BAFF-R antibodies and uses thereof

Abstract

To provide B cell activating factor receptor (BAFF-R) antibodies as well as compositions and methods of making and using the same, which are useful for treatment of cancer and autoimmune diseases.SOLUTION: A BAFF-R antibody is provided which comprises a light chain variable region and a heavy chain variable region, where the light chain variable region comprises a CDR L1, a CDR L2 and a CDR L3 each having specific amino acid sequence, and where the heavy chain variable region comprises a CDR H1, a CDR H2 and a CDR H3 each having specific amino acid sequence. Also provided are pharmaceutical compositions comprising the antibody, and chimeric antigen receptors comprising the antibody.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 346,324, filed June 6, 2016, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] Antibody therapy is one of the most successful immunotherapies available in the clinic for treating hematological malignancies.An example is rituximab, which targets CD20 and induces cytotoxic effect on B-cell lymphoma.However, the main concern about rituximab is the occurrence of rituximab resistance, which is thought to be due to the downregulation of CD20, thus preventing antibody from binding to target cells. Summary of the Invention

[0003] Provided herein is a B-cell activating factor receptor (BAFF-R) antibody comprising a light chain variable region and a heavy chain variable region. 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 a lower K of approximately 4 nM DA humanized B-cell activating factor receptor (BAFF-R) antibody or functional fragment thereof capable of binding to BAFF-R is provided.

[0005] BAFF-R and a lower K of approximately 4 nM D Also provided are humanized B-cell activating factor receptor (BAFF-R) antibodies that bind at

[0006] Also provided are chimeric antigen receptors (CARs) comprising the antibodies or functional fragments thereof provided herein.

[0007] Provided herein are isolated nucleic acids encoding BAFF-R antibodies or functional fragments of the antibodies.

[0008] Also provided is a pharmaceutical composition comprising a therapeutically effective amount of a BAFF-R antibody or functional fragment thereof as disclosed herein and a pharmaceutically acceptable excipient.

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

[0010] Methods for treating cancer in a subject in need thereof are provided, comprising administering to the subject a therapeutically effective amount of a chimeric antigen receptor provided herein, thereby treating the cancer in the subject.

[0011] Further provided is a method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an antibody or functional fragment thereof disclosed herein, thereby treating cancer in the subject.

[0012] Methods for treating an autoimmune disease in a subject in need thereof are provided, comprising administering to the subject a therapeutically effective amount of an antibody or functional fragment thereof as disclosed herein, thereby treating the autoimmune disease in the subject.

[0013] Also provided is a method for inhibiting cell proliferation. The method comprises contacting a cell with a BAFF-R antibody or functional fragment thereof as disclosed herein, thereby forming a contacted cell. The BAFF-R antibody or functional fragment thereof binds to the BAFF-R protein on the contacted cell, thereby inhibiting cell proliferation. Optionally, the cell is a lymphoid cell.

[0014] A method for producing an anti-human BAFF-R antibody or a functional fragment thereof is provided. The method comprises 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 the BAFF-R antibody, thereby producing the anti-BAFF-R antibody. [Brief explanation 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 for 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) 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 / 10 cells. The BAFF-R-negative 293T embryonic kidney cell line was used as a control. Figure 1C shows the binding of chimeric antibodies C55 and C90 to hBAFF-R-expressing L cells at high and low concentrations. Parental L cells and a secondary anti-hIgG-APC antibody alone were used as controls. Figure 1D shows a panel of Alexa Fluor 488-conjugated chimeric antibodies binding to NHL cell lines. Figure 1E shows the binding of chimeric antibodies to three types of NHL primary patient samples. Data are representative of three independent experiments. For all Figures 1B-1E, the top-to-bottom traces shown in the figures correlate with the top-to-bottom values ​​of the variables used (e.g., antibody type or cell type) shown below or adjacent to the figures. [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). Gating for 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) 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 / 10 cells. The BAFF-R-negative 293T embryonic kidney cell line was used as a control. Figure 1C shows the binding of chimeric antibodies C55 and C90 to hBAFF-R-expressing L cells at high and low concentrations. Parental L cells and a secondary anti-hIgG-APC antibody alone were used as controls. Figure 1D shows a panel of Alexa Fluor 488-conjugated chimeric antibodies binding to NHL cell lines. Figure 1E shows the binding of chimeric antibodies to three types of NHL primary patient samples. Data are representative of three independent experiments. For all Figures 1B-1E, the top-to-bottom traces shown in the figures correlate with the top-to-bottom values ​​of the variables used (e.g., antibody type or cell type) shown below or adjacent 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). Gating for 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) 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 / 10 cells. The BAFF-R-negative 293T embryonic kidney cell line was used as a control. Figure 1C shows the binding of chimeric antibodies C55 and C90 to hBAFF-R-expressing L cells at high and low concentrations. Parental L cells and a secondary anti-hIgG-APC antibody alone were used as controls. Figure 1D shows a panel of Alexa Fluor 488-conjugated chimeric antibodies binding to NHL cell lines. Figure 1E shows the binding of chimeric antibodies to three types of NHL primary patient samples. Data are representative of three independent experiments. For all Figures 1B-1E, the top-to-bottom traces shown in the figures correlate with the top-to-bottom values ​​of the variables used (e.g., antibody type or cell type) shown below or adjacent 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). Gating for 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) 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 / 10 cells. The BAFF-R-negative 293T embryonic kidney cell line was used as a control. Figure 1C shows the binding of chimeric antibodies C55 and C90 to hBAFF-R-expressing L cells at high and low concentrations. Parental L cells and a secondary anti-hIgG-APC antibody alone were used as controls. Figure 1D shows a panel of Alexa Fluor 488-conjugated chimeric antibodies binding to NHL cell lines. Figure 1E shows the binding of chimeric antibodies to three types of NHL primary patient samples. Data are representative of three independent experiments. For all Figures 1B-1E, the top-to-bottom traces shown in the figures correlate with the top-to-bottom values ​​of the variables used (e.g., antibody type or cell type) shown below or adjacent 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). Gating for 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) 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 / 10 cells. The BAFF-R-negative 293T embryonic kidney cell line was used as a control. Figure 1C shows the binding of chimeric antibodies C55 and C90 to hBAFF-R-expressing L cells at high and low concentrations. Parental L cells and a secondary anti-hIgG-APC antibody alone were used as controls. Figure 1D shows a panel of Alexa Fluor 488-conjugated chimeric antibodies binding to NHL cell lines. Figure 1E shows the binding of chimeric antibodies to three types of NHL primary patient samples. Data are representative of three independent experiments. For all Figures 1B-1E, the top-to-bottom traces shown in the figures correlate with the top-to-bottom values ​​of the variables used (e.g., antibody type or cell type) shown below or adjacent to the figures. [Figure 2-1]Figures 2A, 2B, and 2C are graphs showing that BAFF-R monoclonal antibodies exhibited specific in vitro cytotoxicity against B-cell tumor lines. Antibody-induced cytotoxicity was measured by chromium-51 release after incubation with C55, C90, or rituximab and effectors (NK cells or complement-containing serum) at a 20:1 NK effector cell:target ratio (E:T). 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 CDC-sensitive (Raji) and CDC-resistant (Raji-2P) cells by antibodies (1:3 dilution) mixed with active, complement-containing human serum. Figure 2C shows the ADCC effect of BAFF-R chimeric antibodies with or without NK effector cells (E:T = 20:1) against NHL lines JeKo-1, SU-DHL-6, Raji, and RL. Data are shown as the mean ± standard deviation of triplicate samples. *P < 0.05 compared to NK cells by two-tailed Student's t-test. [Figure 2-2]Figures 2A, 2B, and 2C are graphs showing that BAFF-R monoclonal antibodies exhibited specific in vitro cytotoxicity against B-cell tumor lines. Antibody-induced cytotoxicity was measured by chromium-51 release after incubation with C55, C90, or rituximab and effectors (NK cells or complement-containing serum) at a 20:1 NK effector cell:target ratio (E:T). 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 CDC-sensitive (Raji) and CDC-resistant (Raji-2P) cells by antibodies (1:3 dilution) mixed with active, complement-containing human serum. Figure 2C shows the ADCC effect of BAFF-R chimeric antibodies with or without NK effector cells (E:T = 20:1) against NHL lines JeKo-1, SU-DHL-6, Raji, and RL. Data are shown as the mean ± standard deviation of triplicate samples. *P < 0.05 compared to NK cells by two-tailed Student's t-test. [Figure 2-3]Figures 2A, 2B, and 2C are graphs showing that BAFF-R monoclonal antibodies exhibited specific in vitro cytotoxicity against B-cell tumor lines. Antibody-induced cytotoxicity was measured by chromium-51 release after incubation with C55, C90, or rituximab and effectors (NK cells or complement-containing serum) at a 20:1 NK effector cell:target ratio (E:T). 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 CDC-sensitive (Raji) and CDC-resistant (Raji-2P) cells by antibodies (1:3 dilution) mixed with active, complement-containing human serum. Figure 2C shows the ADCC effect of BAFF-R chimeric antibodies with or without NK effector cells (E:T = 20:1) against NHL lines JeKo-1, SU-DHL-6, Raji, and RL. Data are shown as the mean ± standard deviation of triplicate samples. *P < 0.05 compared to NK cells by two-tailed Student's t-test. [Figure 3-1] Figures 3A and 3B are graphs showing that BAFF-R monoclonal antibodies induce 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 from rituximab-refractory patients (E:T = 20:1). Data are presented as the mean ± standard deviation of triplicate samples. *P < 0.05 compared to NK cells by two-tailed Student's t-test. [Figure 3-2]Figures 3A and 3B are graphs showing that BAFF-R monoclonal antibodies induce 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 from rituximab-refractory patients (E:T = 20:1). Data are presented as the mean ± standard deviation of triplicate samples. *P < 0.05 compared to NK cells by two-tailed Student's t-test. [Figure 4-1] Figure 4A is a schematic diagram showing the treatment schedule following day 0 tumor challenge with the minimal lethal dose of tumor. Treatment was administered via IV tail vein injection. Figures 4B and 4C are images showing the in vivo therapeutic efficacy of chimeric antibodies targeting human BAFF-R against B cell tumors. Bioluminescence images of mice challenged with luciferase-expressing tumors: JeKo-1 (MCL) (Figure 4B) or RS4;11 (ALL) (Figure 4C). Experimental groups received chimeric BAFF-R mAb treatment (C55 or C90, as indicated). Control mice received 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 day 0 tumor challenge with the minimal lethal dose of tumor. Treatment was administered via IV tail vein injection. Figures 4B and 4C are images showing the in vivo therapeutic efficacy of chimeric antibodies targeting human BAFF-R against B cell tumors. Bioluminescence images of mice challenged with luciferase-expressing tumors: JeKo-1 (MCL) (Figure 4B) or RS4;11 (ALL) (Figure 4C). Experimental groups received chimeric BAFF-R mAb treatment (C55 or C90, as indicated). Control mice received 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 day 0 tumor challenge with the minimal lethal dose of tumor. Treatment was administered via IV tail vein injection. Figures 4B and 4C are images showing the in vivo therapeutic efficacy of chimeric antibodies targeting human BAFF-R against B cell tumors. Bioluminescence images of mice challenged with luciferase-expressing tumors: JeKo-1 (MCL) (Figure 4B) or RS4;11 (ALL) (Figure 4C). Experimental groups received chimeric BAFF-R mAb treatment (C55 or C90, as indicated). Control mice received 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 on drug-resistant lymphoma models. 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 the selected CD20- / - clone #25 was compared to wild-type JeKo-1. ADCC efficacy was measured by chromium-51 release after incubation with C55, C90, or rituximab and effector NK cells (E:T = 20:1). The percentage of cell-specific lysis of target cells is 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 shown as the mean ± standard deviation of triplicate samples. *P<0.05 compared with NK cells by two-tailed 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 on drug-resistant lymphoma models. 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 the selected CD20- / - clone #25 was compared to wild-type JeKo-1. ADCC efficacy was measured by chromium-51 release after incubation with C55, C90, or rituximab and effector NK cells (E:T = 20:1). The percentage of cell-specific lysis of target cells is 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 shown as the mean ± standard deviation of triplicate samples. *P<0.05 compared with NK cells by two-tailed 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 on drug-resistant lymphoma models. 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 the selected CD20- / - clone #25 was compared to wild-type JeKo-1. ADCC efficacy was measured by chromium-51 release after incubation with C55, C90, or rituximab and effector NK cells (E:T = 20:1). The percentage of cell-specific lysis of target cells is 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 shown as the mean ± standard deviation of triplicate samples. *P<0.05 compared with NK cells by two-tailed Student's t-test. [Figure 6-1] Figures 6A and 6B show images and a graph (Figure 6C) showing the in vivo therapeutic efficacy of chimeric antibodies targeting human BAFF-R against drug-resistant B cell tumors. Bioluminescence images of mice challenged with luciferase-expressing tumors, JeKo-1-CD20-KO cells (Figure 6A) or ibrutinib-resistant Z-138 cells (Figure 6B), followed by antibody treatment as 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 of the mice shown in (A) and (B), respectively. Differences in tumor-free rates and survival between experimental and all control groups were analyzed by the log-rank test (**P<0.001). Data are representative of three independent experiments. [Figure 6-2]Figures 6A and 6B show images and a graph (Figure 6C) showing the in vivo therapeutic efficacy of chimeric antibodies targeting human BAFF-R against drug-resistant B cell tumors. Bioluminescence images of mice challenged with luciferase-expressing tumors, JeKo-1-CD20-KO cells (Figure 6A) or ibrutinib-resistant Z-138 cells (Figure 6B), followed by antibody treatment as 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 of the mice shown in (A) and (B), respectively. Differences in tumor-free rates and survival between experimental and all control groups were analyzed by the log-rank test (**P<0.001). Data are representative of three independent experiments. [Figure 6-3] Figures 6A and 6B show images and a graph (Figure 6C) showing the in vivo therapeutic efficacy of chimeric antibodies targeting human BAFF-R against drug-resistant B cell tumors. Bioluminescence images of mice challenged with luciferase-expressing tumors, JeKo-1-CD20-KO cells (Figure 6A) or ibrutinib-resistant Z-138 cells (Figure 6B), followed by antibody treatment as 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 of the mice shown in (A) and (B), respectively. Differences in tumor-free rates and survival between experimental and all control groups were analyzed by the log-rank test (**P<0.001). Data are representative of three independent experiments. [Figure 7-1]Figures 7A and 7B show the generation and clone selection of anti-human BAFF-R monoclonal antibodies. Figure 7A is a schematic diagram showing that L-cell clone D2C (which stably expresses human hBAFF-R with a C-terminal GFP tag on the intracellular domain) was used to immunize BALB / c mice according to the indicated schedule. Spleen tissue was harvested on day 20, and B-cell hybridoma clones were established. 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, whereas clone 37 (representative of other negative clones) did not. [Figure 7-2] Figures 7A and 7B show the generation and clone selection of anti-human BAFF-R monoclonal antibodies. Figure 7A is a schematic diagram showing that L-cell clone D2C (which stably expresses human hBAFF-R with a C-terminal GFP tag on the intracellular domain) was used to immunize BALB / c mice according to the indicated schedule. Spleen tissue was harvested on day 20, and B-cell hybridoma clones were established. 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, whereas clone 37 (representative of other negative clones) did not. [Figure 8] 1 shows flow cytometry results confirming that selected hybridoma clones bind to MCL cells. Binding of the supernatants of hybridoma clones 53, 55, 67, and 90 (dilutions of 1 / 10, 1 / 50, and 1 / 200) to the Mino (mantle cell lymphoma) and 293T (negative control) cell lines was assessed by flow cytometry performed with anti-mouse IgG-APC. [Figure 9]Figure 1 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 serially diluted (1 μg / 10 cells to 1.6 ng / 10 cells) purified mouse mAbs to Mino cells was assessed by flow cytometry with an anti-mouse IgG-APC secondary antibody. [Figure 10] Figure 1 shows the results of a FACS screening analysis demonstrating that hBAFF-R mAbs recognized non-Hodgkin's lymphoma cell lines in vitro. Mouse mAb clones 55 and 90 bound additional cell lines: JeKo-1 (mantle cell lymphoma), SU-DHL-6 (diffuse large B-cell lymphoma), Raji (Burkitt's lymphoma), and RL (follicular lymphoma) at high (2 μg mAb / 10 cells) and low (0.05 μg mAb / 10 cells) doses. Flow cytometry analysis was performed with anti-mouse IgG-APC. The top-to-bottom traces shown in the figure correlate with the top-to-bottom of the variables used (e.g., antibody type or cell type) shown next to the figure. [Figure 11] Figure 1 shows a graph demonstrating that hBAFF-R mAb recognized lymphoma patient samples. Patient samples from mantle cell lymphoma, diffuse large B-cell lymphoma, and follicular lymphoma were stained with mouse mAbs C55 and C90 at high (2 μg / 10 cells) and low (0.05 μg / 10 cells) doses. Flow cytometry analysis was performed with anti-mouse IgG-APC. The top-to-bottom traces shown in the figure correlate with the left-to-right variables used (e.g., antibody type or cell type) shown below the figure. [Figure 12] Graph showing that chimeric antibodies induced ADCC against BAFF-R-expressing L cells. BAFF-R-expressing D2C L cells (targets) were labeled with chromium-51 and subsequently incubated overnight with chimeric mAb + NK cells (effector-to-target ratio, 20:1). Culture supernatants were analyzed for released chromium. [Figure 13]This graph shows the chimeric antibody required by NK cells for cytotoxicity against tumor cells. JeKo-1 cells (targets) were labeled with chromium-51. The cells were incubated with chimeric mAbs (C55, C90, or rituximab) and with or without NK cells (effectors) at an effector-to-target ratio of 20:1. Chimeric antibodies were added at concentrations ranging from 50 to 0.005 μg / mL. Culture supernatants were analyzed for released chromium. [Figure 14] Figure 1 shows FACS results demonstrating that hBAFF-R mAb blocked BAFF / BAFF-R interaction. BAFF-R-expressing D2C L cell clones were incubated with C90 (0-1000 ng / 10 cells) for 45 minutes at 4°C, followed by incubation with recombinant BAFF ligand (0.5 μg / 10 cells) for 90 minutes at 4°C. Flow cytometry was performed and gated for anti-BAFF-PE. Signal plots show the binding signal of BAFF / BAFF-R in the presence of each mAb concentration. The concentrations shown in the signal plots are indicated above each FACS result. [Figure 15] FACS results showing that limited internalization was observed with BAFF-R mAb. Mino cells were incubated with mAb C90 (0.05 μg / 10 cells) for 20 minutes at 4°C, followed by 1 hour at 37°C. Flow cytometry analysis was performed with anti-mouse IgG-APC. Cells were gated for surface-localized antibody (out) and loss of cell surface staining (in). [Figure 16-1]Figure 16A shows FACS results (Figure 16A) and a gel image (Figure 16B) demonstrating that CD20 knockout clones were generated by CRISPR. JeKo-1 CD20 knockout clones were generated using a commercial CRISPR / HDR system to replace RFP at the CD20 locus. In Figure 16A, clones were screened and selected by flow cytometry for CD20- / RFP+ expression. In Figure 16B, Western blots with an anti-CD20 antibody were performed on whole cell lysates from CD20- / RFP+ clones. β-actin was blotted as a loading control. Figure 16C shows FACS results for the same clones as in Figure 16A, which were screened for BAFF-R / RFP+ expression to confirm that BAFF-R expression was not affected by CRISPR / HDR manipulation of CD20. [Figure 16-2] Figure 16A shows FACS results (Figure 16A) and a gel image (Figure 16B) demonstrating that CD20 knockout clones were generated by CRISPR. JeKo-1 CD20 knockout clones were generated using a commercial CRISPR / HDR system to replace RFP at the CD20 locus. In Figure 16A, clones were screened and selected by flow cytometry for CD20- / RFP+ expression. In Figure 16B, Western blots with an anti-CD20 antibody were performed on whole cell lysates from CD20- / RFP+ clones. β-actin was blotted as a loading control. Figure 16C shows FACS results for the same clones as in Figure 16A, which were screened for BAFF-R / RFP+ expression to confirm that BAFF-R expression was not affected by CRISPR / HDR manipulation of CD20. [Figure 16-3]Figure 16A shows FACS results (Figure 16A) and a gel image (Figure 16B) demonstrating that CD20 knockout clones were generated by CRISPR. JeKo-1 CD20 knockout clones were generated using a commercial CRISPR / HDR system to replace RFP at the CD20 locus. In Figure 16A, clones were screened and selected by flow cytometry for CD20- / RFP+ expression. In Figure 16B, Western blots with an anti-CD20 antibody were performed on whole cell lysates from CD20- / RFP+ clones. β-actin was blotted as a loading control. Figure 16C shows FACS results for the same clones as in Figure 16A, which were screened for BAFF-R / RFP+ expression to confirm that BAFF-R expression was not affected by CRISPR / HDR manipulation of CD20. [Figure 17-1] FACS results showing the characterization of BAFF-R binding to normal B cells. PBMCs from healthy donors were co-stained with APC-conjugated C90 chimeric antibody and (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 gated and analyzed for binding to the BAFF-R antibody. [Figure 17-2] FACS results showing the characterization of BAFF-R binding to normal B cells. PBMCs from healthy donors were co-stained with APC-conjugated C90 chimeric antibody and (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 gated and analyzed for binding to the BAFF-R antibody. [Figure 18]Figures 18A and 18B are FACS results showing the characterization of hBAFF-R mAbs on 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 / 10 cells). Flow cytometry analysis was performed with anti-mouse IgG. Myeloid cells from PBMCs were gated for CD66b+ and analyzed for staining with mAbs C55 and C90. The top-to-bottom traces shown in the figures correlate with the top-to-bottom of the variables used (e.g., antibody type or cell type) shown next to the figures. [Figure 19-1] Figures 19A and 19B are immunohistochemistry images. In Figure 19A, immunohistochemistry was performed to identify the tissue specificity of the anti-BAFF-R antibody. A 1:150 dilution of 1 mg / mL of antibody was used to stain tissue samples. Tissue specificity of C55 mAb against human BAFF-R (20x objective): 1:150 dilution of 1 mg / mL stock. In Figure 19B, immunohistochemistry was additionally performed on tonsil tissue and breast tissue to identify the tissue specificity of the anti-BAFF-R antibody. A 1:150 dilution of 1 mg / mL of antibody was used to stain tissue samples. Tissue specificity of mAb against human BAFF-R (upper panel: tonsil tissue; lower panel: breast tissue; 20x objective). [Figure 19-2] Figures 19A and 19B are immunohistochemistry images. In Figure 19A, immunohistochemistry was performed to identify the tissue specificity of the anti-BAFF-R antibody. A 1:150 dilution of 1 mg / mL of antibody was used to stain tissue samples. Tissue specificity of C55 mAb against human BAFF-R (20x objective): 1:150 dilution of 1 mg / mL stock. In Figure 19B, immunohistochemistry was additionally performed on tonsil tissue and breast tissue to identify the tissue specificity of the anti-BAFF-R antibody. A 1:150 dilution of 1 mg / mL of antibody was used to stain tissue samples. Tissue specificity of mAb against human BAFF-R (upper panel: tonsil tissue; lower panel: breast tissue; 20x objective). [Figure 20-1]Figures 20A and 20B are graphs showing functional in vitro assays performed on the humanized variants. In Figure 20A, an ELISA assay was performed on nine humanized variants of C90. The recombinant extracellular domain of human BAFF-R was used as the antigen. Antibodies were administered at varying concentrations from 0.78 to 100 ng / mL, and their absorbance was collected at 450 nm. In Figure 20B, the humanized variants were tested on JeKo-1 cells in a chromium release assay. The cells were allowed to take up chromium and subsequently treated with the humanized C90 variants and effector NK cells. The cells were incubated for 6 hours, and the supernatant was sampled for chromium content. [Figure 20-2] Figures 20A and 20B are graphs showing functional in vitro assays performed on the humanized variants. In Figure 20A, an ELISA assay was performed on nine humanized variants of C90. The recombinant extracellular domain of human BAFF-R was used as the antigen. Antibodies were administered at varying concentrations from 0.78 to 100 ng / mL, and their absorbance was collected at 450 nm. In Figure 20B, the humanized variants were tested on JeKo-1 cells in a chromium release assay. The cells were allowed to take up chromium and subsequently treated with the humanized C90 variants and effector NK cells. The cells were incubated for 6 hours, and the supernatant was sampled for chromium content. [Figure 21-1] Figures 21A and 21B are graphs showing the analysis of humanized antibodies C90-4 and C90-5 for specific cytotoxicity in various lymphoma lines. In Figure 21A, JeKo-1, Z138, and RS4 were subjected to a chromium release assay with 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. Cell supernatants were analyzed for chromium content. In Figure 21B, LY-10, MEC-2, RL, and Raji lymphoma lines were subjected to a chromium release assay with humanized antibodies C90-4 and C90-5. Antibodies were administered to the cell lines at 5 μg / mL and incubated with NK cells at an E:T ratio of 20:1 for 6 hours. Cell supernatants were analyzed for chromium content. [Figure 21-2] Figures 21A and 21B are graphs showing the analysis of humanized antibodies C90-4 and C90-5 for specific cytotoxicity in various lymphoma lines. In Figure 21A, JeKo-1, Z138, and RS4 were subjected to a chromium release assay with 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. Cell supernatants were analyzed for chromium content. In Figure 21B, LY-10, MEC-2, RL, and Raji lymphoma lines were subjected to a chromium release assay with humanized antibodies C90-4 and C90-5. Antibodies were administered to the cell lines at 5 μg / mL and incubated with NK cells at an E:T ratio of 20:1 for 6 hours. Cell supernatants were analyzed for chromium content. [Figure 22-1] Figures 22A and 22B are FACS results and graphs showing the humanized C90 antibody front-runner tested for binding and cytotoxicity against primary MCL samples. In Figure 22A, three primary MCL tumor samples were co-stained with CD20-APC and biotinylated humanized C90, followed by signal detection using PE-conjugated streptavidin. In Figure 22B, the cytotoxicity of humanized C90 against primary tumor samples was assessed by chromium release assay. Cells were incubated with chromium-51 and subsequently treated with antibody and effector NK cells. Following overnight incubation, supernatants were sampled and chromium content was determined. [Figure 22-2]Figures 22A and 22B are FACS results and graphs showing the humanized C90 antibody front-runner tested for binding and cytotoxicity against primary MCL samples. In Figure 22A, three primary MCL tumor samples were co-stained with CD20-APC and biotinylated humanized C90, followed by signal detection using PE-conjugated streptavidin. In Figure 22B, the cytotoxicity of humanized C90 against primary tumor samples was assessed by chromium release assay. Cells were incubated with chromium-51 and subsequently treated with antibody and effector NK cells. Following overnight incubation, supernatants were sampled and chromium content was determined. [Figure 23] Figure 1 shows FACS results 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 the granulocyte marker CD66b-PerCP-Cy5.5, the monocyte marker CD14-PE-Cy7, the B cell marker CD20-APC, the T cell marker CD3-PE-Cy5, and the NK cell marker CD56-FITC. PBMCs were analyzed by flow cytometry. [Figure 24] Images showing in vivo tumor therapy of BAFF-R chimeric antigen receptor (CAR) T cells. Donor T cells were engineered to express a chimeric C55 anti-BAFF-R single-chain variable fragment (sFv) on a T cell receptor signaling domain with a 4-1BB motif. NSG mice were challenged with a minimally lethal dose (1 × 10 cells) of NHL JeKo-1-Luci cells. Tumor cells were allowed to engraft until tumors were detectable by bioluminescence imaging (day 9). At days 9 and 15 after tumor challenge, mice received either T cell therapy (5 × 10 CAR-T cells) or control. Mice were closely monitored and imaged every 3 days to track tumor progression. DETAILED DESCRIPTION OF THE INVENTION

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

[0017] As referred to herein, BAFF-R, BAFF receptor, or BAFF-R protein includes any recombinant or naturally occurring form of B-cell activating factor receptor (BAFF-R), also known as tumor necrosis factor receptor superfamily member 13C (TNFRSF13C), or a variant or homolog thereof, that maintains BAFF-R activity (e.g., activity within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of that of BAFF-R. Optionally, the variant or homolog has 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 50, 100, 150, or 200 contiguous amino acid portion) relative to naturally occurring BAFF-R. Optionally, BAFF-R is substantially identical to a protein identified by UniProt reference number Q96RJ3 or a variant or homolog having substantial identity thereto. Optionally, BAFF-R is substantially identical to a protein identified by UniProt reference number Q9D8D0 or a variant or homolog having substantial identity thereto. Optionally, BAFF-R is substantially identical to a protein identified by NCBI reference number GI:16445027 or a variant or homolog having substantial identity thereto. Optionally, BAFF-R is substantially identical to a protein identified by NCBI reference number GI:16306481 or a variant or homolog having substantial identity thereto.

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

[0019] The humanized antibodies as provided herein can bind to BAFF-R protein and comprise at least one murine CDR of the BAFF-R antibodies provided herein, or a functional fragment or variant thereof (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 entire CDR amino acid sequence, but an antibody or fragment thereof containing the functional fragment can still bind to an 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 thereof containing the functional variant can still bind to an antigen (e.g., BAFF-R). For example, a functional variant of a nucleic acid sequence encoding a CDR can contain one or more changes but still encode the same amino acid sequence of the CDR. Furthermore, functional variants of the polypeptide sequence of a CDR can contain one or more amino acid changes, so long as the antibody or functional fragment thereof binds to the antigen. Thus, functional fragments or variants of a CDR typically contain the amino acid residues required for antibody binding to the antigen (e.g., BAFF-R). When a humanized antibody contains at least one CDR, at least one CDR or functional fragment thereof is derived from a donor antibody. Optionally, the donor antibody is a mouse antibody. Those skilled in the art will readily recognize that a humanized antibody containing at least one mouse CDR is a humanized antibody with at least one mouse CDR derived from the donor antibody, with the additional CDR derived from the acceptor antibody (e.g., when 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 comprise 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 comprise 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 comprise six combined CDRs, at least one of which is a mouse CDR. When the humanized light chain variable region and the humanized heavy chain variable region comprise a combined mouse CDR, the humanized light chain variable region or the humanized heavy chain variable region comprises one mouse CDR. For example, a humanized antibody may comprise CDR L3 (e.g., mouse, also referred to herein as mouse CDR L3) derived from the donor antibody and CDR L1, CDR L2, CDR H1, CDR H2, and CDR H3 (i.e., human) derived from the acceptor antibody.

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

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

[0023] The humanized light chain variable region and the humanized heavy chain variable region may comprise four combined mouse CDRs. When the humanized light chain variable region and the humanized heavy chain variable region comprise four combined mouse CDRs, the humanized light chain variable region comprises one mouse CDR and the humanized heavy chain variable region comprises three mouse CDRs (i), the humanized light chain variable region comprises three mouse CDRs and the humanized heavy chain variable region comprises one mouse CDR (ii), or the humanized light chain variable region comprises two mouse CDRs and the humanized heavy chain variable region comprises two mouse CDRs (iii). For example, a humanized antibody may comprise CDRs L3, H3, L2, and L1 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 H2 from an acceptor antibody (i.e., human).

[0024] The humanized light chain variable region and the humanized heavy chain variable region may each comprise at least one murine CDR. When the humanized light chain variable region and the humanized heavy chain variable region each comprise at least one murine CDR, the humanized light chain variable region comprises at least one murine CDR, and the humanized heavy chain variable region comprises at least one murine CDR. Thus, the humanized light chain variable region may comprise murine CDR L1, and the humanized heavy chain may comprise murine CDR H1. Optionally, the murine CDR L1 comprises the amino acid sequence of SEQ ID NO: 1, and the murine CDR H1 comprises the amino acid sequence of SEQ ID NO: 4. Optionally, the murine CDR L1 is the amino acid sequence of SEQ ID NO: 1, and the murine CDR H1 is the amino acid sequence of SEQ ID NO: 4. Optionally, the humanized light chain variable region comprises murine CDR L2, and the humanized heavy chain variable region comprises murine CDR H2. Optionally, the murine CDR L2 comprises the amino acid sequence of SEQ ID NO: 2, and the murine CDR H2 comprises the amino acid sequence of SEQ ID NO: 5. Optionally, the murine CDR L2 has the amino acid sequence of SEQ ID NO: 2, and the murine CDR H2 has the amino acid sequence of SEQ ID NO: 5. Optionally, the humanized light chain variable region comprises a murine CDR L3, and the humanized heavy chain variable region comprises a murine CDR H3. Optionally, the murine CDR L3 has the amino acid sequence of SEQ ID NO: 3, and the murine CDR H3 has the amino acid sequence of SEQ ID NO: 6. Optionally, the CDR L3 has the amino acid sequence of SEQ ID NO: 3, and the murine CDR H3 has the amino acid sequence of SEQ ID NO: 6.

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

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

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

[0028] The positions of CDRs and FRs can be defined by the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., USDapartment of Health and Human Services, US Government Printing Office (1991)). Similarly, the positions occupied by individual residues in the light or heavy chain of an antibody can be defined by the Kabat numbering system. Thus, the locations of residues required for binding in the humanized light chain and humanized heavy chain 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 can be an antibody having CDRs from a donor antibody (e.g., mouse) and variable region framework regions (FRs) from a human antibody. The framework regions (FRs) are intended to keep the CDRs in the correct place in the humanized antibody. Proceeding from the amino terminus, these regions are designated 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. Provided herein are humanized antibodies comprising one or more residues within the framework regions. 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 to herein as connecting framework region residues. Connecting framework region residues may be present in the framework regions of the humanized light chain variable region (i.e., FR L1, FR L2, FR L3, FR L4), or they may be present in the framework of the humanized heavy chain variable region (i.e., FR H1, FR H2, FR H3, FR H4). Connecting framework residues present in the FR L3 region of the humanized light chain are referred to herein as FR L3 connecting framework region residues. Thus, the joining framework region residues present in the FR H3 region of the humanized heavy chain are referred to herein as FR H3 joining framework region residues.

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

[0030] The humanized light chain variable region comprises at least one binding framework region residue (e.g., 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 residues), The humanized heavy chain variable region can include at least one binding framework region residue (e.g., 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 residues). The positions of the binding framework region residues in the humanized antibody can be defined by the Kabat numbering system, similar to the positions of the CDR residues.

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

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

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

[0034] Optionally, the light chain variable region comprises a connecting framework region residue that is a serine at a position corresponding to Kabat position 7, a proline at a position corresponding to Kabat position 8, a valine at a position corresponding to Kabat position 15, a threonine at a position corresponding to Kabat position 22, a glutamine or serine at a position corresponding to Kabat position 24, a glycine at a position corresponding to Kabat position 41, a lysine at a position corresponding to Kabat position 42, an alanine or threonine at a position corresponding to Kabat position 43, a proline at a position corresponding to Kabat position 44, a threonine at a position corresponding to Kabat position 56, a threonine at a position corresponding to Kabat position 72, a phenylalanine or lysine at a position corresponding to Kabat position 73, a glutamine at a position corresponding to Kabat position 79, or a valine at a position corresponding to Kabat position 104.

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

[0036] Optionally, the heavy chain variable region contains a threonine or alanine at a position corresponding to Kabat position 10, a lysine at a position corresponding to Kabat position 11, a valine at a position corresponding to Kabat position 12, a threonine at a position corresponding to Kabat position 15, a threonine at a position corresponding to Kabat position 19, a threonine at a position corresponding to Kabat position 23, a proline at a position corresponding to Kabat position 41, an alanine, a proline at a position corresponding to Kabat position 44, a serine or threonine at a position corresponding to Kabat position 61, an arginine at a position corresponding to Kabat position 66, a valine at a position corresponding to Kabat position 17, a threonine at a position corresponding to Kabat position 19, a threonine at a position corresponding to Kabat position 23, a proline at a position corresponding to Kabat position 41, an alanine, a proline at a position corresponding to Kabat position 44, a serine or threonine at a position corresponding to Kabat position 61, an arginine at a position corresponding to Kabat position 66, a valine at a position corresponding to Kabat position 18, a valine ...valine at a position corresponding to Kabat position 23, a valine at a position corresponding to Kabat position 24, a valine at a position and a threonine at a position corresponding to Kabat position 70, a lysine at a position corresponding to Kabat position 75, a valine at a position corresponding to Kabat position 79, a threonine or lysine at a position corresponding to Kabat position 81, a methionine at a position corresponding to Kabat position 82, an asparagine at a position corresponding to Kabat position 82B, a methionine at a position corresponding to Kabat position 82C, a proline at a position corresponding to Kabat position 84, a valine at a position corresponding to Kabat position 85, a lysine at a position corresponding to Kabat position 108, or a valine at a position corresponding to Kabat position 109.

[0037] Optionally, the heavy chain variable region contains a threonine or alanine at a position corresponding to Kabat position 10, a lysine at a position corresponding to Kabat position 11, a valine at a position corresponding to Kabat position 12, a threonine at a position corresponding to Kabat position 15, a threonine at a position corresponding to Kabat position 19, a threonine at a position corresponding to Kabat position 23, a proline at a position corresponding to Kabat position 41, an alanine, a proline at a position corresponding to Kabat position 44, a serine or threonine at a position corresponding to Kabat position 61, an arginine at a position corresponding to Kabat position 66, a K and a valine at a position corresponding to Kabat position 109.

[0038] Optionally, the heavy chain variable region contains a threonine or alanine at a position corresponding to Kabat position 10, a lysine at a position corresponding to Kabat position 11, a valine at a position corresponding to Kabat position 12, a threonine at a position corresponding to Kabat position 15, a threonine at a position corresponding to Kabat position 19, a threonine at a position corresponding to Kabat position 23, a proline at a position corresponding to Kabat position 41, an alanine, a proline at a position corresponding to Kabat position 44, a serine or threonine at a position corresponding to Kabat position 61, an arginine at a position corresponding to Kabat position 66, an arginine at a position corresponding to Kabat position 70, an arginine at a position corresponding to Kabat position 72, an arginine at a position corresponding to Kabat position 74, an arginine at a position corresponding to Kabat position 76, an arginine at a position corresponding to Kabat position 78, an arginine at a position corresponding to Kabat position 79, an arginine at a position corresponding to Kabat position 80, an arginine at a position corresponding to Kabat position 81, an arginine at a position corresponding to Kabat position 82, an arginine at a position corresponding to Kabat position 83, an arginine at a position corresponding to Kabat position 84, an arginine at a position corresponding to Kabat position 85, an arginine at a position corresponding to Kabat position 86, an arginine at a position corresponding to Kabat position 87, an arginine at a position corresponding to Kabat position 88, an arginine at a position corresponding to Kabat position 89, an arginine at a position corresponding to Kabat position 90, an arginine at a position corresponding to Kabat position 91, an arginine at a position corresponding to Kabat position and 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] Humanized BAFF-R antibodies are provided, comprising a humanized light chain variable region comprising murine CDR L1, murine CDR L2, or murine CDR L3, and a humanized heavy chain variable region comprising murine CDR H1, murine CDR H2, or murine CDR H3. The humanized light chain variable region may comprise murine CDR L1 as specified in SEQ ID NO: 1, murine CDR L2 as specified in SEQ ID NO: 2, or murine CDR L3 as specified in SEQ ID NO: 3. The humanized light chain variable region may comprise murine CDR L1 as specified in SEQ ID NO: 1, murine CDR L2 as specified in SEQ ID NO: 2, and murine CDR L3 as specified in SEQ ID NO: 3. The humanized heavy chain variable region may comprise murine CDR H1 as specified in SEQ ID NO: 4, murine CDR H2 as specified in SEQ ID NO: 5, or murine CDR H3 as specified in SEQ ID NO: 6. The humanized heavy chain variable region may comprise murine CDR H1 as specified in SEQ ID NO:4, murine CDR H2 as specified in SEQ ID NO:5, and murine CDR H3 as specified in SEQ ID NO:6. Optionally, the humanized light chain variable region comprises murine CDR L1 as specified in SEQ ID NO:1. Optionally, the humanized light chain variable region comprises murine CDR L2 as specified in SEQ ID NO:2. Optionally, the humanized light chain variable region comprises murine CDR L3 as specified in SEQ ID NO:3. Optionally, the humanized heavy chain variable region comprises murine CDR H1 as specified in SEQ ID NO:4. Optionally, the humanized heavy chain variable region comprises murine CDR H2 as specified in SEQ ID NO:5. Optionally, the humanized light chain variable region comprises murine CDR H3 as specified in SEQ ID NO:6. In a further embodiment, the humanized light chain variable region comprises at least one binding framework region residue. In other further embodiments, the humanized heavy chain variable region comprises at least one binding framework region residue.

[0040] Humanized BAFF-R antibodies are provided, comprising a humanized light chain variable region comprising murine CDR L1, murine CDR L2, or murine CDR L3, and a humanized heavy chain variable region comprising murine CDR H1, murine CDR H2, or murine CDR H3. The humanized light chain variable region may comprise murine CDR L1 as specified in SEQ ID NO:7, murine CDR L2 as specified in SEQ ID NO:8, or murine CDR L3 as specified in SEQ ID NO:9. The humanized light chain variable region may comprise murine CDR L1 as specified in SEQ ID NO:7, murine CDR L2 as specified in SEQ ID NO:8, and murine CDR L3 as specified in SEQ ID NO:9. The humanized heavy chain variable region may comprise murine CDR H1 as specified in SEQ ID NO:10, murine CDR H2 as specified in SEQ ID NO:11, or murine CDR H3 as specified in SEQ ID NO:12. The humanized heavy chain variable region may comprise murine CDR H1 as specified in SEQ ID NO: 10, murine CDR H2 as specified in SEQ ID NO: 11, and murine CDR H3 as specified in SEQ ID NO: 12. Optionally, the humanized light chain variable region comprises murine CDR L1 as specified in SEQ ID NO: 7. Optionally, the humanized light chain variable region comprises murine CDR L2 as specified in SEQ ID NO: 8. Optionally, the humanized light chain variable region comprises murine CDR L3 as specified in SEQ ID NO: 9. Optionally, the humanized heavy chain variable region comprises murine CDR H1 as specified in SEQ ID NO: 10. Optionally, the humanized heavy chain variable region comprises murine CDR H2 as specified in SEQ ID NO: 11. Optionally, the humanized light chain variable region comprises murine CDR H3 as specified in SEQ ID NO: 12. In a further embodiment, the humanized light chain variable region comprises at least one binding framework region residue. In other further embodiments, the humanized heavy chain variable region comprises at least one binding framework region residue.

[0041] Optionally, the light chain variable region comprises the sequence of SEQ ID NO: 18, SEQ ID NO: 20, or SEQ ID NO: 22. Optionally, the light chain variable region comprises the sequence of SEQ ID NO: 18. Optionally, the light chain variable region comprises the sequence of SEQ ID NO: 20. Optionally, the light chain variable region is the sequence of SEQ ID NO: 18. Optionally, the light chain variable region is the sequence of SEQ ID NO: 20. Optionally, the light chain variable region is the sequence of SEQ ID NO: 22. Optionally, the heavy chain variable region comprises the sequence of SEQ ID NO: 24, SEQ ID NO: 26, or SEQ ID NO: 28. Optionally, the heavy chain variable region comprises the sequence of SEQ ID NO: 24. Optionally, the heavy chain variable region comprises the sequence of SEQ ID NO: 26. Optionally, the heavy chain variable region comprises the sequence of SEQ ID NO: 28. Optionally, the heavy chain variable region is the sequence of SEQ ID NO: 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. Thus, in another embodiment, a humanized BAFF-R antibody is provided comprising a humanized light chain variable region and a humanized heavy chain variable region, wherein the humanized light chain variable region comprises the sequence of SEQ ID NO: 18 and the heavy chain variable region comprises the sequence of SEQ ID NO: 24. In another embodiment, a humanized BAFF-R antibody is provided comprising a humanized light chain variable region and a humanized heavy chain variable region, wherein the humanized light chain variable region comprises the sequence of SEQ ID NO: 20 and the heavy chain variable region comprises the sequence of SEQ ID NO: 26. In another embodiment, a humanized BAFF-R antibody is provided comprising a humanized light chain variable region and a humanized heavy chain variable region, wherein the humanized light chain variable region comprises the sequence of SEQ ID NO: 22 and the heavy chain variable region comprises the sequence of SEQ ID NO: 28.

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

[0043] Optionally, the light chain variable region comprises the sequence of SEQ ID NO: 30. Optionally, the heavy chain variable region comprises 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. Thus, in another aspect, a chimeric BAFF-R antibody is provided comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises the sequence of SEQ ID NO: 30 and the heavy chain variable region comprises the sequence of SEQ ID NO: 32.

[0044] In each instance where antibodies are recited herein, functional fragments may be used. Thus, for example, Fab' fragments are provided, which may contain a heavy chain (e.g., including a constant region and a variable region) and a light chain (e.g., including a constant region and a variable region). Optionally, the Fab' fragment contains a humanized heavy chain (e.g., including a constant region and a variable region) and a humanized light chain (e.g., including a constant region and a variable region).

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

[0046] Optionally, the BAFF-R antibody or fragment thereof is a single-chain antibody. Single-chain antibodies comprise a variable light chain and a variable heavy chain. Those skilled in the art will readily recognize that, in contrast to immunoglobulin antibodies (which comprise two identical pairs of polypeptide chains, each pair having one light chain and one heavy chain), single-chain antibodies comprise a single light chain and a single heavy chain. Each light chain and heavy chain then consists of two regions: a variable ("V") region (i.e., a variable light chain and a variable heavy chain) involved in binding to a 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 may be linked via a linker peptide. Examples of linker peptides for single-chain antibodies are described in Bird, RE, et al., Science. 242(4877):423-6 (1988). Methods for producing scFv antibodies have also been 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 from B cells from immunized animals is isolated and cDNA is prepared. Specific primers for the variable regions of immunoglobulin heavy and light chains are used to amplify the cDNA. The PCR product is purified, and the nucleic acid sequences are ligated. If a linker peptide is desired, a nucleic acid sequence encoding the peptide is inserted between the heavy and light chain nucleic acid sequences. The nucleic acid encoding the scFv is inserted into a vector and expressed in a suitable host cell.

[0047] The ability of an antibody or functional fragment thereof to bind to a specific epitope (e.g., BAFF-R) is determined by the equilibrium dissociation constant (K D ) as defined herein. D ) is the ratio of the dissociation rate (Koff) and the association rate (Kon) of the BAFF-R antibody to the BAFF-R protein. It is calculated using the following formula: K D Optionally, the BAFF-R antibody has an equilibrium dissociation constant (K) of less than about 5 nM.D ) to the BAFF-R protein. Optionally, the BAFF-R antibody can bind to the BAFF-R protein with an equilibrium dissociation constant (K) of less than about 4.5 nM. D ) to the BAFF-R protein. Optionally, the BAFF-R antibody can bind to the BAFF-R protein with an equilibrium dissociation constant (K) of less than about 4 nM. D ) to the BAFF-R protein. Optionally, the BAFF-R antibody can bind to the BAFF-R protein with an equilibrium dissociation constant (K) of less than about 3.5 nM. D ) to the BAFF-R protein. Optionally, the BAFF-R antibody can bind to the BAFF-R protein with an equilibrium dissociation constant (K) of less than about 3 nM. D ) to the BAFF-R protein. Optionally, the BAFF-R antibody can bind to the BAFF-R protein with an equilibrium dissociation constant (K) of less than about 2.5 nM. D ) to the BAFF-R protein. Optionally, the BAFF-R antibody can bind to the BAFF-R protein with an equilibrium dissociation constant (K) of less than about 2 nM. D ) to the BAFF-R protein. Optionally, the BAFF-R antibody can bind to the BAFF-R protein with an equilibrium dissociation constant (K) of less than about 1.5 nM. D ) to the BAFF-R protein. Optionally, the BAFF-R antibody can bind to the BAFF-R protein with an equilibrium dissociation constant (K) of less than about 1 nM. D ) to the BAFF-R protein. Optionally, the BAFF-R antibody can bind to the BAFF-R protein with an equilibrium dissociation constant (K) of less than about 0.5 nM. D ) can bind to the BAFF-R protein.

[0048] Optionally, the BAFF-R antibody or functional fragment thereof has an equilibrium dissociation constant (K D Optionally, the BAFF-R antibody or functional fragment thereof can bind to the BAFF-R protein with an equilibrium dissociation constant (K) of about 1 nM. D Optionally, the BAFF-R antibody or functional fragment thereof can bind to the BAFF-R protein with an equilibrium dissociation constant (K) of about 1.5 nM. D Optionally, the BAFF-R antibody or functional fragment thereof can bind to the BAFF-R protein with an equilibrium dissociation constant (K) of about 2 nM. DOptionally, the BAFF-R antibody or functional fragment thereof can bind to the BAFF-R protein with an equilibrium dissociation constant (K) of about 2.5 nM. D Optionally, the BAFF-R antibody or functional fragment thereof can bind to the BAFF-R protein with an equilibrium dissociation constant (K) of about 3 nM. D Optionally, the BAFF-R antibody or functional fragment thereof can bind to the BAFF-R protein with an equilibrium dissociation constant (K) of about 3.5 nM. D Optionally, the BAFF-R antibody or functional fragment thereof can bind to the BAFF-R protein with an equilibrium dissociation constant (K) of about 4 nM. D Optionally, the BAFF-R antibody or functional fragment thereof can bind to the BAFF-R protein with an equilibrium dissociation constant (K) of about 4.5 nM. D ) to the BAFF-R protein. Optionally, the BAFF-R antibody or functional fragment thereof can bind to the BAFF-R protein with an equilibrium dissociation constant (K) of about 5 nM. D Optionally, the BAFF-R antibody or functional fragment thereof can bind to the BAFF-R protein with an equilibrium dissociation constant (K) of about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 nM. D ) can bind to the BAFF-R protein.

[0049] Optionally, a K of less than about 4 nM with BAFF-R D

[0013] Humanized B-cell activating factor receptor (BAFF-R) antibodies are provided that can bind to BAFF-R with a K of less than about 4 nM. D Humanized B-cell activating factor receptor (BAFF-R) antibodies are provided that bind to BAFF-R at 1000 ng / mL. Optionally, the antibodies do not induce BAFF-R activity.

[0050] Optionally, the BAFF-R antibody is bound to a BAFF-R protein. Optionally, the BAFF-R protein is a 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 lymphoid cell. Optionally, the cell is a B cell. Optionally, the cell is a cancer cell. Optionally, the cancer cell is a lymphoma cell.

[0051] A variety of diagnostic and therapeutic moieties and combinations thereof can be conjugated to the BAFF-R antibodies or functional fragments thereof (including embodiments thereof) provided herein, thereby providing highly stable and / or versatile drug delivery and / or diagnostic compositions. Optionally, the BAFF-R antibodies or functional fragments thereof include a therapeutic or diagnostic moiety. Optionally, the therapeutic or diagnostic moiety is attached to the BAFF-R antibodies or functional fragments thereof via a chemical linker. Optionally, the chemical linker is a covalent or non-covalent linker. Techniques for conjugating therapeutic moieties to antibodies are well known (e.g., Arnon et al., Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy (Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985)); Hellstrom et al., Antibodies For Drug Delivery in Controlled Drug Delivery (2nd ed.), Robinson et al. (eds.), 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, Pinchera et al. (eds.), pp. 475-506 (1985); and Thorpe et al., The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates, Immunol. Rev., 62:119-58 (1982). As used herein, the term antibody drug conjugate or ADC refers to a therapeutic moiety conjugated or otherwise covalently attached to an antibody or functional fragment thereof.

[0052] The term "therapeutic moiety" as provided herein is used according to its plain and ordinary meaning and refers to a monovalent compound that has a therapeutic benefit (e.g., prevention, eradication, or amelioration of the underlying disorder being treated) when administered to a subject in need thereof. Therapeutic moieties as 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, ricin, doxorubicin, daunorubicin, taxol, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxy anthracin dione, actinomycin D, diphtheria toxin, Pseudomonas aeruginosa exotoxin (PE) A, PE40, abrin, and glucocorticoids. Optionally, the therapeutic moiety is an anticancer or chemotherapeutic agent as described herein. Optionally, the therapeutic moiety is a nucleic acid moiety, a peptide moiety, or a small molecule drug moiety. Optionally, the therapeutic moiety is a nucleic acid moiety. Optionally, the therapeutic moiety is an antibody moiety. Optionally, the therapeutic moiety is a peptide moiety. Optionally, the therapeutic moiety is a small molecule drug moiety. Optionally, the therapeutic moiety is a nuclease. Optionally, the therapeutic moiety is an immunostimulant. Optionally, the therapeutic moiety is a toxin. Optionally, the therapeutic moiety is a nuclease.

[0053] Also provided herein are chimeric antigen receptors (CARs) comprising the antibodies or functional fragments thereof provided herein.

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

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

[0056] Optionally, the isolated nucleic acid comprises the sequence 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 comprises the sequence of SEQ ID NO: 17 and the sequence of SEQ ID NO: 23. Optionally, the isolated nucleic acid comprises the sequence of SEQ ID NO: 19 and the sequence of SEQ ID NO: 25. Optionally, the isolated nucleic acid comprises the sequence of SEQ ID NO: 21 and the sequence of SEQ ID NO: 27.

[0057] Pharmaceutical compositions are provided that include a therapeutically effective amount of a BAFF-R antibody or functional fragment thereof provided herein and a pharmaceutically acceptable excipient.

[0058] A therapeutically effective amount as provided herein refers to an amount effective to achieve its intended purpose. The actual amount effective for a particular application will depend, inter alia, on the condition being treated. When administered in a method of treating a disease, the pharmaceutical compositions described herein will contain an amount of active humanized antibody effective to achieve the desired result (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)). Determination of a therapeutically effective amount of a BAFF-R antibody provided herein is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure herein.

[0059] Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed and typically include buffers (such as phosphate, citrate, or acetate) at a pH of 5.0-8.0 (optionally 6.0-7.0); salts for isotonicity (such as sodium chloride, potassium chloride, and the like); antioxidants; preservatives; low molecular weight polypeptides; proteins; hydrophilic polymers (such as polysorbate 80); amino acids (such as glycine); carbohydrates; chelating agents; sugars; and other standard components known to those skilled in the art (Remington: The Science and Practice of Pharmacy, 22nd ed., Loyd V. Allen et al., eds., Pharmaceutical Press (2012)). The mAb may be present at a concentration 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 can be administered by a variety of methods known in the art. The route and / or mode of administration varies depending on the desired results. Optionally, administration is intravenous, intramuscular, intraperitoneal, or subcutaneous, or administered proximal to the target site. Pharmaceutically acceptable excipients can be suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion).

[0061] Pharmaceutical compositions of antibodies or functional fragments thereof can be prepared according to well-known, routine methods practiced in the art. See, for example, Remington: The Science and Practice of Pharmacy, 22nd ed., Loyd V. Allen et al., ed., Pharmaceutical Press (2012); and Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978. 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 can 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., a therapeutic response). For example, a single bolus can be administered, multiple divided doses can be administered over time, or the dose can be proportionally reduced or increased as indicated by the requirements of the therapeutic situation. It can be advantageous to formulate the humanized antibody in combination with other therapies or drugs. For ease of administration and uniformity of dosage, it may be advantageous to formulate parenteral compositions in dosage unit form. Dosage unit form, as used herein, refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of a humanized antibody intended to produce a desired therapeutic effect in association 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 to achieve the desired therapeutic response for a particular patient, composition, and mode of administration without toxicity to the patient. The selected dosage level will depend on various pharmacokinetic factors, including the activity of the particular composition employed, the route of administration, the time of administration, the rate of excretion of the particular antibody employed, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular composition employed, the age, sex, weight, condition, overall health, and prior medical history of the patient being treated, and the like.

[0063] A physician or veterinarian may start the dosage of an antibody or functional fragment thereof used in a pharmaceutical composition at a level lower than that required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. Generally, the effective dosage of a composition may vary depending on various factors, including the specific disease or condition being treated, the means of administration, the target site, the physiological condition of the patient, whether the patient is human or animal, other medications being administered, and whether the treatment is prophylactic or therapeutic. Therapeutic dosages should be titrated to optimize safety and efficacy. For administration with antibodies, the dosage ranges from about 0.0001 to 100 mg / kg (usually 0.01 to 5 mg / kg) of host body weight. For example, the dosage may be 1 mg / kg body weight or 10 mg / kg body weight, or within the range of 1 to 10 mg / kg. An exemplary treatment regime involves administration once every 2 or 3 weeks, once a month, or once every 3 to 6 months.

[0064] The BAFF-R antibody or functional fragment thereof provided herein may be administered on multiple occasions. The interval between single doses may be weekly, monthly, or yearly. The interval may also be irregular, as indicated by measuring the blood level of the humanized antibody in the patient. In some methods, the dosage is adjusted to achieve a plasma antibody concentration 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 dosage and frequency vary depending on the half-life of the antibody in the patient. In general, humanized antibodies exhibit a longer half-life than chimeric and non-human antibodies. The dosage and frequency of administration may vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, relatively low dosages are administered at relatively infrequent intervals over an extended period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, sometimes relatively high dosages are required at relatively short intervals until the progression of the disease is reduced or halted, and preferably until the patient shows partial or complete improvement in the symptoms of the disease, after which the patient can be administered a prophylactic regime.

[0065] A mouse fibroblast cell expressing a human BAFF-R protein or a fragment thereof is provided, and the human BAFF-R protein or a fragment thereof is expressed on the cell surface of the cell. Optionally, the human BAFF-R protein or a fragment thereof comprises a detectable moiety. Optionally, the detectable moiety is a fluorescent moiety. Optionally, the detectable moiety is enhanced green fluorescent protein (eGFP).

[0066] Methods for treating cancer in a subject in need thereof are provided, comprising administering to the subject a therapeutically effective amount of a chimeric antigen receptor provided herein, thereby treating the cancer in the subject.

[0067] In another embodiment, a method of treating cancer in a subject in need thereof 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, the cancer is lymphoma, leukemia, or myeloma. Optionally, the cancer is lymphoma. Optionally, the lymphoma is mantle cell lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, marginal zone lymphoma, or Burkitt lymphoma. Optionally, the lymphoma is mantle cell lymphoma. Optionally, the lymphoma is follicular lymphoma. Optionally, the lymphoma is diffuse large B-cell lymphoma. Optionally, the lymphoma is marginal zone lymphoma. Optionally, the lymphoma is Burkitt lymphoma.

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

[0069] Optionally, the cancer is myeloma. Optionally, the 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 its conventional sense to a monoclonal antibody against the protein CD20, identified by the ATC code L01XC02.

[0071] Also provided is a method for treating an autoimmune disease in a subject in need thereof. The method comprises administering to the subject a therapeutically effective amount of an antibody or functional fragment thereof as provided herein, thereby treating the autoimmune disease in the subject. Optionally, the autoimmune disease is rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, glomerulonephritis, Sjogren'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 Sjogren'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 aspect, a method for inhibiting cell proliferation is provided. The method includes contacting a cell with a BAFF-R antibody or functional fragment thereof (including embodiments thereof) as provided herein, thereby forming a contacted cell. The BAFF-R antibody or functional fragment thereof binds to a BAFF-R protein on the contacted cell, thereby inhibiting cell proliferation. Optionally, the cell is a lymphoid cell. Optionally, the cell is a B cell. Optionally, the cell is a cancer cell. Optionally, the cell is a lymphoma cell.

[0073] In another embodiment, a method for producing an anti-human BAFF-R antibody is provided. The method includes administering mouse fibroblasts 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 the anti-BAFF-R antibody. Optionally, the anti-BAFF-R antibody is an antibody as provided herein.

[0074] While various embodiments and aspects have been shown and described herein, it will be apparent to those skilled in the art that such embodiments and aspects are provided by way of example only. Numerous variations, changes, and substitutions will now occur to 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 (approximately 150,000 Da molecular weight or approximately 1,320 amino acids) with complex internal structures. Natural antibody molecules contain two identical pairs of polypeptide chains, each pair consisting of one light chain and one heavy chain. Each light and heavy chain consists of two regions: a variable ("V") region, which is responsible for binding the target antigen, and a constant ("C") region, which interacts with other components of the immune system. The light and heavy chain variable regions combine in three-dimensional space to form the variable region that binds the antigen (e.g., a receptor on the surface of a cell). Within each light or heavy chain variable region, there are three short segments (averaging 10 amino acids in length) called complementarity-determining regions ("CDRs"). The six CDRs in an 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 positions and lengths of the CDRs were precisely defined by Kabat, E. et al., Sequences of Proteins of Immunological Interest, USDapartment of Health and Human Services, 1983, 1987. The parts of the variable region that are not contained in the CDRs are called the framework ("FR"), which forms the environment for the CDRs.

[0076] The term antibody is used according to its normal meaning known in the art. Antibodies exist, for example, as intact immunoglobulins. However, whenever the term antibody(ies) is recited 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 antibodies below the disulfide bonds in the hinge region to produce F(ab)'2 (Fab (light chains separated by disulfide bonds) H -C H1 The F(ab)'2 can be reduced under mild conditions that disrupt the disulfide bond in the hinge region, thereby converting the F(ab)'2 dimer to a Fab' monomer. Fab' monomers are essentially Fab with part of the hinge region (see Fundamental Immunology (Paul, ed., 3rd ed. (1993))). While various antibody fragments are defined in terms of the digestion of intact antibodies, those skilled in the art will recognize that such fragments can be synthesized de novo, either chemically or by using recombinant DNA methodology. Thus, the term antibody, as used herein, is exemplary and may be used to describe antibody fragments produced by the modification of whole antibodies, or those synthesized de novo using recombinant DNA methodology (e.g., single-chain Fv) or those identified using phage display libraries (see, e.g., McCafferty et al., Nature 348:552-554 (1990)).

[0077] Any technique known in the art can be used for the preparation of monoclonal or polyclonal antibodies (see, e.g., 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 the production of single-chain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce antibodies to the polypeptides described herein. Transgenic mice or other organisms (such as other mammals) can also be used to express humanized antibodies. Alternatively, phage display technology can be used to identify antibodies and heteromeric Fab fragments that specifically bind to a selected antigen (see, e.g., McCafferty et al., Nature 348:552-554 (1990); Marks et al., Biotechnology 10:779-783 (1992)).

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

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

[0080] A label or detectable moiety is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. For example, useful labels include: 32 Examples of suitable labeling agents include P, fluorescent dyes, electron-dense reagents, enzymes (such as those commonly used in ELISA), biotin, digoxigenin, or haptens and proteins or other entities that can be made detectable by incorporating a radioisotope label into, for example, a peptide or antibody specifically reactive with the target peptide. Any suitable method known in the art for conjugating an antibody to a label can be used (e.g., using the methods described in Hermanson, Bioconjugate Techniques 1996, Academic Press, Inc., San Diego).

[0081] Contacting is used according to its plain and ordinary meaning to refer to a process that allows at least two distinct entities (e.g., chemical compounds, including biomolecules or cells) to come into sufficient proximity to react, interact, or come into physical contact. However, it should be recognized that the resulting reaction product may be produced directly from the reaction between the added reagents, or from an intermediate from one or more of the added reagents that may be produced in the reaction mixture.

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

[0083] As used herein, treating a condition, disease, or disorder, or symptoms associated with a condition, disease, or disorder, or their treatment, refers to an approach to achieving beneficial or desired results (including clinical results). Beneficial or desired clinical results may include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, reduction in the severity of the condition, disorder, or disease, stabilization of the condition, disorder, or disease state, prevention of the onset of the condition, disorder, or disease, prevention of the spread of the condition, disorder, or disease, delay or slowing of the progression of the condition, disorder, or disease, delay or slowing of the onset of the condition, disorder, or disease, amelioration or alleviation of the condition, disorder, or disease state, and remission (whether partial or total). Treatment may also refer to prolonged survival of a subject beyond that expected in the absence of treatment. In some cases, treatment encompasses permanently halting the progression of the condition, disorder, or disease, but may also refer to inhibiting the progression of the condition, disorder, or disease, or temporarily slowing the progression of the condition, disorder, or disease. As used herein, the terms "treatment," "treating," or "treating" refer to a method of reducing the effects of one or more symptoms of a disease or condition characterized by protease expression, or a symptom of a disease or condition characterized by protease expression. Thus, in the disclosed methods, treatment can refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of an established disease, condition, or symptom of a disease or condition. For example, a method of treating a disease is considered therapeutic if there is a 10% reduction in one or more symptoms of the disease in a subject compared to a control. Thus, the reduction can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percentage reduction between 10% and 100% compared to native or control levels. It is understood that treatment does not necessarily refer to a cure or complete elimination of the disease, condition, or symptoms of the disease or condition.Furthermore, as used herein, references to decrease, reduction or inhibition include a change of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to a control level, and such terms can, but do not necessarily, include complete elimination.

[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 moiety other than amino acids. The terms apply to naturally occurring and non-naturally occurring amino acid polymers, as well as to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of corresponding naturally occurring amino acids. A fusion protein refers to a chimeric protein encoding two or more separate protein sequences that are recombinantly expressed as a single moiety. The terms peptidyl and peptidyl moiety refer to a monovalent peptide.

[0085] As used herein, the term amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics 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 amino acids 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 a naturally occurring amino acid (i.e., an α-carbon bonded to a hydrogen, a carboxyl group, an amino group, and an R group). Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refer to chemical compounds that have a structure that differs from the general chemical structure of an amino acid but functions in a manner similar to a naturally occurring amino acid. The terms non-naturally occurring amino acids and unnatural amino acids refer to amino acid analogs, synthetic amino acids, and amino acid mimetics that are not found in nature.

[0086] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides may also be referred to by their commonly accepted single-letter codes.

[0087] "Conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to a particular nucleic acid sequence, conservatively modified variants refer to nucleic acids that encode identical or essentially identical amino acid sequences. Because of the degeneracy of the genetic code, many nucleic acid sequences will encode any given protein. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at every position where alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are silent variations, which are one species of conservatively modified variation. Every nucleic acid sequence herein that encodes a polypeptide also describes every possible silent variation of the nucleic acid. Those of skill 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 nucleic acid that encodes a polypeptide 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 a nucleic acid, peptide, polypeptide, or protein sequence (which alter, add, or delete a single amino acid or a small percentage of amino acids in the encoded sequence) are conservatively modified variants when the alteration results in the substitution of an amino acid with a chemically similar amino acid. Catalogs of conservative substitutions that provide functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to, and do not exclude, polymorphic variants, interspecies homologs, and alleles.

[0089] The following eight 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 contain amino acids that are conservative substitutions for one another (see, e.g., Creighton, Proteins (1984)).

[0090] The percentage of sequence identity is determined by comparing two optimally aligned sequences over a comparison window, where, due to 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 the reference sequence (which does not contain additions or deletions). The percentage is calculated by determining the number of positions where the identical nucleic acid base or amino acid residue occurs in both sequences to obtain the number of matched positions, dividing the number of matched 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 identity refer to two or more sequences or subsequences that, when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using 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, e.g., over a specified region of an entire polypeptide sequence or a distinct domain of a polypeptide). Such sequences are then said to be substantially identical. This definition also refers to the complement of a test sequence. Optionally, identity exists over a region that is at least about 50 nucleotides in length, more preferably over a region that is 100 to 500 or 1000 or more nucleotides in length. The present description includes polypeptides substantially identical to any of SEQ ID NOs: 30-51.

[0092] For sequence comparison, typically, one sequence serves as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence relative to the reference sequence based on the program parameters.

[0093] As used herein, a comparison window includes reference to, for example, a full-length sequence or any segment of a number of contiguous 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 a sequence can be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods for aligning sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be accomplished, 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 for similarity method 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, e.g., Ausubel et al., Current Protocols in Molecular Biology (1995 supplement)).

[0094] Examples of suitable algorithms for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, 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 words of length W in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing the seed. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0) for nucleotide sequences. For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of word hits in each direction is stopped when the cumulative alignment score falls by an amount X from its maximum achieved value; the cumulative score falls below 0 due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. For nucleotide sequences, the BLASTN program uses as defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands.For amino acid sequences, the BLASTP program uses as defaults a word length of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915), an alignment (B) of 50, an expectation (E) of 10, M=5, N=−4, and a 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 smallest sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, in a comparison of a test nucleic acid to a reference nucleic acid, if the smallest 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 determined to be similar to the reference sequence.

[0096] An indication 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 generated against the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, when the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below. Yet another indication 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 in the antibody as the given residue. For example, a selected residue in a comparison antibody corresponds to position 48 (according to the Kabat numbering system as described herein) in an antibody provided herein if the selected residue occupies the same essential spatial or structural relationship to Kabat position 48 as assessed using methods available in the art. For example, the comparison antibody can be aligned with the antibody provided herein for maximum sequence similarity, and positions in the aligned comparison antibody that align with Kabat position 48 can be determined to correspond thereto. Alternatively, instead of (or in addition to) a primary sequence alignment as described above, three-dimensional structural alignment can also be used, for example, when the structure of the comparison antibody is aligned with the antibody provided herein for maximum correspondence and the overall structures are compared. In this case, amino acids occupying the same essential position as Kabat position 48 in the structural model can be said to correspond.

[0098] The term "isolated," when applied to a protein, denotes that the protein is essentially free of other cellular components with which it is naturally associated. It can be in either a dry or aqueous solution, but is preferably in a homogeneous state. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. The predominant species of protein present in a preparation is substantially purified. The term "isolated" denotes that the protein gives rise to essentially one band in an 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, when referring to a protein or peptide, refer to a binding reaction that is determinative of the protein's presence in a heterogeneous population of proteins and other biologics. Thus, under specified immunoassay conditions, a given antibody will bind to a particular protein at least twice background and will not substantially bind in significant amounts to other proteins present in the sample. Typically, a specific or selective reaction will be at least twice background signal or noise, and more typically 10-100 times background or more.

[0100] As used herein, a 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, including, for example, the presence of an intact membrane, staining with specific dyes, the ability to produce progeny, or, in the case of gametes, the ability to combine with a second gamete to produce viable offspring. Cells can include prokaryotic and eukaryotic cells. Prokaryotic cells include, but are not limited to, bacteria. Eukaryotic cells include, but are not limited to, yeast cells and cells derived from plants and animals, such as mammalian cells, insect (e.g., Spodoptera) cells, and human cells.

[0101] As defined herein, the terms inhibit, inhibit, inhibiting, and the like, with respect to protein inhibitor (e.g., a BAFF-R antibody provided herein) interaction, mean negatively affecting (e.g., decreasing) the activity or function of a protein (e.g., decreasing the activity of BAFF-R) compared to the activity or function of the protein in the absence of the inhibitor (e.g., a BAFF-R antibody). Inhibition includes the reduction of disease or symptoms of disease (e.g., cancer or autoimmune disease). Thus, inhibition includes at least partially, partially, or completely blocking stimulation, reducing, preventing, delaying activation, inactivating, desensitizing, or downregulating signal transduction or enzymatic activity or protein amount. 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 (e.g., antibodies) provided herein are often administered as pharmaceutical compositions containing an active therapeutic agent and various other pharmaceutically acceptable components. See Remington: The Science and Practice of Pharmacy, 22nd ed., 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 contain a pharmaceutically acceptable, non-toxic carrier or diluent (usually defined as a vehicle used in formulating pharmaceutical compositions for animal or human administration). The diluent is selected so as not to affect the biological activity of the combined entity. Examples of such diluents are distilled water, physiological phosphate-buffered saline, Ringer's solution, dextrose solution, and Hank's solution. In addition, the pharmaceutical composition or formulation may also include other carriers, adjuvants, or non-toxic, non-therapeutic, non-immunogenic stabilizing substances and the like.

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

[0104] Formulations suitable for oral administration may consist of (a) solutions (such as an effective amount of an antibody provided herein 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, granules, or gelatin); (c) a suspension in a suitable liquid; and (d) a suitable emulsion. Tablet forms may contain one or more of lactose, sucrose, mannitol, sorbitol, calcium phosphate, corn starch, potato starch, microcrystalline cellulose, gelatin, colloidal silicon dioxide, talc, magnesium stearate, stearic acid and other excipients, coloring substances, fillers, binding substances, diluents, buffers, wetting agents, preservatives, flavoring agents, dyes, disintegrants, and pharmaceutically compatible carriers. Lozenge forms contain the active ingredient in a flavoring (e.g., sucrose); similarly, troches contain the active ingredient in an inert base (gelatin and glycerin or sucrose and acacia emulsion, gel, etc.); and the like may contain carriers known in the art in addition to the active ingredient.

[0105] Pharmaceutical compositions may also include large, slowly metabolized macromolecules such as proteins, polysaccharides (such as chitosan), polylactic acids, polyglycolic acids and copolymers (such as latex, functionalized Sepharose™, agarose, cellulose, and the like), polymeric amino acids, amino acid copolymers, and lipid aggregates (such as oil droplets or liposomes). In addition, these carriers may function as immunostimulating agents (i.e., adjuvants).

[0106] Suitable preparations for rectal administration include, for example, suppositories (composed of nucleic acid packaged in a suppository base). Suitable suppository bases include natural or synthetic triglycerides or paraffin hydrocarbons. In addition, gelatin rectal capsules can be used, which are made up of a combination of a selected compound with a base (for example, liquid triglycerides, polyethylene glycol, and paraffin hydrocarbons).

[0107] Suitable formulations for parenteral administration (e.g., by intra-articular (intra-articular), intravenous, intramuscular, intratumoral, intradermal, intraperitoneal, and subcutaneous routes, etc.) include aqueous and non-aqueous isotonic sterile injection solutions (which may contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient), and aqueous and non-aqueous sterile suspensions (which may contain suspending agents, solubilizing agents, thickening agents, stabilizing agents, and preservatives). Compositions may be administered, for example, by intravenous infusion, orally, topically, intraperitoneally, intravesically, or intrathecally. Parenteral, oral, and intravenous administration are preferred methods of administration. Formulations of the compounds may be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials.

[0108] Injection solutions and suspensions may be prepared from sterile powders, granules, and tablets of the kind previously described. Nucleic acid-transduced cells for ex vivo therapy may also be administered intravenously or parenterally as described above.

[0109] The pharmaceutical preparation can be in unit dosage form. In such form, the preparation is subdivided into unit doses containing appropriate amounts of the active ingredient. The unit dosage form can be a packaged preparation, the package containing discrete quantities of the preparation (such as tablets, capsules, and powders, dispensed in vials or ampoules). Further, the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form. The composition can also contain other compatible therapeutic agents, if desired.

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

[0111] The effective dose of the compositions provided herein may vary depending on different factors, including the means of administration, the target site, the physiological condition of the patient, whether the patient is human or animal, other medications being administered, and whether the treatment is prophylactic or therapeutic. However, one of ordinary skill in the art will readily recognize appropriate and / or equivalent doses, taking into account the dosages of compositions approved for treating and preventing cancer for guidance.

[0112] The term disease or condition refers to a condition that is treated by the compounds, pharmaceutical compositions or methods provided herein, or the health status of a patient or subject that can be treated in this way.Optionally, the 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).The disease can 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 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, carcinoma, and sarcoma. Exemplary cancers that can be treated by the compounds, pharmaceutical compositions, or methods provided herein include lymphoma, sarcoma, bladder cancer, bone cancer, brain cancer, 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, ductal carcinoma, lobular carcinoma, primary, metastatic), and ovarian cancer. , pancreatic cancer, liver cancer (e.g., hepatocellular carcinoma), lung cancer (e.g., non-small cell lung cancer, squamous cell lung carcinoma, adenocarcinoma, large cell lung carcinoma, small cell lung carcinoma, carcinoid, sarcoma), glioblastoma multiforme, 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 esophageal), colorectal cancer, leukemia, acute myeloid leukemia, lymphoma, B-cell lymphoma, or multiple myeloma. Additional examples include thyroid cancer, cancer of the endocrine system, brain tumors, breast cancer, cervical cancer, colon 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's lymphoma, multiple myeloma, neuroblastoma, glioma, glioblastoma multiforme, ovarian cancer, rhabdomyosarcoma, primary thrombocythemia, primary macroglobulinemia, primary brain tumors, cancer, malignant pancreatic insulin tumors include thyroid cancer, lymphoma, malignant carcinoid, bladder cancer, premalignant skin lesions, testicular cancer, lymphoma, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary cancer, malignant hypercalcemia, endometrial cancer, adrenocortical carcinoma, neoplasms of the endocrine or exocrine pancreas, medullary thyroid carcinoma, melanoma, colorectal cancer, papillary thyroid carcinoma, hepatocellular carcinoma, Paget's disease of the breast, phyllodes tumor, lobular carcinoma, ductal carcinoma, carcinoma of the pancreatic stellate cells, carcinoma of the hepatic stellate cells, or prostate cancer.

[0114] The term leukemia broadly refers to progressive, malignant diseases of the blood-forming organs and is generally characterized by distorted proliferation and development of white blood cells and their precursors in the blood and bone marrow. Leukemias are generally classified clinically based on: (1) the duration and character of the disease (acute or chronic); (2) the type of cells involved: myeloid (myelogenic), lymphoid (lymphogenic), or monocytic; and (3) the increased or absent 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 nonlymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, non-leukemic leukemia, leukocytic leukemia, and leukocytic leukemia. leukemia), basophilic leukemia, blast cell leukemia, bovine leukemia, chronic myeloid leukemia, leukemia cutis, stem cell leukemia, eosinophilic leukemia, gross leukemia, hairy cell leukemia, hemoblastic leukemia, hemoblastic leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenic leukemia, lymphocytic leukemia, lymphoblastic leukemia, lymphocytic leukemia, lymphoid leukemia, These include lymphosarcoma cell leukemia, mast cell leukemia, megakaryocytic leukemia, small myeloblastic leukemia, monocytic leukemia, myeloblastic leukemia, myeloid leukemia, myelogranulocytic leukemia, myelomonocytic leukemia, Naegeli leukemia, plasma cell leukemia, multiple myeloma, plasma cell leukemia, promyelocytic leukemia, Leder cell leukemia, Schilling leukemia, stem cell leukemia, subleukemia, or anaplastic cell leukemia.

[0115] As used herein, the terms metastasis and metastatic cancer are used interchangeably and can refer to the spread of a proliferative disease or disorder (e.g., cancer) from one organ or another non-adjacent organ or body part. Cancer begins at a site of origin (e.g., the breast), which is called a 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 surrounding normal tissue in the local area and / or penetrate the walls of the circulatory lymphatic or vascular system via the system to other sites and tissues in the body. A second, clinically detectable tumor formed from cancer cells of the primary tumor is called a metastatic tumor or secondary tumor. When cancer cells metastasize, the metastatic tumor and its cells are expected to resemble those of the original tumor. Thus, if lung cancer metastasizes to the breast, the secondary tumor at the breast site will consist of abnormal lung cells rather than abnormal breast cells. A secondary tumor in the breast is called 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 the subject suffering from 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 which the subject suffers from or has a history of a primary lung tumor, and suffers from one or more secondary tumors at a second location or multiple locations (for example, in the breast).

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

[0117] As used herein, an autoimmune disease refers to a disease or disorder resulting from an altered immune response by a subject's immune system against, for example, tissues and / or cells normally present in the subject's body. Autoimmune diseases include, but are not limited to, arthritis, rheumatoid arthritis, psoriatic arthritis, juvenile idiopathic arthritis, scleroderma, systemic sclerosis, multiple sclerosis, systemic lupus erythematosus (SLE), myasthenia gravis, juvenile diabetes, type 1 diabetes, 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 abnormal or altered inflammation. Inflammation is a biological response initiated by the immune system as part of the 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 myopathy.

[0119] Humanized antibodies are genetically engineered antibodies in which at least one CDR (or a functional fragment or variant thereof) from a mouse antibody (the "donor antibody," which can also be rat, hamster, or other non-human species) is grafted onto a human antibody framework (the "acceptor antibody"). Optionally, more than one mouse CDR is grafted (e.g., all six mouse CDRs are grafted). The acceptor antibody sequence can 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 can be an antibody having one or more CDRs and variable region frameworks (FRs) from the donor antibody. The FRs can form part of the constant and / or variable regions in a human antibody. Additionally, to retain high binding affinity, amino acids in the human acceptor sequence can be replaced by the corresponding amino acid from the donor sequence, e.g., (1) the amino acid is in a CDR, or (2) the amino acid is in a human framework region (e.g., the amino acid is immediately adjacent to one of the CDRs). See U.S. Patent Nos. 5,530,101 and 5,585,089 (incorporated herein by reference), which provide detailed instructions for the construction of humanized antibodies. Humanized antibodies often incorporate all six CDRs from a murine antibody (e.g., as defined by Kabat, but often including hypervariable loop H1 as defined by Chothia), although they can also be generated with fewer murine CDRs and / or less than the entire murine CDR sequence (e.g., functional fragments of the 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) from a murine antibody (also referred to herein as murine CDRs) and a human variable region framework; and (ii) a heavy chain variable region comprising at least one CDR (often three CDRs) from a murine antibody and a human variable region framework (FR). The light and heavy chain variable region frameworks (FR) 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 of a human antibody variable region framework sequence (or a fragment thereof). Optionally, the humanized antibody comprises a light chain variable region as described in (i) and a heavy chain variable region as described in (ii), together with a human light chain constant region and a human heavy chain constant region.

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

[0122] Other approaches to designing humanized antibodies can be used to achieve the same results as the methods in U.S. Patent Nos. 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 the methods of Studnicak et al., Protein Eng. 7:805, 1994. Additionally, other approaches to producing genetically engineered mAbs of reduced immunogenicity include reshaping, hyperchimerization, and veneering / resurfacing, as described, for example, in Vaswami et al., Annals of Allergy, Asthma and Immunology 81:105, 1998; Roguska et al., Protein Eng. 9:895, 1996; and U.S. Pat. Nos. 6,072,035 and 5,639,641.

[0123] Disclosed are materials, compositions, and components that can be used for, used in conjunction with, used in preparation for, or are products of the disclosed methods and compositions. These and other materials are disclosed herein, and when combinations, subsets, interactions, groups, etc. of these materials are disclosed, it is understood that specific reference to each of the various individual and collective combinations and permutations of these compounds may not be explicitly disclosed, but each is specifically contemplated and described herein. For example, if a method is disclosed and contemplated, and many modifications (including methods) that can be made to many molecules are contemplated, each and every combination and permutation of the methods and possible modifications is specifically contemplated unless specifically indicated to the contrary. Likewise, any subset or combination thereof is also specifically contemplated and disclosed. This concept applies to all aspects of the present disclosure, including, but not limited to, steps in methods using the disclosed compositions. Thus, if there are a variety of additional steps that may be performed, it is understood that each of these additional steps may be performed by any specific method step or combination of method steps of the disclosed methods, and that each such combination or subset of combinations should be considered specifically contemplated and disclosed.

[0124] The following examples are intended to further illustrate certain aspects of the methods and compositions described herein, and are not intended to limit the scope of the claims. [Example]

[0125] Example 1. Novel BAFF receptor antibodies to natively folded recombinant protein eliminate drug-resistant human B-cell malignancies in vivo. Traditional recombinant immunogen proteins produced in bacteria for mAb development lack post-translational modifications and fold simply because prokaryotes lack chaperone proteins and an oxidizing environment compared to eukaryotes. As a result, such proteins may differ in conformational structure from the corresponding plasma membrane-anchored native protein. Furthermore, antibodies can be raised against off-target domains (such as the transmembrane or intracellular domains of the target protein). As described herein, a strategy for generating mAbs against natively folded and glycosylated immunogens expressed on eukaryotic cells was applied. In particular, human BAFF-R is expressed as a native protein on mouse fibroblasts, and engineered cell clones were used as immunogens in mice. The generation of novel mAbs that specifically bind and lyse human malignant B-cell lines and primary lymphomas in vitro and inhibit the growth of drug-resistant lymphoma cell lines in vivo in a xenogeneic tumor model is described herein.

[0126] Materials and Methods Animals, cell lines, and primary human tumor samples. BALB / c mice and NOD scidγ (NSG) breeding pairs for antibody development were purchased from the Jackson Laboratory (Bar Harbor, ME). The NSG breeding colony was maintained by the City of Hope Animal Resource Center. Mice were housed in a pathogen-free animal facility 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 line was provided by Dr. Michael Wang (MD Anderson Cancer Center). The ibrutinib-resistant SP49-IR line 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 increasing doses of ibrutinib. The IC50 was 5 nM for parental SP49 compared to >100 nM for SP49-IR. At 100 nM ibrutinib, approximately 5% of SP49 cells were viable 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 lymphomas were obtained from the MD Anderson Cancer Center Lymphoma Satellite Tissue Bank under an Institutional Review Board-approved protocol (IRB: 2005-0656) as viable single-cell suspensions cryopreserved in 10% DMSO. Primary patient samples included leukapheresis or blood from patients with mantle cell lymphoma (MCL) or chronic lymphocytic leukemia (CLL), as well as resected 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% for leukapheresis or blood and 50% to 60% for 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. Human BAFF-R (hBAFF-R) cDNA was derived from human B cells and cloned in frame with the GFP gene into the 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 subsequently cloned into a lentiviral gene delivery system (pLenti6 / V5-DEST Gateway Vector kit, Life Technologies, Grand Island, NY) to produce the hBAFF-R-GFP fusion protein when transduced into mouse fibroblast (L) cells. Single-cell clones were established from sorted GFP-positive L cells, and the (h)BAFF-R-GFP-expressing L cell clone D2C was used in further studies.

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

[0129] Chimeric antibody production. cDNAs from selected hybridomas encoding the antibody light and heavy chain variable regions were engineered onto expression vectors containing the respective human IgG1 constant regions. The vectors were cotransfected into the FreeStyle 293 expression system (Life Technologies, Carlsbad, CA) according to the manufacturer's instructions. Antibodies in the culture supernatant were purified using a 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, and primary patient samples) were labeled with chromium-51 (51Cr, Perkin Elmer, Waltham, MA) for 51Cr release assays. Briefly, antibodies and effectors (NK cells or complement serum standards [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 using a Wizard Automatic Gamma Counter (Perkin Elmer).

[0131] Generation of JeKo-1-CD20-KO. FACS-sorted stable JeKo-1-CD20-KO 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 blot.

[0132] In vivo studies. For tumor models, stable luciferase-expressing tumor lines were established for bioluminescence imaging in mouse models. Briefly, the luciferase gene was introduced into the tumor lines by 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 titration. Tumor cells were injected intravenously (IV), and mice were monitored by in vivo bioluminescence imaging to ensure engraftment of the minimum tumor dose. The minimum lethal tumor dose was 1 × 10 6 JeKo-1 cells, 5 x 10 5 RS4;11 cells, 5 x 10 5 JeKo-1-CD20-KO cells, or 2.5 x 10 4 The cells were Z-138 cells.

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

[0134] Antibody studies: Mice (n=5 per group) were challenged with tumors IV 3 days before four treatments, one every 5 days. Treatments consisted of a 300 μL IV injection (200 μg of therapeutic antibody, 10×10 6 of effector human NK-92-176V cells, and 5 x 10 4 The injection volume was 1.25 mL / min. The injection volume was 1.25 mL / min. IL-2 (Prometheus Laboratories, San Diego, CA). Control groups received the same volume of injections with or without control antibody and / or NK cells. Bioluminescence imaging was performed weekly up to day 80. Survival was followed up to 100 days after tumor challenge.

[0135] result Generation of monoclonal antibodies against human BAFF-R. To generate therapeutic antibodies to biologically relevant epitopes of BAFF-R, a eukaryotic cell surface expression system was used, in which endogenous cell surface proteins are presented in their native conformation with appropriate post-translational modifications. Mouse fibroblast (L) cell clones were engineered to express cell surface GFP-tagged human BAFF-R. BAFF-R-expressing L cell clones were generated and characterized for GFP expression (Figure 1A). Clone D2C was expanded and used to successfully immunize BALB / c mice according to the immunization schedule in Methods and Figure 7A.

[0136] After generating and screening hybridoma clones, four clones (53, 55, 67, and 90) were identified that produced antibodies that specifically bound 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 the BAFF-R-expressing Mino cell line (MCL) in a dose-dependent manner. Antibody binding was not detected in the BAFF-R-negative control cell line (293T) (Figure 8).

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

[0138] Analysis of the complementarity determining regions (CDRs) on the 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 detected at high concentrations (2 μg / 10 6 cells) and low concentrations (0.05 μg / 10 6 In the 10-well platelet-free (100-well platelet-free) cells, JeKo-1 (MCL), SU-DHL-6 (DLBCL), Raji (Burkitt's lymphoma), and RL (FL) were effectively bound (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 (designated C55 and C90, respectively). 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 exhibited direct binding to the non-Hodgkin's lymphoma (NHL) lines JeKo-1, SU-DHL-6, Raji, and RL (Figure 1D). Importantly, the chimeric mAbs readily bound primary tumor samples from patients with MCL, DLBCL, and FL (Figure 1E and Figure 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 a BAFF-R-negative human multiple myeloma line (U266) (Figures 2A and 12). In contrast, the antibodies did not induce complement-dependent 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, the chimeric antibodies induced ADCC against primary patient tumor samples (Figure 3A).

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

[0142] NSG mice were challenged in vivo with the luciferase knock-in JeKo-1 MCL cell line, followed by antibody treatment. Treatment followed the schedule in Figure 4A. Compared with PBS or NK cell-only control groups, mice treated with either C55 or C90 demonstrated significant tumor growth delay (Figure 4B). Similarly, C55 and C90 significantly delayed tumor growth in NSG mice challenged with RS4;11 (acute lymphoblastic leukemia (ALL)) compared with no inhibition by rituximab or control (Figure 4C).

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

[0144] To create a drug-resistant lymphoma model, we generated stable CD20 knockout (KO) clones of JeKo-1 using the CRISPR / HDR system. CD20-KO clones were confirmed for the absence of CD20 surface expression by flow cytometry and Western blot (Figure 5A and Figures 16A and 16B) and the presence of 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 of drug-resistant lymphoma, the chimeric BAFF-R mAb was tested for ADCC against a naturally ibrutinib-resistant human MCL line (Z-138) and an induced ibrutinib-resistant MCL line (SP49-IR, resistance to ibrutinib was induced in vitro (see Methods)). Significant in vitro ADCC was observed with the antibody against both ibrutinib-resistant lines (Figure 5C).

[0146] Finally, 3 days after in vivo challenge with JeKo-1-CD20-KO tumor cells, NSG mice (n = 5 per group) were administered BAFF-R antibody treatment (C55 or C90) or rituximab as described in the methods and according to the schedule in Figure 4A. Bioluminescence imaging on 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 with controls (PBS or NK only) (Figure 6B-C).

[0147] BAFF-R mAb also binds to normal B cells. When tested on 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 (FIG. 17). The positive staining results were verified with purified B cells (FIG. 18). Furthermore, purified T cells, NK cells, and gated myeloid cells showed no binding.

[0148] Expanding the scope of this study, immunohistochemistry studies showed positive staining of the antibody of the present invention in tonsil and spleen samples, with no staining in all other vital organs, including heart, lung, kidney, and brain (Figures 19A and 19B).

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

[0150] The distinct properties of the BAFF-R mAbs may be due to the approach used to generate them. The proposed approach 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 likely that the antibody binds an accessible human BAFF-R epitope distinct from other described antibodies. Thus, a technical strategy was demonstrated for the generation of a monoclonal antibody against natively folded and eukaryotically glycosylated human BAFF-R that 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 of CDC was observed. Both antibodies were able to competitively inhibit the binding of BAFF ligand to BAFF-R (Figure 14).

[0151] One clinically relevant mechanism of resistance to rituximab is downregulation of CD20. This phenomenon of drug resistance was modeled using a CRISPR-edited MCL line (JeKo-1, which lacks CD20). The significant in vivo antitumor effect of C55 or C90 on this line, as well as on naturally ibrutinib-resistant Z-138 MCL (but not on rituximab treatment), suggests their efficacy against drug-resistant lymphomas (Figure 5). Combined with the in vitro cytotoxicity of these antibodies against primary tumors from lymphoma patients who had previously been treated with rituximab and progressed in response, these data suggest the potential of C55 and C90 as a therapeutic strategy to overcome drug resistance (Figure 3).

[0152] Example 2. Humanization of BAFF-R mAb. A chimeric antibody (clone 90) was humanized while retaining its binding specificity and cytotoxic effect. Through computational analysis of the CDRs and predicted structures, three variants of the heavy chain and three variants of the light chain were generated with varying degrees of similarity to human antibodies. A total of nine combinatorial variants were constructed from the humanized heavy and light chains. All of these variants had K values ​​ranging from 2.6 to 5.0 nM. D values ​​and demonstrated binding affinities comparable to those of the parent chimeric antibody (Table 1). [Table 1]

[0153] Nine candidate antibodies were further evaluated to determine the leading candidate. The binding of the humanized antibodies was observed to be specific for BAFF-R, with all exhibiting similar relative binding in a dose-dependent manner (Figure 20A). In addition, the ADCC efficacy of the humanized antibodies was assessed. Again, 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 with a fluorescent streptavidin probe. Their binding to various non-Hodgkin's lymphoma, lymphoblastic leukemia, and multiple myeloma lines, including JeKo-1, Ly-10, MEC-2, RL, RS4, Raji, Z138, and U266, was assessed (Figure 22A). Flow cytometry results reveal significant binding to each of these cell lines. Further flow analysis of the humanized variants against normal PBMCs demonstrates specificity in binding. When assessed for binding to granulocytes, monocytes, B cells, T cells, and NK cells in normal, healthy PBMCs, the antibody binds exclusively to the B cell population (Figure 22B).

[0155] The two variants were further assessed for their ability to initiate ADCC. After a period of chromium uptake, varying concentrations of antibody were administered to the JeKo-1, Z138, and RS4 cell lines. The cells and antibody were incubated with effector NK cells. Supernatants were analyzed 6 hours after treatment (Figure 21A). The antibody had a clear cytotoxic effect on the tumor lines, demonstrating dose-dependence with 10-fold dilutions of each. Results are comparable to those of rituximab, but can also be observed in RS4 acute lymphoblastic lymphoma, for which rituximab is inactive. Further assays with LY-10, MEC-2, RL, and Raji (Figure 21B) were conducted to demonstrate the efficacy of humanized antibody therapy. All results observed were comparable to those of current conventional therapy with rituximab.

[0156] Example 3. Chimeric antigen receptor T cells. Antibodies with high binding affinity and biological activity were used to construct chimeric antigen receptor (CAR) T cells for in vivo studies. DNA sequences for the heavy and light chain variable domains were arranged into a single-chain (sFv) format, and the T cell signaling domain (δ chain) was engineered with a 4-1BB motif. The engineered CAR gene, along with co-expressed GFP, was introduced into purified CD8+ T cells from healthy donors via lentivirus. CAR-T cells were cell-sorted for GFP expression and expanded in vitro with CD3 and CD28 beads for animal studies. NSG mice were challenged with the luciferase-expressing JeKo-1 MCL line (JeKo-1-luci). Tumors were allowed to develop and monitored by bioluminescence imaging until a visible cluster of tumor cells was observed, approximately 9 days after tumor challenge. Mice received 5 x 10 6 Mice were treated with two doses of CAR-T cells (anti-BAFF-R and anti-CD19). Control groups received untreated T cells or saline (PBS). To assess the therapeutic antitumor effects of CAR-T therapy, mice were closely monitored and imaged every three days to track tumor progression.

[0157] The 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 majority of tumor cells expressing BAFF-R. Flow cytometry results reveal distinct populations of these primary tumor cells bound by the humanized antibodies of the present invention (Figure 22A). Furthermore, chromium release cytotoxicity assays on the same primary tumor samples revealed highly specific killing compared to controls. Results were comparable to the effects of rituximab and consistent with previously developed chimeric antibodies (Figure 22B). The cell type specificity of the humanized antibodies was determined by assessing their binding to normal PBMCs. No appreciable binding was noted for major populations 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 are also consistent with previously characterized chimeric antibodies.

[0158] The anti-BAFF-R mAb was further used to generate chimeric antigen receptor (CAR) T cells. The experiment utilized a chimeric C55 variable region engineered into a single-chain (sFv) format. The anti-BAFF-R C55 sFv was attached to a T cell receptor signaling domain containing a 4-1BB motif and successfully transduced into healthy normal human donor CD8+ T cells isolated from PBMCs. The CAR-T cells were administered to tumor-bearing mice with appreciable tumor burden (Figure 24). Mice treated with anti-BAFF-R CAR-T cells experienced significant tumor clearance compared to either saline or unengineered T cell control groups. Additionally, the anti-tumor efficacy of the CAR-T cells of the present invention was comparable to that of the anti-CD19 CAR-T treatment group.

[0159] The chimeric anti-BAFF-R antibody C90 was humanized with multiple variants. The humanization process took into account analysis of the variable regions of the chimeric antibody and in particular the CDRs. From there, three variants for each heavy and light chain were developed with degrees of similarity to humans ranging from 1 (most human) to 3 (most conservative to the chimera). The variants were combined to produce nine variants. Biacore analysis was performed on each variant as well as the chimeric parent C90 to determine their equilibrium dissociation constants, K D The antigen was the extracellular domain of commercial recombinant human BAFF-R.

Claims

1. 1. A B-cell activating factor receptor (BAFF-R) antibody comprising a light chain variable region and a heavy chain variable region, the light chain variable region comprising 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 comprising a CDR H1 as specified in SEQ ID NO: 10, a CDR H2 as specified in SEQ ID NO: 11, and a CDR H3 as specified in SEQ ID NO: 12; The antibody.

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

3. The antibody of claim 1 , wherein the antibody is a chimeric antibody.

4. The antibody of claim 1, wherein the light chain variable region comprises the sequence of SEQ ID NO:

30.

5. The antibody of claim 1, wherein the heavy chain variable region comprises the sequence of SEQ ID NO:

32.

6. The antibody of any one of claims 1 to 5, wherein the antibody is an IgG.

7. The antibody of claim 6, wherein the antibody is an IgG1.

8. The antibody of any one of claims 1 to 5, wherein the antibody is a Fab' fragment.

9. The antibody of any one of claims 1 to 5, wherein the antibody is a single chain antibody (scFv).

10. The antibody of any one of claims 1 to 9, wherein the antibody is capable of binding to a BAFF-R protein with an equilibrium dissociation constant (KD) of less than 5 nM.

11. The antibody of any one of claims 1 to 9, wherein the antibody is capable of binding to a BAFF-R protein with an equilibrium dissociation constant (KD) of less than 4 nM.

12. The antibody of any one of claims 1 to 11, wherein the antibody binds to a BAFF-R protein.

13. The antibody of claim 12, wherein the BAFF-R protein is a human BAFF-R protein.

14. The antibody of claim 13, wherein the BAFF-R protein forms part of a cell.

15. The antibody of claim 14, wherein the BAFF-R protein is expressed on the surface of the cell.

16. The antibody of claim 14 or 15, wherein the cell is a lymphoid cell.

17. The antibody of any one of claims 14 to 16, wherein the cell is a B cell.

18. The antibody according to any one of claims 14 to 16, wherein the cell is a cancer cell.

19. The antibody of claim 18, wherein the cancer cells are lymphoma cells.

20. An isolated nucleic acid encoding the antibody of any one of claims 1 to 11.

21. A pharmaceutical composition comprising a therapeutically effective amount of the antibody of any one of claims 1 to 19 and a pharmaceutically acceptable excipient.

22. A chimeric antigen receptor (CAR) comprising the antibody or functional fragment thereof according to any one of claims 1 to 19.

23. 23. A pharmaceutical composition for treating cancer in a subject in need thereof, comprising a therapeutically effective amount of the chimeric antigen receptor of claim 22.

24. 20. A pharmaceutical composition for treating cancer in a subject in need thereof, comprising a therapeutically effective amount of the antibody of any one of claims 1 to 19.

25. 25. The pharmaceutical composition of claim 23 or 24, wherein the cancer is lymphoma, leukemia, or myeloma.

26. 26. The pharmaceutical composition of claim 25, wherein the lymphoma is mantle cell lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, marginal zone lymphoma, or Burkitt's lymphoma.

27. 26. The pharmaceutical composition of claim 25, wherein the leukemia is lymphoblastic leukemia, chronic lymphocytic leukemia, or hairy cell leukemia.

28. 26. The pharmaceutical composition of claim 25, wherein the myeloma is multiple myeloma.

29. The pharmaceutical composition of any one of claims 23 to 28, further comprising a second therapeutic agent.

30. 30. The pharmaceutical composition of claim 29, wherein the therapeutic agent is a chimeric monoclonal antibody capable of binding to the CD20 antigen.

31. 31. The pharmaceutical composition of claim 29 or 30, wherein the therapeutic agent is rituximab.

32. 1. An in vitro method for inhibiting cell proliferation, comprising: (i) contacting a cell with a BAFF-R antibody of any one of claims 1 to 19, thereby forming a contacted cell; (ii) causing the BAFF-R antibody to bind to BAFF-R protein on the contacted cells, thereby inhibiting the proliferation of the cells; The method comprising:

33. 33. The method of claim 32, wherein the cells are lymphoid cells.

34. 34. The method of claim 32 or 33, wherein the cell is a B cell.

35. The method of any one of claims 32 to 34, wherein the cells are cancer cells.

36. The method of any one of claims 32 to 35, wherein the cells are lymphoma cells.

Images