanized antibody as provided herein may comprise (i) a light chain variable region comprising at least one CDR (often three CDRs) (also referred to herein as mouse CDRs) and a human variable region framework from a mouse antibody; and (ii) a heavy chain variable region comprising at least one CDR (often three CDRs) and a human variable region framework (FR) from a mouse antibody. The light chain and heavy chain variable region frameworks (FRs) may each be a mature human antibody variable region framework sequence (or a fragment thereof), a germline variable region framework sequence (in combination with a J region sequence) (or a fragment thereof), or a consensus sequence (or a fragment thereof) of a human antibody variable region framework sequence. Optionally, a humanized antibody may comprise a light chain variable region as described in (i) and a heavy chain variable region as described in (ii), together with the light chain human constant region and the heavy chain human constant region.

[0121] Chimeric antibodies are antibodies in which the variable region of a mouse (or other rodent) antibody is combined with the constant region of a human antibody; the construction of chimeric antibodies using genetic engineering is well known. Such antibodies retain the binding specificity of the mouse antibody, while approximately two-thirds are human. The ratio of non-human sequences present in mouse antibodies, chimeric antibodies, and humanized antibodies suggests that the immunogenicity of chimeric antibodies is intermediate between that of mouse antibodies and humanized antibodies. Other types of genetically modified antibodies that may have reduced immunogenicity compared to mouse antibodies include human antibodies produced using phage display (Dower et al., WO91 / 17271; McCafferty et al., WO92 / 001047; Winter, WO92 / 20791; and Winter, FEBS Lett. 23:92, 1998, each incorporated herein by reference) or using genetically modified animals (Lonberg et al., WO93 / 12227; Kucherlapati WO91 / 10741, each incorporated herein by reference).

[0122] Other approaches to designing humanized antibodies can achieve the same results as those described in U.S. Patents No. 5,530,101 and 5,585,089, such as hyperhumanization as described in Tan et al. J.Immunol. 169:1119, 2002 and U.S. Patent No. 6,881,557 or Studnicak et al., Protein Eng. 7:805, 1994. Furthermore, other approaches to producing genetically engineered, reduced-immunogenic mAbs include, for example, reshaping, hyperchimerization, and veneering / resurfacing, as described in Vaswami et al., Annals of Allergy, Asthma and Immunology 81:105, 1998; Roguska et al. Protein Eng. 9:895, 1996; and U.S. Patents No. 6,072,035 and No. 5,639,641.

[0123] Materials, compositions, and components that may be used for, in combination with, or in preparation of the methods and compositions disclosed herein, or are products thereof. These and other materials are disclosed herein, and where combinations, subsets, interactions, and groups of these materials are disclosed, specific references to various individual and collective combinations and permutations of each of these compounds are not explicitly disclosed, but each is understood to be specifically intended and described herein. For example, if a method is disclosed and examined, and many modifications (including methods) that can be made to many molecules are examined, then each and all combinations and permutations of the methods and possible modifications are specifically intended unless specifically pointed out to be in conflict. Similarly, any subset or combination thereof is also specifically intended and disclosed. This concept applies to all aspects of this disclosure, including, but not limited to, steps in methods using the disclosed compositions. Therefore, if there are various additional steps that can be performed, it should be understood that each of these additional steps can be performed by any specific method step or combination of method steps of the disclosed method, and that such combination or subset of combinations should each be deemed to have been specifically contemplated and disclosed.

[0124] The following examples are intended to further illustrate specific embodiments of the methods and compositions described herein and are not intended to limit the scope of the claims. [Examples]

[0125] Example 1. A novel BAFF receptor antibody against a natively folded recombinant protein clears drug-resistant human B-cell malignancies in vivo. Conventional recombinant immunogenic proteins produced in bacteria for mAb development lack post-translational modifications, and prokaryotes, compared to eukaryotes, lack chaperone proteins and oxidative environments, resulting in simplified folding. Consequently, such proteins may differ conformally from the corresponding plasma membrane-anchored native proteins. Furthermore, antibodies can be produced against off-target domains (such as the transmembrane or intracellular domain of the target protein). As described herein, strategies were applied to generate mAbs against natively folded and glycosylated immunogens expressed on eukaryotic cells. In particular, human BAFF-R is a native protein on mouse fibroblasts, and cell clones engineered as immunogens in mice were used. The generation of novel mAbs that specifically bind and lyse human malignant B cell lines and primary lymphoma cells in vitro, and inhibit the proliferation of drug-resistant lymphoma cell lines in vivo in heterologous tumor models, is described herein.

[0126] Materials and methods Animals, cell lines, and primary human tumor samples. BALB / c mice and NOD scidγ (NSG) breeding pairs were purchased from Jackson Laboratory (Bar Harbor, ME) for antibody development. NSG breeding colonies were maintained by the Animal Resource Center at City of Hope. Mice were housed in pathogen-free animal facilities in accordance with institutional guidelines. All animal studies were approved by the Institutional Animal Care and Use Committee (IACUC:15020). JeKo-1, SU-DHL-6, Raji, U266, and RL were purchased from ATCC (Manassas (VA)). The Z-138 strain was provided by Dr. Michael Wang (MD Anderson Cancer Center). The ibrutinib-resistant SP49-IR strain was developed and provided by Dr. Jianguo Tao (University of South Florida). The ibrutinib-resistant SP49 cell line (SP49-IR) was established by treating cells with escalating doses of ibrutinib. IC50 was 5 nM in parental SP49 compared to >100 nM for SP49-IR. Approximately 5% of SP49 cells were viable with 100 nM ibrutinib, compared to >90% of SP49-IR cells. Human NK-92 176V cells were obtained from Conkwest Inc. (San Diego, CA). For human blood and tumor samples, uncultured primary human lymphoma cells were obtained from the Lymphoma Satellite Tissue Bank at MD Anderson Cancer Center as viable single-cell suspensions cryopreserved in 10% DMSO under an institutional review board approved protocol (IRB:2005-0656). Primary patient samples included leukocytapheresis transfusions or blood from patients with mantle cell lymphoma (MCL) or chronic lymphocytic leukemia (CLL), as well as excised lymph nodes from patients with diffuse large B-cell lymphoma (DLBCL) or follicular lymphoma (FL).Tumor cells in each sample ranged from 80% to 98% in leukocytapheresis transfusions or blood samples, and from 50% to 60% in lymph node biopsies. Peripheral blood mononuclear cells (PBMCs) were provided by the Michael Amini Transfusion Medicine Center at City of Hope (IRB:15283).

[0127] Generation of human BAFF-R expressing mouse fibroblasts. The human BAFF-R (hBAFF-R) cDNA was derived from human B cells and cloned in-frame with the GFP gene on a pEGFP-N1 vector (Takara / Clotech, Mountain View, CA). The hBAFF-R cDNA sequence was confirmed against the NCBI gene sequence database (gene identification number: 115650). The cDNA encoding the hBAFF-R-GFP fusion was then cloned into a lentiviral gene delivery system (pLenti6 / V5-DEST Gateway Vector kit, Life Technologies, Grand Island, NY) to induce the production of the hBAFF-R-GFP fusion protein when transduced into mouse fibroblast (L) cells. Single-cell clones were established from selected GFP-positive L cells, and the (h)BAFF-R-GFP expressing L cell clone D2C was used in further research.

[0128] Antibody-producing hybridomas. Two 6-week-old BALB / c mice were immunized every 3 days by subcutaneous injection of D2C cells into five leg pads. Blood samples were obtained from both mice, and serum antibodies against D2C were measured by ELISA. Spleen tissue was harvested on day 20. Harvested spleen cells were fused with Sp2 / 0 myeloma cells to establish hybridomas, and antibody screening was performed using plates coated with D2C or parental L cells via ELISA. Immunization and hybridoma procedures were performed at the Antibody Core Facility of MD Anderson Cancer Center.

[0129] Chimeric antibody production. cDNA from selected hybridomas encoding the variable regions of the antibody's light and heavy chains was manipulated onto expression vectors containing the respective human IgG1 constant regions. The vectors were co-transfected into a FreeStyle 293 expression system (Life Technologies, Carlsbad, CA) according to the manufacturer's instructions. The antibodies in the culture supernatant were purified by HiTrap protein A affinity chromatography column (GE Healthcare, Marlborough, MA) according to the manufacturer's instructions.

[0130] Cytotoxicity assay. Target cells (L cells, human tumor lines, primary patient samples) were labeled with chromium-51 (51Cr, Perkin Elmer, Waltham, MA) for the 51Cr release assay. Briefly, the antibody and effector (NK cells or complement serum standard [Sigma Aldrich (St. Louis), MO]) were added to the labeled target cells and incubated for up to 18 hours. NK cells were enriched from PBMCs (NK cell enrichment kit, Stemcell Technologies, Vancouver, Canada). 51Cr released into the supernatant was detected by a Wizard Automatic Gamma Counter (Perkin Elmer).

[0131] Generation of JeKo-1-CD20-KO. Stable JeKo-1-CD20-KO, selected via FACS, was generated using CD20-CRISPR / Cas9 and HDR Plasmid Systems (Santa Cruz Biotechnology, Santa Cruz, CA) according to the manufacturer's instructions. CD20 knockout was verified by flow cytometry and Western blotting.

[0132] In vivo research. For tumor models, stable luciferase-expressing tumor cells were established for bioluminescence imaging in mouse models. Briefly, the luciferase gene was introduced into tumor cells using a lentiviral gene delivery system (pLenti7.3 / V5-DEST Gateway Vector Kit, Life Technologies, Carlsbad, CA). The minimum lethal dose per mouse was determined for each tumor cell line by dose-titer measurement. Tumor cells were injected intravenously (IV), and mice were monitored by in vivo bioluminescence imaging to ensure engraftment at the minimum tumor dose. The minimum lethal tumor dose was 1 × 10⁻⁶. 6 JeKo-1 cells, 5 × 10 5 RS4;11 cells, 5 × 10 5 JeKo-1-CD20-KO cells, or 2.5 × 10⁶ cells. 4 These were Z-138 cells.

[0133] Bioluminescence imaging: Mice were anesthetized with isoflurane and administered 150 mg / kg of D-luciferin (Life Technologies, Carlsbad, CA) via intraperitoneal (IP) injection 10 minutes prior to imaging. Imaging was performed on the AmiX imaging system (Spectral Instruments Imaging, Tucson, AZ).

[0134] Antibody study: Mice (n=5 per group) were given an intravenous injection to attack tumors 3 days before four treatments, once every 5 days. The treatment consisted of a 300 μL intravenous injection (200 μg of therapeutic antibody, 10 × 10⁻¹⁴). 6 Effector human NK-92-176V cells, and 5 × 10 4 The treatment involved IU of IL-2 (Prometheus Laboratories, San Diego, CA). The control group received the same volume of injection with or without the control antibody and / or NK cells. Bioluminescence imaging was performed weekly until day 80. Survival was followed up until 100 days after tumor-attack administration.

[0135] result Generation of monoclonal antibodies against human BAFF-R. To generate therapeutic antibodies against the biologically significant epitope of BAFF-R, a eukaryotic cell surface expression system was used, where endogenous cell surface proteins are provided in their native conformations through appropriate post-translational modifications. Mouse fibroblast (L) cell clones were engineered to express human BAFF-R with cell surface GFP tagging. BAFF-R-expressing L cell clones were generated and characterized for GFP expression (Figure 1A). Clones D2C were amplified and used to successfully immunize BALB / c mice according to the method and immunization schedule in Figure 7A.

[0136] After generating and screening hybridoma clones, four clones (53, 55, 67, and 90) were identified as producing antibodies that specifically bind to BAFF-R-expressing L cells but not to parental L cells (Figure 7B). The supernatants of all four clones contained antibodies that bound to BAFF-R-expressing Mino cell lines (MCL) in a dose-dependent manner. Antibody binding was not detected in BAFF-R-negative control cell lines (293T) (Figure 8).

[0137] Antibodies from four hybridoma supernatants were purified by protein A affinity chromatography. The purified antibodies were dose-dependently conjugated to Mino cells (Figure 9) as well as other human MCL strains (including JeKo-1, REC-1, and ibrutinib-resistant JVM-13 and Z-138) (Figure 1B).

[0138] Analysis of the complementarity-determining regions (CDRs) on four antibodies revealed that clones 53, 55, and 67 have nearly identical sequences, while clone 90 is unique. Therefore, clones 55 and 90 were selected for further investigation. Both clones 55 and 90 were found at high concentrations (2 μg / 10 6 Cells) and low concentrations (0.05 μg / 10 6 JeKo-1 (MCL), SU-DHL-6 (DLBCL), Raji (Burkitt lymphoma), and RL (FL) were effectively bound to cells (Figure 10).

[0139] Chimeric mAbs against human BAFF-R induced antitumor effects in vitro and in vivo. Clones 55 and 90 were further developed into chimeric mAbs containing the human IgG1 constant region (referred to as C55 and C90). The chimeric antibodies retained specific dose-dependent binding to BAFF-R-expressing L cells (Figure 1C). C55 and C90 were conjugated to Alexa Fluor 488 and represented direct binding to JeKo-1, SU-DHL-6, Raji, and RL in non-Hodgkin lymphoma (NHL) strains (Figure 1D). Importantly, the chimeric mAbs readily bound to patient primary tumor samples of MCL, DLBCL, and FL (Figures 1E and 11).

[0140] C55 and C90 specifically induced antibody-dependent cell-mediated cytotoxicity (ADCC) against BAFF-R-expressing L cells and JeKo-1, but not against BAFF-R-negative L cells or BAFF-R-negative human multiple myeloma cell line (U266) (Figures 2A and 12). In contrast, the antibodies did not induce complement-dependent cell-mediated cytotoxicity (CDC) in vitro (Figure 2B). As shown for SU-DHL-6, Raji, and RL lymphoma cell lines, cytotoxicity required the addition of NK cells (Figures 2C and 13), suggesting ADCC as the primary mechanism of antibody-mediated cytotoxicity. Importantly, chimeric antibodies induced ADCC against primary patient tumor samples (Figure 3A).

[0141] The antibodies inhibited BAFF / BAFF-R binding in a dose-dependent manner (Figure 14), suggesting the potential disruption of BAFF / BAFF-R survival signaling in tumor cells. Furthermore, C55 and C90 showed limited internalization upon BAFF-R binding (Figure 15).

[0142] In vivo, NSG mice were administered luciferase knock-in JeKo-1 MCL cell line as an attack, followed by antibody treatment. The treatment schedule was as shown in Figure 4A. Mice administered with either C55 or C90 demonstrated a significant delay in tumor growth compared to control groups treated with PBS or NK cells alone (Figure 4B). Similarly, C55 and C90 significantly delayed tumor growth in NSG mice attacked with RS4;11 (acute lymphoblastic leukemia (ALL)) compared to those without inhibition by rituximab or control (Figure 4C).

[0143] Chimeric mAbs induced potent antitumor effects against drug-resistant lymphomas both in vitro and in vivo. Antibodies were further tested against primary CLL (n=3) and MCL (n=2) samples from patients previously treated with rituximab. All five primary samples were susceptible to ADCC killing by C55 and C90, suggesting their efficacy against clinically advanced tumors after rituximab exposure (Figure 3B).

[0144] To create a model of drug-resistant lymphoma, stable CD20 knockout (KO) clones of JeKo-1 were generated using a CRISPR / HDR system. The CD20-KO clones were confirmed to lack CD20 surface expression by flow cytometry and Western blotting (Figure 5A and Figures 16A and 16B), and to have BAFF-R surface expression by flow cytometry (Figure 16C). JeKo-1-CD20-KO clone 25, selected for further study, retained sensitivity to C55 and C90-mediated ADCC but became insensitive to anti-CD20 rituximab-mediated cytotoxicity (Figure 5B).

[0145] As a second model for drug-resistant lymphoma, chimeric BAFF-R mAb was tested for ADCC against a naturally occurring ibrutinib-resistant human MCL strain (Z-138) and an induced ibrutinib-resistant MCL strain (SP49-IR, ibrutinib resistance induced in vitro (see Methods)). Significant in vitro ADCC was observed by antibody against both ibrutinib-resistant strains (Figure 5C).

[0146] Finally, three days after in vivo IV-mediated attack administration of JeKo-1-CD20-KO tumor cells, NSG mice (n=5 per group) were administered either BAFF-R antibody therapy (C55 or C90) or rituximab, as described in the methods and according to the schedule in Figure 4A. Bioluminescence imaging at day 20 revealed substantial tumor burden in control and rituximab-treated mice, but no visible tumors in the BAFF-R antibody-treated group (Figure 6A). Tumor-free monitoring and long-term overall survival confirmed significant antitumor effects of both BAFF-R antibodies, but not rituximab (Figure 6C). Similarly, significant effects were observed following treatment of ibrutinib-resistant Z-138 tumor-bearing mice with either BAFF-R antibody compared to controls (PBS or NK only) (Figures 6B-C).

[0147] BAFF-R mAbs also bind to normal B cells. When tested against normal PBMCs, the anti-BAFF-R antibody C90 showed specific binding to B cells as expected, without staining any T cells, NK cells, granulocytes, or monocytes (Figure 17). Positive staining results were verified with purified B cells (Figure 18). Furthermore, it was shown that purified T cells, NK cells, and gated myeloid cells did not bind.

[0148] Expanding the scope of this study, immunohistochemical studies showed positive staining of the antibody of the present invention in tonsil and spleen samples, while all other vital organs, including the heart, lungs, kidneys, and brain, were not stained (Figures 19A and 19B).

[0149] Consideration The provided BAFF-R mAb induced steady in vivo antitumor effects as a monotherapy against multiple B-cell tumor types, including NHL, CLL, and ALL. Furthermore, the antibody eradicated established tumors, leading to long-term tumor-free survival in vivo.

[0150] The distinctive properties of the BAFF-R mAbs may be due to the approach used for their production. The approach provided involves expressing human BAFFF-R as a native surface protein on mouse fibroblasts for immunization, increasing the likelihood of presenting a natively folded and glycosylated immunogen. Therefore, it is highly probable that the antibody binds to an accessible human BAFF-R epitope different from the other antibodies described. Thus, a technical strategy was demonstrated for the production of a monoclonal antibody against natively folded and eukaryotically glycosylated human BAFF-R (which specifically binds, lyses, and inhibits B-cell tumors in vivo). These results suggest that the primary antitumor mechanism of the mAbs of the present invention is ADCC, as NK cells were required in addition to the mAb for in vitro activity (Figure 2); no evidence for CDC was observed. Both antibodies were able to competitively inhibit the binding of BAFF ligands to BAFF-R (Figure 14).

[0151] One clinically significant mechanism of resistance to rituximab is CD20 downregulation. This phenomenon of drug resistance was modeled by a CRISPR-edited MCL strain (JeKo-1, which lacks CD20). Significant in vivo antitumor effects of C55 or C90 against this strain, and similarly against naturally occurring ibrutinib-resistant Z-138 MCL (whereas rituximab treatment did not have this effect), suggest efficacy against drug-resistant lymphoma (Figure 5). When combined with in vitro cytotoxicity of these antibodies against primary tumors from lymphoma patients previously treated with rituximab and whose response progressed, these data suggest the potential of C55 and C90 as therapeutic strategies to overcome drug resistance (Figure 3).

[0152] Example 2. Humanization of BAFF-R mAb. The chimeric antibody (clone 90) was humanized while retaining its binding specificity and cytotoxic effects. Three heavy chain variants and three light chain variants were produced via computer analysis of CDRs and predicted structures, based on their similarity to the fluctuating human antibody. A total of nine combinations of variants were constructed from the humanized heavy and light chains. All of these variants had a K content in the range of 2.6–5.0 nM. D The value was such that it was proven to be comparable to the parent chimeric antibody in terms of binding affinity (Table 1). [Table 1]

[0153] Nine candidate antibodies were further evaluated, and the most promising candidate was determined. The humanized antibodies exhibited specificity to BAFF-R binding, and all were observed to have similar relative binding in a dose-dependent manner (Figure 20A). In addition, the ADCC effect of the humanized antibodies was assessed. In this case as well, the humanized candidates were found to maintain specific cytotoxicity and perform equally well compared to the chimeric control and rituximab (Figure 20B).

[0154] Humanized clone 90 variants 4 and 5 were selected for further in vitro testing. The humanized antibody variants were biotinylated and visualized using a fluorescent streptavidin probe. Their binding to various non-Hodgkin lymphoma, lymphoblastic leukemia, and multiple myeloma strains (including JeKo-1, Ly-10, MEC-2, RL, RS4, Raji, Z138, and U266) was assessed (Figure 22A). Flow cytometry results revealed significant binding to each of these cell lines. Further flow analysis of the humanized variants against normal PBMCs demonstrated specificity in binding. When assessing binding to granulocytes, monocytes, B cells, T cells, and NK cells in normal, healthy PBMCs, the antibody bound only to the B cell population (Figure 22B).

[0155] Two variants were further assessed for their ability to initiate ADCC. After a period of chromium uptake, JeKo-1, Z138, and RS4 cell lines were administered antibodies at varying concentrations. Cells and antibodies were incubated with effector NK cells. The supernatant was analyzed 6 hours after treatment (Figure 21A). The antibodies exhibited clear cytotoxic effects against the tumor lines, demonstrating dose-dependence with each 10-fold dilution. The results were comparable to those of rituximab, but could also be observed in RS4 acute lymphoblastic lymphoma, where rituximab is inactive. Further assays with LY-10, MEC-2, RL, and Raji (Figure 21B) were continued to demonstrate the efficacy of humanized antibody therapy. All results found were comparable to those of current conventional treatment with rituximab.

[0156] Example 3. Chimeric antigen receptor T cells. Chimeric antigen receptor (CAR) T cells for in vivo studies were constructed using antibodies with high binding affinity and bioactivity. DNA sequences for the heavy and light chain variable domains were arranged in a single-stranded (sFv) format and engineered into the T cell signaling domain (δ chain) along with the 4-1BB motif. The engineered CAR gene, along with co-expressed GFP, was introduced into purified healthy donor CD8+ T cells via lentivirus. CAR-T cells were sorted for GFP expression and augmented in vitro with CD3 and CD28 beads for animal studies. NSG mice were attacked with JeKo-1 MCL strain expressing luciferase (JeKo-1-luci). Tumor development was induced and monitored by bioluminescence imaging until a visually observable population of tumor cells was observed, approximately 9 days after tumor attack administration. Mice were subjected to 5 × 10⁶ fluorescein bioluminescence imaging 9 and 15 days after tumor attack administration. 6 Two doses of CAR-T cells (anti-BAFF-R and anti-CD19) were administered. The control group received either untreated T cells or saline (PBS). To evaluate the therapeutic antitumor effect of CAR-T therapy, tumor development was tracked by closely monitoring and imaging the mice every three days.

[0157] Humanized anti-BAFF-R mAbs were further assessed for their binding and cytotoxicity against primary patient tumor samples. Three mantle cell lymphoma patient samples were characterized by the expression of BAFF-R in the majority of tumor cells. Flow cytometry results revealed distinct populations of these primary tumor cells bound by the humanized antibodies of the present invention (Figure 22A). Furthermore, chromium-releasing cytotoxicity assays on the same primary tumor samples revealed highly specific killing compared to controls. The results were comparable to the effect of rituximab and consistent with previously developed chimeric antibodies (Figure 22B). Cell type specificity of the humanized antibodies was determined by assessing their binding to normal PBMCs. Sensible binding was not noted for the major groups of PBMCs (including granulocytes, monocytes, T cells, and NK cells). The B cell population was the only detectable population bound by the antibody (Figure 23). The results of this assay were also consistent with previously characterized chimeric antibodies.

[0158] Chimeric antigen receptor (CAR) T cells were created using anti-BAFF-R mAbs. The experiment utilized a chimeric C55 variable region engineered into a single-strand (sFv) format. Anti-BAFF-R C55 sFv was attached to a T cell receptor signaling domain containing a 4-1BB motif and successfully introduced into healthy, normal human donor CD8+ T cells isolated from PBMCs. CAR-T cells were administered to tumor-bearing mice with a detectable tumor burden (Figure 24). Mice treated with anti-BAFF-R CAR-T cells had significant tumor clearance compared to either a saline or unengineered T cell control group. In addition, the antitumor effect of the CAR-T cells of this invention is comparable to that of anti-CD-19 CAR-T therapy.

[0159] The chimeric anti-BAFF-R antibody C90 was humanized using multiple variants. The humanization process took into account the analysis of the variable region of the chimeric antibody, particularly the CDR. From this, three variants were developed for each heavy and light chain, with similarity to humans ranging from 1 (most human) to 3 (most conserved to the chimeric form). Nine variants were produced by combining these variants. Biacore analysis was performed on each variant in addition to the parent chimeric C90 to determine their equilibrium dissociation constants K D The antigen was determined to be the extracellular domain of commercially recombinant human BAFF-R.

Claims

1. A B-cell activator receptor (BAFF-R) antibody comprising a light chain variable region and a heavy chain variable region, The light chain variable region is Includes CDR L1 as specified in Sequence ID 1, CDR L2 as specified in Sequence ID 2, and CDR L3 as specified in Sequence ID 3; The aforementioned heavy chain variable region is Includes CDR H1 as specified in Sequence ID No. 4, CDR H2 as specified in Sequence ID No. 5, and CDR H3 as specified in Sequence ID No. 6, The antibody.

2. The antibody according to claim 1, wherein the antibody is a humanized antibody.

3. The antibody according to any one of claims 1 or 2, wherein the light chain variable region includes the sequence of SEQ ID NO: 18, SEQ ID NO: 20, or SEQ ID NO:

22.

4. The antibody according to any one of claims 1 to 5, wherein the heavy chain variable region includes the sequence of SEQ ID NO: 24, SEQ ID NO: 26, or SEQ ID NO:

28.

5. The antibody according to claim 1, wherein the antibody is a chimeric antibody.

6. The antibody according to claim 5, wherein the light chain variable region includes the sequence of sequence number 14.

7. The antibody according to any one of claims 5 or 6, wherein the heavy chain variable region includes the sequence of SEQ ID NO:

16.

8. A B-cell activator receptor (BAFF-R) antibody comprising a light chain variable region and a heavy chain variable region, The light chain variable region is Including CDR L1 as specified in Sequence ID 7, CDR L2 as specified in Sequence ID 8, and CDR L3 as specified in Sequence ID 9; The aforementioned heavy chain variable region is Includes CDR H1 as specified in Sequence ID 10, CDR H2 as specified in Sequence ID 11, and CDR H3 as specified in Sequence ID 12, The antibody.

9. The antibody according to claim 8, wherein the antibody is a humanized antibody.

10. The antibody according to claim 8, wherein the antibody is a chimeric antibody.

11. The antibody according to claim 10, wherein the light chain variable region includes the sequence of SEQ ID NO:

30.

12. The antibody according to any one of claims 10 or 11, wherein the heavy chain variable region includes the sequence of SEQ ID NO:

32.

13. The antibody according to any one of claims 1 to 12, wherein the antibody is IgG.

14. The antibody according to any one of claims 1 to 13, wherein the antibody is IgG1.

15. The antibody according to any one of claims 1 to 12, wherein the antibody is a Fab' fragment.

16. The antibody according to any one of claims 1 to 12, wherein the antibody is a single-chain antibody (scFv).

17. The aforementioned antibody has an equilibrium dissociation constant (K) lower than approximately 5 nM. Ｄ The antibody according to any one of claims 1 to 16, which can bind to the BAFF-R protein.

18. The aforementioned antibody has an equilibrium dissociation constant (K) lower than approximately 4 nM. Ｄ The antibody according to any one of claims 1 to 17, which can bind to the BAFF-R protein.

19. The antibody according to any one of claims 1 to 18, wherein the antibody is bound to the BAFF-R protein.

20. The antibody according to claim 19, wherein the BAFF-R protein is human BAFF-R protein.

21. The antibody according to any one of claims 18 or 19, wherein the BAFF-R protein forms part of a cell.

22. The antibody according to claim 21, wherein the BAFF-R protein is expressed on the surface of the cell.

23. The antibody according to any one of claims 21 or 22, wherein the cells are lymphocyte-like cells.

24. The antibody according to any one of claims 21 to 23, wherein the cell is a B cell.

25. The antibody according to any one of claims 21 to 24, wherein the cells are cancer cells.

26. The antibody according to claim 25, wherein the cancer cells are lymphoma cells.

27. An isolated nucleic acid encoding an antibody according to any one of claims 1 to 26.

28. A pharmaceutical composition comprising a therapeutically effective amount of the antibody described in any one of claims 1 to 26 and a pharmaceutically acceptable excipient.

29. A chimeric antigen receptor (CAR) comprising an antibody or a functional fragment thereof according to any one of claims 1 to 26.

30. A method for treating cancer in a subject in need thereof, comprising administering a therapeutically effective amount of the chimeric antigen receptor described in claim 29 to the subject, thereby treating the cancer in the subject.

31. A method for treating cancer in a subject in need thereof, comprising administering a therapeutically effective amount of an antibody according to any one of claims 1 to 26 to the subject, thereby treating the cancer in the subject.

32. The method according to any one of claims 30 or 31, wherein the cancer is lymphoma, leukemia, or myeloma.

33. The method according to claim 32, wherein the lymphoma is mantle cell lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, marginal zone lymphoma, or Burkitt lymphoma.

34. The method according to claim 32, wherein the leukemia is lymphoblastic leukemia, chronic lymphocytic leukemia, or hairy cell leukemia.

35. The method according to claim 32, wherein the myeloma is multiple myeloma.

36. The method according to any one of claims 30 to 32, further comprising administering a second therapeutic agent to the subject.

37. The method according to claim 36, wherein the therapeutic agent is a chimeric monoclonal antibody capable of binding to the CD20 antigen.

38. The method according to either claim 36 or 37, wherein the therapeutic agent is rituximab.

39. A method for treating an autoimmune disease in a subject in need thereof, comprising administering a therapeutically effective amount of an antibody according to any one of claims 1 to 26 to the subject, thereby treating the autoimmune disease in the subject.

40. The method according to claim 39, wherein the autoimmune disease is rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, glomerulonephritis, Sjögren's syndrome, or autoimmune hemolytic anemia.

41. The method according to claim 40, further comprising administering a second therapeutic agent to the subject.

42. A method for inhibiting cell division and proliferation, (i) Contacting a cell with the BAFF-R antibody according to any one of claims 1 to 26, thereby forming the contacted cell; (ii) The BAFF-R antibody binds to the BAFF-R protein on the contacted cells, thereby inhibiting the division and proliferation of the cells. The method, including the method described above.

43. The method according to claim 42, wherein the cells are lymphocyte-like cells.

44. The method according to any one of claims 42 or 43, wherein the cell is a B cell.

45. The method according to any one of claims 42 to 44, wherein the cells are cancer cells.

46. The method according to any one of claims 42 to 45, wherein the cells are lymphoma cells.

47. BAFF-R and K is lower than approximately 4 nM Ｄ A humanized B-cell activator receptor (BAFF-R) antibody that can bind to it.

48. BAFF-R has a K value lower than approximately 4 nM. Ｄ A humanized B-cell activator receptor (BAFF-R) antibody that binds to it.

49. The humanized antibody according to any one of claims 47 or 48, wherein the antibody does not induce BAFF-R activity.

50. Mouse fibroblasts expressing human BAFF-R protein, wherein the human BAFF-R protein is expressed on the cell surface of the mouse fibroblasts.

51. The mouse fibroblast according to claim 50, comprising a portion in which the human BAFF-R protein can be detected.

52. The mouse fibroblast according to claim 51, wherein the detectable portion is a fluorescent portion.

53. The mouse fibroblast according to any one of claims 51 or 52, wherein the detectable portion is enhanced green fluorescent protein (eGFP).

54. A method for producing an anti-human BAFF-R antibody, (i) administering mouse fibroblasts according to any one of claims 50 to 53 to a mouse to form an immunized BAFF-R mouse; (ii) fusing spleen cells from the immunized BAFF-R mouse with human myeloma cells to form BAFF-R hybridoma cells; (iii) The BAFF-R hybridoma cells express BAFF-R antibodies, thereby producing anti-BAFF-R antibodies, The method, including the method described above.

55. The method of claim 54, wherein the anti-BAFF-R antibody is the antibody described in any one of claim 1 or claim 8.

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