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 Jun. 6, 2016, the entirety of which is 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 exemplary case is rituximab, which targets CD20 and induces cytotoxic effects on B-cell lymphoma.However, the main concern with rituximab is the emergence of rituximab resistance, which is believed to be due to CD20 being downregulated, 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 K lower than approximately 4 nM DA humanized B-cell activating factor receptor (BAFF-R) antibody or functional fragment thereof capable of binding to

[0005] BAFF-R and K lower than approximately 4 nM D Also provided is a humanized B-cell activating factor receptor (BAFF-R) antibody that binds

[0006] Also provided is a chimeric antigen receptor (CAR) comprising an antibody or functional fragment thereof provided herein.

[0007] Provided herein are isolated nucleic acids encoding a BAFF-R antibody or a functional fragment of the antibody.

[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 pharma- ceutically acceptable excipient.

[0009] A mouse fibroblast cell expressing a human BAFF-R protein, or a functional fragment thereof, is provided, wherein the human BAFF-R protein, or a functional fragment thereof, is expressed on the cell surface of the cell.

[0010] Methods of 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 includes administering mouse fibroblast cells 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 allowed to express BAFF-R antibodies, thereby producing anti-BAFF-R antibodies. [Brief description of the drawings]

[0015] [Figure 1-1]Figures 1A, 1B, 1C, 1D and 1E are FACS images showing the generation and specificity of novel monoclonal antibodies against human BAFF-R. Figure 1A is a FACS analysis of cell surface expression of hBAFF-R-GFP fusion protein in mouse fibroblasts (L cells). Engineered L cell clones (right plot) are compared to parental L cells (left plot) by gating on GFP positive cells. Clone D2C was selected for further studies. Figures 1B, 1C, 1D and 1E are FACS traces of fluorescence counts of anti-BAFF-R antibodies binding to cell lines and patient samples. Figure 1B shows affinity purified hybridoma mAbs (C90, C67, C55 and C53) binding to BAFF-R positive human MCL lines including Mino, JeKo-1, REC-1, JVM-13 and Z-138 at a concentration of 0.05 μg mAb / 106 cells. A BAFF-R negative 293T embryonic kidney cell line was used as a control. Figure 1C shows chimeric antibodies C55 and C90 binding to hBAFF-R expressing L cells at high and low concentrations. Parental L cells and secondary anti-hIgG-APC antibody alone were used as controls. Figure 1D shows a panel of alexa fluor 488 conjugated chimeric antibodies binding NHL cell lines. Figure 1E shows chimeric antibodies binding to three types of NHL primary patient samples. Data are representative of three independent experiments. For all of Figures 1B-1E, the top-to-bottom traces as shown in the figures correlate with the top-to-bottom of the variables used (e.g. antibody type or cell type) shown below or next 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). Engineered L cell clones (right plot) are compared to parental L cells (left plot) by gating on GFP positive cells. Clone D2C was selected for further studies. Figures 1B, 1C, 1D and 1E are FACS traces of fluorescence counts of anti-BAFF-R antibodies binding to cell lines and patient samples. Figure 1B shows affinity purified hybridoma mAbs (C90, C67, C55 and C53) binding to BAFF-R positive human MCL lines including Mino, JeKo-1, REC-1, JVM-13 and Z-138 at a concentration of 0.05 μg mAb / 106 cells. A BAFF-R negative 293T embryonic kidney cell line was used as a control. Figure 1C shows chimeric antibodies C55 and C90 binding to hBAFF-R expressing L cells at high and low concentrations. Parental L cells and secondary anti-hIgG-APC antibody alone were used as controls. Figure 1D shows a panel of alexa fluor 488 conjugated chimeric antibodies binding NHL cell lines. Figure 1E shows chimeric antibodies binding to three types of NHL primary patient samples. Data are representative of three independent experiments. For all of Figures 1B-1E, the top-to-bottom traces as shown in the figures correlate with the top-to-bottom of the variables used (e.g. antibody type or cell type) shown below or next to the figures. [Figure 1-3]Figures 1A, 1B, 1C, 1D and 1E are FACS images showing the generation and specificity of novel monoclonal antibodies against human BAFF-R. Figure 1A is a FACS analysis of cell surface expression of hBAFF-R-GFP fusion protein in mouse fibroblasts (L cells). Engineered L cell clones (right plot) are compared to parental L cells (left plot) by gating on GFP positive cells. Clone D2C was selected for further studies. Figures 1B, 1C, 1D and 1E are FACS traces of fluorescence counts of anti-BAFF-R antibodies binding to cell lines and patient samples. Figure 1B shows affinity purified hybridoma mAbs (C90, C67, C55 and C53) binding to BAFF-R positive human MCL lines including Mino, JeKo-1, REC-1, JVM-13 and Z-138 at a concentration of 0.05 μg mAb / 106 cells. A BAFF-R negative 293T embryonic kidney cell line was used as a control. Figure 1C shows chimeric antibodies C55 and C90 binding to hBAFF-R expressing L cells at high and low concentrations. Parental L cells and secondary anti-hIgG-APC antibody alone were used as controls. Figure 1D shows a panel of alexa fluor 488 conjugated chimeric antibodies binding NHL cell lines. Figure 1E shows chimeric antibodies binding to three types of NHL primary patient samples. Data are representative of three independent experiments. For all of Figures 1B-1E, the top-to-bottom traces as shown in the figures correlate with the top-to-bottom of the variables used (e.g. antibody type or cell type) shown below or next to the figures. [Figure 1-4]Figures 1A, 1B, 1C, 1D and 1E are FACS images showing the generation and specificity of novel monoclonal antibodies against human BAFF-R. Figure 1A is a FACS analysis of cell surface expression of hBAFF-R-GFP fusion protein in mouse fibroblasts (L cells). Engineered L cell clones (right plot) are compared to parental L cells (left plot) by gating on GFP positive cells. Clone D2C was selected for further studies. Figures 1B, 1C, 1D and 1E are FACS traces of fluorescence counts of anti-BAFF-R antibodies binding to cell lines and patient samples. Figure 1B shows affinity purified hybridoma mAbs (C90, C67, C55 and C53) binding to BAFF-R positive human MCL lines including Mino, JeKo-1, REC-1, JVM-13 and Z-138 at a concentration of 0.05 μg mAb / 106 cells. A BAFF-R negative 293T embryonic kidney cell line was used as a control. Figure 1C shows chimeric antibodies C55 and C90 binding to hBAFF-R expressing L cells at high and low concentrations. Parental L cells and secondary anti-hIgG-APC antibody alone were used as controls. Figure 1D shows a panel of alexa fluor 488 conjugated chimeric antibodies binding NHL cell lines. Figure 1E shows chimeric antibodies binding to three types of NHL primary patient samples. Data are representative of three independent experiments. For all of Figures 1B-1E, the top-to-bottom traces as shown in the figures correlate with the top-to-bottom of the variables used (e.g. antibody type or cell type) shown below or next to the figures. [Figure 1-5]Figures 1A, 1B, 1C, 1D and 1E are FACS images showing the generation and specificity of novel monoclonal antibodies against human BAFF-R. Figure 1A is a FACS analysis of cell surface expression of hBAFF-R-GFP fusion protein in mouse fibroblasts (L cells). Engineered L cell clones (right plot) are compared to parental L cells (left plot) by gating on GFP positive cells. Clone D2C was selected for further studies. Figures 1B, 1C, 1D and 1E are FACS traces of fluorescence counts of anti-BAFF-R antibodies binding to cell lines and patient samples. Figure 1B shows affinity purified hybridoma mAbs (C90, C67, C55 and C53) binding to BAFF-R positive human MCL lines including Mino, JeKo-1, REC-1, JVM-13 and Z-138 at a concentration of 0.05 μg mAb / 106 cells. A BAFF-R negative 293T embryonic kidney cell line was used as a control. Figure 1C shows chimeric antibodies C55 and C90 binding to hBAFF-R expressing L cells at high and low concentrations. Parental L cells and secondary anti-hIgG-APC antibody alone were used as controls. Figure 1D shows a panel of alexa fluor 488 conjugated chimeric antibodies binding NHL cell lines. Figure 1E shows chimeric antibodies binding to three types of NHL primary patient samples. Data are representative of three independent experiments. For all of Figures 1B-1E, the top-to-bottom traces as shown in the figures correlate with the top-to-bottom of the variables used (e.g. antibody type or cell type) shown below or next to the figures. [Figure 2-1]Figures 2A, 2B and 2C are graphs showing that BAFF-R monoclonal antibodies displayed 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). NK effector cell:target ratio (E:T) of 20:1. The percentage of cell-specific lysis of target cells is as follows: the first panel shows BAFF-R expressing L cells or control parental L cells; the second and third panels show BAFF-R positive JeKo-1 MCL or BAFF-R negative U266 multiple myeloma cells, shown as dose-response curves with varying antibody concentrations. Figure 2B shows 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 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 displayed 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). NK effector cell:target ratio (E:T) of 20:1. The percentage of cell-specific lysis of target cells is as follows: the first panel shows BAFF-R expressing L cells or control parental L cells; the second and third panels show BAFF-R positive JeKo-1 MCL or BAFF-R negative U266 multiple myeloma cells, shown as dose-response curves with varying antibody concentrations. Figure 2B shows 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 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 displayed 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). NK effector cell:target ratio (E:T) of 20:1. The percentage of cell-specific lysis of target cells is as follows: the first panel shows BAFF-R expressing L cells or control parental L cells; the second and third panels show BAFF-R positive JeKo-1 MCL or BAFF-R negative U266 multiple myeloma cells, shown as dose-response curves with varying antibody concentrations. Figure 2B shows 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 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. Antibody-dependent cell-mediated cytotoxicity (ADCC) effects were measured by chromium-51 release after incubation with C55, C90 or rituximab and effector (NK cells). The percentage of cell-specific lysis of target cells is as follows: Figure 3A shows NHL patient samples (E:T=20:1 or 10:1); Figure 3B shows primary MCL and CLL samples (E:T=20:1) from rituximab-refractory patients. Data are shown as mean ± standard deviation of 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. Antibody-dependent cell-mediated cytotoxicity (ADCC) effects were measured by chromium-51 release after incubation with C55, C90 or rituximab and effector (NK cells). The percentage of cell-specific lysis of target cells is as follows: Figure 3A shows NHL patient samples (E:T=20:1 or 10:1); Figure 3B shows primary MCL and CLL samples (E:T=20:1) from rituximab-refractory patients. Data are shown as mean ± standard deviation of 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 a minimal lethal dose of tumor. Treatment was given via IV tail vein injection. Figures 4B and 4C are images showing chimeric antibody-induced in vivo therapeutic efficacy against B cell tumors targeting human BAFF-R. Bioluminescence images of mice challenged with luciferase-expressing tumors: JeKo-1 (MCL) (Figure 4B) or RS4;11 (ALL) (Figure 4C). Experimental groups were administered treatment with chimeric BAFF-R mAb (C55 or C90 as indicated). Control mice were administered 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 a minimal lethal dose of tumor. Treatment was given via IV tail vein injection. Figures 4B and 4C are images showing chimeric antibody-induced in vivo therapeutic efficacy against B cell tumors targeting human BAFF-R. Bioluminescence images of mice challenged with luciferase-expressing tumors: JeKo-1 (MCL) (Figure 4B) or RS4;11 (ALL) (Figure 4C). Experimental groups were administered treatment with chimeric BAFF-R mAb (C55 or C90 as indicated). Control mice were administered 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 a minimal lethal dose of tumor. Treatment was given via IV tail vein injection. Figures 4B and 4C are images showing chimeric antibody-induced in vivo therapeutic efficacy against B cell tumors targeting human BAFF-R. Bioluminescence images of mice challenged with luciferase-expressing tumors: JeKo-1 (MCL) (Figure 4B) or RS4;11 (ALL) (Figure 4C). Experimental groups were administered treatment with chimeric BAFF-R mAb (C55 or C90 as indicated). Control mice were administered 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 chimeric BAFF-R antibodies induce 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 selected CD20- / - clone number 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 presented 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 chimeric BAFF-R antibodies induce 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 selected CD20- / - clone number 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 presented 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 chimeric BAFF-R antibodies induce 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 selected CD20- / - clone number 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 presented 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] Images (FIGS. 6A and 6B) and graphs (FIG. 6C) showing 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 (FIG. 6A) or ibrutinib-resistant Z-138 cells (FIG. 6B) followed by antibody treatment as in FIG. 4. Control mice were administered PBS, NK cells alone, or rituximab on the same schedule. FIG. 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 log-rank test (**P<0.001). Data are representative of three independent experiments. [Figure 6-2]Images (FIGS. 6A and 6B) and graphs (FIG. 6C) showing 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 (FIG. 6A) or ibrutinib-resistant Z-138 cells (FIG. 6B) followed by antibody treatment as in FIG. 4. Control mice were administered PBS, NK cells alone, or rituximab on the same schedule. FIG. 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 log-rank test (**P<0.001). Data are representative of three independent experiments. [Figure 6-3] Images (FIGS. 6A and 6B) and graphs (FIG. 6C) showing 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 (FIG. 6A) or ibrutinib-resistant Z-138 cells (FIG. 6B) followed by antibody treatment as in FIG. 4. Control mice were administered PBS, NK cells alone, or rituximab on the same schedule. FIG. 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 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 the 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 did not (representative of other negative clones). [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 the 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 did not (representative of other negative clones). [Figure 8] 1 shows the results of flow cytometry 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 Mino (mantle cell lymphoma) and 293T (negative control) cell lines was assessed by flow cytometry performed with anti-mouse IgG-APC. [Figure 9]Graph showing 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 / 106 cells to 1.6 ng / 106 cells) purified mouse mAbs to Mino cells was assessed by flow cytometry with an anti-mouse IgG-APC secondary antibody. [Figure 10] Results of FACS screening analysis showing 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 / 106 cells) and low doses (0.05 μg mAb / 106 cells). Flow cytometry analysis was performed with anti-mouse IgG-APC. The top-to-bottom traces as 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] Graph showing that hBAFF-R mAb recognized lymphoma patient samples. Mantle cell lymphoma, diffuse large B cell lymphoma, and follicular lymphoma patient samples were stained with mouse mAbs C55 and C90 at high (2 μg / 106 cells) and low (0.05 μg / 106 cells) doses. Flow cytometry analysis was performed with anti-mouse IgG-APC. The top-to-bottom traces as shown in the figure correlate with the left-to-right of the 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]Graph showing chimeric antibody required by NK cells for cytotoxicity against tumor cells. JeKo-1 cells (targets) were labeled with chromium-51. 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] FACS results showing that hBAFF-R mAbs blocked BAFF / BAFF-R interaction. BAFF-R expressing D2C L cell clones were incubated with C90 (0-1000ng / 106 cells) for 45 minutes at 4°C, followed by incubation with recombinant BAFF ligand (0.5μg / 106 cells) for 90 minutes at 4°C. Flow cytometry was performed and gated on 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 on top of each of the FACS results. [Figure 15] FACS results showing that limited internalization was observed with BAFF-R mAb. Mino cells were incubated with mAb C90 (0.05 μg / 106 cells) for 20 min at 4° C., followed by 1 h incubation 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]FACS results (FIG. 16A) and gel images (16B) showing that CD20 knockout clones were generated by CRISPR. CD20 knockout clones of JeKo-1 were generated by a commercial CRISPR / HDR system replacing RFP at the CD20 locus. In FIG. 16A, clones were screened and selected by flow cytometry for CD20- / RFP+ expression. In FIG. 16B, Western blot with anti-CD20 antibody was performed on total cell lysates from CD20- / RFP+ clones. β-actin was blotted as a loading control. FIG. 16C shows FACS results for the same clones in FIG. 16A that were screened for BAFF-R / RFP+ expression to confirm that the expression of BAFF-R was not affected by CRISPR / HDR manipulation of CD20. [Figure 16-2] FACS results (FIG. 16A) and gel images (16B) showing that CD20 knockout clones were generated by CRISPR. CD20 knockout clones of JeKo-1 were generated by a commercial CRISPR / HDR system replacing RFP at the CD20 locus. In FIG. 16A, clones were screened and selected by flow cytometry for CD20- / RFP+ expression. In FIG. 16B, Western blot with anti-CD20 antibody was performed on total cell lysates from CD20- / RFP+ clones. β-actin was blotted as a loading control. FIG. 16C shows FACS results for the same clones in FIG. 16A that were screened for BAFF-R / RFP+ expression to confirm that the expression of BAFF-R was not affected by CRISPR / HDR manipulation of CD20. [Figure 16-3]FACS results (FIG. 16A) and gel images (16B) showing that CD20 knockout clones were generated by CRISPR. CD20 knockout clones of JeKo-1 were generated by a commercial CRISPR / HDR system replacing RFP at the CD20 locus. In FIG. 16A, clones were screened and selected by flow cytometry for CD20- / RFP+ expression. In FIG. 16B, Western blot with anti-CD20 antibody was performed on total cell lysates from CD20- / RFP+ clones. β-actin was blotted as a loading control. FIG. 16C shows FACS results for the same clones in FIG. 16A that were screened for BAFF-R / RFP+ expression to confirm that the expression of BAFF-R was not affected by CRISPR / HDR manipulation of CD20. [Figure 17-1] FACS results showing 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 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 by commercial specific cell type isolation kits and stained with C55 and C90 (0.05 μg mAb / 106 cells). Flow cytometry analysis was performed with anti-mouse IgG. Myeloid cells from PBMC were gated for CD66b+ and analyzed for staining with mAbs C55 and C90. The top-to-bottom traces as 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 the antibody was used to stain the 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 and breast tissue to identify the tissue specificity of the anti-BAFF-R antibody. A 1:150 dilution of 1 mg / mL of the antibody was used to stain the 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 the antibody was used to stain the 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 and breast tissue to identify the tissue specificity of the anti-BAFF-R antibody. A 1:150 dilution of 1 mg / mL of the antibody was used to stain the 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. Recombinant extracellular domain of human BAFF-R was used as 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, humanized variants were tested on JeKo-1 cells in a chromium release assay. Cells were allowed to take up chromium and subsequently treated with humanized C90 variants and effector NK cells. Cells were incubated for 6 hours and the supernatant was sampled for chromium 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. Recombinant extracellular domain of human BAFF-R was used as 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, humanized variants were tested on JeKo-1 cells in a chromium release assay. Cells were allowed to take up chromium and subsequently treated with humanized C90 variants and effector NK cells. Cells were incubated for 6 hours and the supernatant was sampled for chromium content. [Figure 21-1] Figures 21A and 21B are graphs showing the analysis of humanized antibodies C90-4 and C90-5 for specific cytotoxicity of various lymphoma lines. In Figure 21A, JeKo-1, Z138 and RS4 were subjected to 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 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 of various lymphoma lines. In Figure 21A, JeKo-1, Z138 and RS4 were subjected to 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 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 top candidates 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 evaluated by chromium release assay. Cells were incubated with chromium-51, followed by treatment with antibodies 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 top candidates 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 evaluated by chromium release assay. Cells were incubated with chromium-51, followed by treatment with antibodies 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] 13 is an image 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 (sFv) on top of a T cell receptor signaling domain with a 4-1BB motif. NSG mice were challenged with a minimal lethal dose (1×106 cells) of NHL JeKo-1-Luci cells. Tumor cells were allowed to engraft until tumors were detectable by bioluminescence imaging (day 9). Mice were administered either T cell therapy (5×106 CAR-T cells) or control 9 and 15 days after tumor challenge. Mice were closely monitored and imaged every 3 days to follow tumor progression. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] In particular, BAFF-R antibodies are provided herein that include 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 cells expressing BAFF-R (e.g., B cells). Optionally, the light chain variable region and the 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% compared to 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 portion of 50, 100, 150 or 200 contiguous amino acids) compared to naturally occurring BAFF-R. Optionally, BAFF-R is substantially identical to a protein identified by UniProt reference number Q96RJ3 or a variant or homologue having substantial identity thereto. Optionally, BAFF-R is substantially identical to a protein identified by UniProt reference number Q9D8D0 or a variant or homologue having substantial identity thereto. Optionally, BAFF-R is substantially identical to a protein identified by NCBI reference number GI:16445027 or a variant or homologue having substantial identity thereto. Optionally, BAFF-R is substantially identical to a protein identified by NCBI reference number GI:16306481 or a variant or homologue 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 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. And 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. Optionally, 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. And 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.

[0019] The humanized antibody as provided herein can bind to BAFF-R protein and includes at least one mouse CDR or functional fragment or variant thereof of the BAFF-R antibody provided herein (e.g., CDR L1 of SEQ ID NO: 1 or 7, CDR L2 of SEQ ID NO: 2 or 8, CDR L3 of SEQ ID NO: 3 or 9, CDR H1 of SEQ ID NO: 4 or 10, CDR H2 of SEQ ID NO: 5 or 11, CDR H3 of SEQ ID NO: 7 or 13). A functional fragment of a CDR is a portion of the complete CDR amino acid sequence, but an antibody or a 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 a 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 may include one or more changes, but still encode the same amino acid sequence of a CDR. Furthermore, functional variants of the polypeptide sequence of the CDR can include 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 the CDR typically include the amino acid residues required for antibody binding to the antigen (e.g., BAFF-R). When a humanized antibody comprises 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 that comprises at least one mouse CDR is a humanized antibody with at least one mouse CDR derived from a donor antibody, and additional CDRs are derived from an acceptor antibody (e.g., when the light chain comprises a total of three CDRs and the heavy chain comprises 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 one 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, where at least one of the six CDRs is a mouse CDR. When the humanized light chain variable region and the humanized heavy chain variable region comprise one combined mouse CDR, the humanized light chain variable region or the humanized heavy chain variable region comprises one mouse CDR. For example, the humanized antibody may comprise CDR L3 (e.g., mouse, also referred to herein as mouse CDR L3) from the donor antibody, and CDR L1, CDR L2, CDR H1, CDR H2 and CDR H3 (i.e., human) 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, the humanized antibody may comprise CDR L3 and CDR H3 (e.g., mouse, also referred to herein as mouse CDR L3 and mouse CDR H3, respectively) from the donor antibody, and CDR L1, CDR L2, CDR H1, and CDR H2 (i.e., human) from the 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, the humanized antibody may comprise CDR L3, CDR H3, and CDR 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 CDR L1, CDR H1, and CDR 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, the humanized antibody may comprise CDR L3, CDR H3, CDR L2 and CDR L1 (e.g., mouse, also referred to herein as mouse CDR L3, mouse CDR H3, mouse CDR L2 and mouse CDR L1, respectively) derived from the donor antibody, and CDR H1 and CDR H2 (i.e., human) derived from the acceptor antibody.

[0024] The humanized light chain variable region and the humanized heavy chain variable region may each comprise at least one mouse CDR. When the humanized light chain variable region and the humanized heavy chain variable region each comprise at least one mouse CDR, the humanized light chain variable region comprises at least one mouse CDR and the humanized heavy chain variable region comprises at least one mouse CDR. Thus, the humanized light chain variable region may comprise mouse CDR L1 and the humanized heavy chain may comprise mouse CDR H1. Optionally, the mouse CDR L1 comprises the amino acid sequence of SEQ ID NO: 1 and the mouse CDR H1 comprises the amino acid sequence of SEQ ID NO: 4. Optionally, the mouse CDR L1 is the amino acid sequence of SEQ ID NO: 1 and the mouse CDR H1 is the amino acid sequence of SEQ ID NO: 4. Optionally, the humanized light chain variable region comprises mouse CDR L2 and the humanized heavy chain variable region comprises mouse CDR H2. Optionally, the mouse CDR L2 comprises the amino acid sequence of SEQ ID NO: 2 and the mouse CDR H2 comprises the amino acid sequence of SEQ ID NO: 5. Optionally, the murine CDR L2 is the amino acid sequence of SEQ ID NO: 2 and the murine CDR H2 is 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 comprises the amino acid sequence of SEQ ID NO: 3 and the murine CDR H3 comprises the amino acid sequence of SEQ ID NO: 6. Optionally, the CDR L3 is the amino acid sequence of SEQ ID NO: 3 and the murine CDR H3 is 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 mouse CDR L3 and mouse CDR H3 may be sufficient for the binding of the humanized antibody to BAFF-R. Thus, the humanized antibody may not contain mouse CDR L1, mouse CDR L2, CDR H1 or mouse CDR H2. If the humanized antibody does not contain mouse CDR L1, mouse CDR L2, mouse CDR H1 or mouse CDR H2, the humanized antibody will contain CDR L1, CDR L2, CDR H1 or CDR H2 from the acceptor antibody (i.e. human). Thus, a humanized antibody that does not contain mouse CDR L1, mouse CDR L2, mouse CDR H1 or mouse CDR H2 will not contain CDR L1, CDR L2, CDR H1 or CDR H2 from the donor antibody (e.g. mouse, rat, rabbit), but will contain CDR L1, CDR L2, CDR H1 or CDR 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 a murine CDR L2 and a murine CDR L3, and the humanized heavy chain variable region comprises a murine CDR H2 and a murine CDR H3. Optionally, the humanized light chain variable region comprises a murine CDR L1, a murine CDR L2 and a murine CDR L3, and the humanized heavy chain variable region comprises a murine CDR H1, a murine CDR H2 and a murine CDR H3. Optionally, the humanized light chain variable region comprises a murine CDR L1 as specified in SEQ ID NO: 1, a murine CDR L2 as specified in SEQ ID NO: 2, and a murine CDR L3 as specified in SEQ ID NO: 3, and the humanized heavy chain variable region comprises a murine CDR H1 as specified in SEQ ID NO: 4, a murine CDR H2 as specified in SEQ ID NO: 5, and a murine CDR H3 as specified in SEQ ID NO: 6. Optionally, the humanized light chain variable region comprises a murine CDR L1 as specified in SEQ ID NO:7, a murine CDR L2 as specified in SEQ ID NO:8, and a murine CDR L3 as specified in SEQ ID NO:9, and the humanized heavy chain variable region comprises a murine CDR H1 as specified in SEQ ID NO:10, a murine CDR H2 as specified in SEQ ID NO:11, and a murine CDR H3 as specified in SEQ ID NO:12.

[0028] The positions of CDRs and FRs may be defined by the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., USDepartment of Health and Human Services, USGovernment Printing Office (1991)). Similarly, the positions occupied by individual residues in the light or heavy chains of an antibody may be defined by the Kabat numbering system. Thus, the locations of residues required for binding in the humanized light and heavy chains of a humanized antibody may be defined by the positions of the residues according to the Kabat numbering system, as is well known in the art. As described above, a humanized antibody may be an antibody having CDRs from a donor antibody (e.g., mouse) and variable region frameworks (FRs) from a human antibody. The framework regions (FRs) are assumed 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 that include 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 (or important) in epitope binding (e.g., BAFF-R binding) are referred to herein as binding framework region residues. The binding 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). The binding framework residues present in the FR L3 region of the humanized light chain are referred to herein as FR L3 binding framework region residues. Accordingly, the binding framework region residues present in the FR H3 region of the humanized heavy chain are referred to herein as FR H3 binding framework region residues.

[0029] Optionally, the humanized antibody 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) and The humanized heavy chain variable region may comprise 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 may 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 binding 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 an 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 includes 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 valine 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, 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 at a position corresponding to Kabat position 19, a valine at a position corresponding to Kabat position 23, a valine at a position corresponding to Kabat position 24, a valine at a position corresponding to Kabat position 25, a valine at a position corresponding to Kabat position 26, a valine at a position corresponding to Kabat position 27, a valine at a position corresponding to Kabat position 29, a valine at a position corresponding to Kabat position 30, a valine at a position corresponding to Kabat position 31, a valine at a position corresponding to Kabat position 32, a valine at a position corresponding to Kabat position 33, a valine at a position corresponding to Kabat position 34 and / 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 Kabat position 109.

[0038] Optionally, the heavy chain variable region comprises 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 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 92, an arginine at a position corresponding to Kabat position and binding framework region residues which are a threonine at the corresponding position, a lysine at the position corresponding to Kabat position 75, a valine at the position corresponding to Kabat position 79, a threonine or lysine at the position corresponding to Kabat position 81, a methionine at the position corresponding to Kabat position 82, an asparagine at the position corresponding to Kabat position 82B, a methionine at the position corresponding to Kabat position 82C, a proline at the position corresponding to Kabat position 84, a valine at the position corresponding to Kabat position 85, a lysine at the position corresponding to Kabat position 108, or a valine at the position corresponding to Kabat position 109.

[0039] A humanized BAFF-R antibody is provided, comprising a humanized light chain variable region comprising a murine CDR L1, a murine CDR L2 or a murine CDR L3, and a humanized heavy chain variable region comprising a murine CDR H1 or a murine CDR H2 or a murine CDR H3. The humanized light chain variable region may comprise a murine CDR L1 as specified in SEQ ID NO: 1, a murine CDR L2 as specified in SEQ ID NO: 2, or a murine CDR L3 as specified in SEQ ID NO: 3. The humanized light chain variable region may comprise a murine CDR L1 as specified in SEQ ID NO: 1, a murine CDR L2 as specified in SEQ ID NO: 2, and a murine CDR L3 as specified in SEQ ID NO: 3. The humanized heavy chain variable region may comprise a murine CDR H1 as specified in SEQ ID NO: 4, a murine CDR H2 as specified in SEQ ID NO: 5, or a murine CDR H3 as specified in SEQ ID NO: 6. The humanized heavy chain variable region may comprise a murine CDR H1 as specified in SEQ ID NO:4, a murine CDR H2 as specified in SEQ ID NO:5, and a murine CDR H3 as specified in SEQ ID NO:6. Optionally, the humanized light chain variable region comprises a murine CDR L1 as specified in SEQ ID NO:1. Optionally, the humanized light chain variable region comprises a murine CDR L2 as specified in SEQ ID NO:2. Optionally, the humanized light chain variable region comprises a murine CDR L3 as specified in SEQ ID NO:3. Optionally, the humanized heavy chain variable region comprises a murine CDR H1 as specified in SEQ ID NO:4. Optionally, the humanized heavy chain variable region comprises a murine CDR H2 as specified in SEQ ID NO:5. Optionally, the humanized light chain variable region comprises a 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] A humanized BAFF-R antibody is provided, comprising a humanized light chain variable region comprising a murine CDR L1, a murine CDR L2 or a murine CDR L3, and a humanized heavy chain variable region comprising a murine CDR H1 or a murine CDR H2 or a murine CDR H3. The humanized light chain variable region may comprise a murine CDR L1 as specified in SEQ ID NO:7, a murine CDR L2 as specified in SEQ ID NO:8, or a murine CDR L3 as specified in SEQ ID NO:9. The humanized light chain variable region may comprise a murine CDR L1 as specified in SEQ ID NO:7, a murine CDR L2 as specified in SEQ ID NO:8, and a murine CDR L3 as specified in SEQ ID NO:9. The humanized heavy chain variable region may comprise a murine CDR H1 as specified in SEQ ID NO:10, a murine CDR H2 as specified in SEQ ID NO:11, or a murine CDR H3 as specified in SEQ ID NO:12. The humanized heavy chain variable region may comprise a murine CDR H1 as specified in SEQ ID NO: 10, a murine CDR H2 as specified in SEQ ID NO: 11, and a murine CDR H3 as specified in SEQ ID NO: 12. Optionally, the humanized light chain variable region comprises a murine CDR L1 as specified in SEQ ID NO: 7. Optionally, the humanized light chain variable region comprises a murine CDR L2 as specified in SEQ ID NO: 8. Optionally, the humanized light chain variable region comprises a murine CDR L3 as specified in SEQ ID NO: 9. Optionally, the humanized heavy chain variable region comprises a murine CDR H1 as specified in SEQ ID NO: 10. Optionally, the humanized heavy chain variable region comprises a murine CDR H2 as specified in SEQ ID NO: 11. Optionally, the humanized light chain variable region comprises a 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 comprises the sequence of SEQ ID NO: 22. 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 of the sequence of SEQ ID NO: 26. Optionally, the heavy chain variable region is of 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 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: 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 case where an antibody is recited herein, a functional fragment may be used. Thus, for example, a Fab' fragment is 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. A single chain antibody comprises a variable light chain and a variable heavy chain. Those skilled in the art will immediately 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), a single chain antibody comprises a single light chain and a single heavy chain. And each light chain and heavy chain consists of two regions: a variable ("V") region (i.e., a variable light chain and a variable heavy chain) that is involved in binding the target antigen, and a constant ("C") region that interacts with other components of the immune system. The variable light chain and the 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 making scFv antibodies are 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 the heavy and light immunoglobulin chains are used to amplify the cDNA. The PCR product is purified and the nucleic acid sequences are linked. 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 Optionally, the BAFF-R antibody can bind to the BAFF-R protein with an equilibrium dissociation constant (K D Optionally, the BAFF-R antibody can bind to the BAFF-R protein with an equilibrium dissociation constant (K D Optionally, the BAFF-R antibody can bind to the BAFF-R protein with an equilibrium dissociation constant (K D Optionally, the BAFF-R antibody can bind to the BAFF-R protein with an equilibrium dissociation constant (K D Optionally, the BAFF-R antibody can bind to the BAFF-R protein with an equilibrium dissociation constant (K D Optionally, the BAFF-R antibody can bind to the BAFF-R protein with an equilibrium dissociation constant (K D Optionally, the BAFF-R antibody can bind to the BAFF-R protein with an equilibrium dissociation constant (K D Optionally, the BAFF-R antibody can bind to the BAFF-R protein with an equilibrium dissociation constant (K D Optionally, the BAFF-R antibody can bind to the BAFF-R protein with an equilibrium dissociation constant (K D ) which 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 D Optionally, the BAFF-R antibody or functional fragment thereof can bind to the BAFF-R protein with 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 DOptionally, the BAFF-R antibody or functional fragment thereof can bind to the BAFF-R protein with 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 D Optionally, the BAFF-R antibody or functional fragment thereof can bind to the BAFF-R protein with 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 D Optionally, the BAFF-R antibody or functional fragment thereof can bind to the BAFF-R protein with 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 D Optionally, the BAFF-R antibody or functional fragment thereof can bind to the BAFF-R protein with an equilibrium dissociation constant (K D ) which can bind to the BAFF-R protein.

[0049] Optionally, BAFF-R and a K lower than about 4 nM D

[0023] 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 6-fold. 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 said 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 linker or a non-covalent linker. Techniques for conjugating therapeutic moieties to antibodies are well known (see, 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, 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 anti-cancer 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 is a chimeric antigen receptor (CAR) comprising an antibody or functional fragment thereof provided herein.

[0054] Provided herein is an isolated nucleic acid encoding the BAFF-R antibody or functional fragment thereof (including embodiments thereof) provided herein. The BAFF-R antibody or functional fragment thereof encoded by the isolated nucleic acid is described in detail throughout this application (including the above description and example section). For example, the nucleic acid may encode at least one CDR, specific residues involved in binding the epitope, 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] Provided is a pharmaceutical composition comprising a therapeutically effective amount of a BAFF-R antibody or functional fragment thereof provided herein and a pharma- ceutically 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 a disease symptom (e.g., cancer, autoimmune disease)). Determination of a therapeutically effective amount of a BAFF-R antibody provided herein is well within the capabilities of one of ordinary skill in the art, especially in light of the detailed disclosure herein.

[0059] Acceptable carriers, excipients or stabilizing substances are non-toxic to recipients at the dosages and concentrations employed and typically include buffers (such as phosphates, citrates or acetates) 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 of skill in the art (Remington: The Science and Practice of Pharmacy, 22nd Edition, edited by Loyd V. Allen et al., Pharmaceutical Press (2012)). The mAb may be present in 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 an antibody (e.g., a humanized antibody) or a functional fragment 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. The pharmaceutically acceptable excipient can be suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, epidermal administration (e.g., by injection or infusion).

[0061] Pharmaceutical compositions of antibodies or functional fragments thereof can be prepared according to well-known routinely practiced methods in the art. See, for example, Remington: The Science and Practice of Pharmacy, 22nd Edition, edited by Loyd V. Allen et al., Pharmaceutical Press (2012); and Sustained and Controlled Release Drug Delivery Systems, edited by JR Robinson, Marcel Dekker, Inc., New York, 1978. Pharmaceutical compositions are preferably manufactured under GMP conditions. Typically, a therapeutically effective dose or effective dose of humanized antibodies is used in the pharmaceutical composition. Provided humanized antibodies can be formulated into pharma- ceutically acceptable dosage forms by conventional methods known to those skilled in the art. Dosage regimens are adjusted to provide the optimum desired response (e.g., 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 humanized antibodies 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 amount of humanized antibody intended to produce the desired therapeutic effect in association with the required pharmaceutical excipients.

[0062] The actual dosage level of the active ingredient in the 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 a variety of 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 begin the dosage of the antibody or functional fragment thereof used in the 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. In general, the effective dosage of the composition may vary depending on different 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 pharmaceutical agents being administered, and whether the treatment is prophylactic or therapeutic. Therapeutic dosages should be titrated to optimize safety and efficacy. For administration with antibodies, dosages range from about 0.0001 to 100 mg / kg (usually 0.01 to 5 mg / kg) of host body weight. For example, dosages may be 1 mg / kg body weight or 10 mg / kg body weight, or may be within the range of 1 to 10 mg / kg. Exemplary treatment regimes involve administration once every 2 or 3 weeks, or 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 dosages may be weekly, monthly or yearly. The interval may also be irregular, as indicated by measuring the blood levels 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 will 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 over a long period of time at relatively infrequent intervals. 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 terminated, and preferably until the patient shows partial or complete improvement of the symptoms of the disease. Thereafter, the patient may 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 of 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 to the subject a therapeutically effective amount of an antibody or functional fragment thereof provided herein, 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 a 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 of 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 to the subject a second therapeutic agent.

[0072] In another aspect, a method of 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 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 fibroblast cells 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 allowed to express BAFF-R antibodies, thereby producing anti-BAFF-R antibodies. 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 1320 amino acids) with a complex internal structure. Natural antibody molecules contain two identical pairs of polypeptide chains, each pair having one light chain and one heavy chain. Each light and heavy chain then 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 come together in three-dimensional space to form a 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 (average 10 amino acids in length) called complementarity determining regions ("CDRs"). The six CDRs (three from the light chain and three from the heavy chain) in the antibody variable domain fold together in three-dimensional space to form the actual antibody binding site (paratope), which docks onto the target antigen (epitope). The position and length of the CDRs were precisely defined by Kabat, E. et al., Sequences of Proteins of Immunological Interest, USDepartment 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 and known meaning 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 into V H -C H1 The F(ab)'2 can be reduced under mild conditions that break the disulfide bond in the hinge region, thereby converting the F(ab)'2 dimer into a Fab' monomer. The Fab' monomer is essentially a Fab with a portion of the hinge region (see Fundamental Immunology (Paul, ed., 3rd ed. (1993)). Although various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill 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 antibody fragments produced by modification of whole antibodies, or those synthesized de novo using recombinant DNA methodology (e.g., single chain Fv) or those identified using a phage display library (see, e.g., McCafferty et al., Nature 348:552-554 (1990)) can be used to describe antibodies.

[0077] For preparation of monoclonal or polyclonal antibodies, any technique known in the art may be used (see, for example, Kohler & Milstein, Nature 256:495-497 (1975); Kozbor et al., Immunology Today 4:72 (1983); Cole et al. (Monoclonal Antibodies and Cancer Therapy (1985) at pages 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 the binding of the other by at least 30%, but preferably 50%, 75%, 90% or even up to 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 the binding of one antibody reduce or eliminate the binding of the other. Two antibodies have overlapping epitopes if some amino acid mutations that reduce or eliminate the binding of one antibody reduce or eliminate the 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 such labels 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, for example, by incorporating a radioisotope label into a peptide or antibody that is 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 biological molecules or cells) to come close enough to react, interact, or be in 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 or treating a condition, disease or disorder, or a symptom associated with a condition, disease or disorder refers to an approach to obtain a beneficial or desired result (including clinical results). Beneficial or desired clinical results may include, but are not limited to, alleviation or amelioration of one or more symptoms or condition, reduction of the severity of a condition, disorder or disease, stabilization of the condition, disorder or disease state, prevention of the onset of a condition, disorder or disease, prevention of the spread of a condition, disorder or disease, delay or slowing of the progression of a condition, disorder or disease, delay or slowing of the onset of a condition, disorder or disease, amelioration or alleviation of a condition, disorder or disease, and remission (whether partial or total) of a condition, disorder or disease state. Treatment may also refer to the prolonged survival of a subject beyond that expected in the absence of treatment. Treatment may in some cases include permanently halting the progression of a condition, disorder or disease, but may also refer to inhibition of the progression of a condition, disorder or disease, or temporary slowing of the progression of a condition, disorder or disease. As used herein, the terms treatment, treat, or treating refer to a method of reducing the effect of one or more symptoms of a disease or condition characterized by the expression of a protease, or a symptom of a disease or condition characterized by the expression of a protease. 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 determined to be 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 a disease or condition.Additionally, as used herein, references to decrease, reduction or inhibition include a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater change 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 a polymer of amino acid residues, where the polymer may be conjugated to a moiety that does not consist of amino acids. The term applies 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 the corresponding naturally occurring amino acid. 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] The term amino acid, as used herein, 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 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) (e.g., homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium). 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 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 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 code for the same or essentially the same amino acid sequence. Because of the degeneracy of the genetic code, many nucleic acid sequences will code for any given protein. For example, the codons GCA, GCC, GCG, and GCU all code for the amino acid alanine. Thus, at every position where alanine is specified by a codon, the codon can be changed to any of the corresponding codons described without changing the encoded polypeptide. Such nucleic acid variations are silent variations, which are one type of conservatively modified variation. Every nucleic acid sequence herein that codes for a polypeptide also describes every possible silent variation of the nucleic acid. Those skilled in the art will recognize that each codon in a nucleic acid (except AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan) can be modified to result in a functionally identical molecule. Thus, each silent variation of a nucleic acid that codes for a polypeptide is implicit in each described sequence.

[0088] With respect to amino acid sequences, one of skill 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 replacement 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, cross-species 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 comparison of 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 term identical or percent identity refers 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 defined percentage of identical amino acid residues or nucleotides (i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity, for example, over a defined region of an entire polypeptide sequence or individual 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 100-500 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 sequence comparison algorithm, test and reference sequences are input into 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 compared to the reference sequence based on the program parameters.

[0093] A comparison window, as used herein, includes reference to, for example, a full length sequence, or any one 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, Wis.)), or by manual alignment and visual inspection (see, for example, Ausubel et al., Current Protocols in Molecular Biology (1995 supplement)).

[0094] Examples of suitable algorithms for determining percent sequence identity and sequence similarity are the BLAST algorithm and the BLAST 2.0 algorithm, which are described in Altschul et al. (1977) Nuc. Acids Res. 25:3389-3402 and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively. Software for performing BLAST real-time analysis is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ). This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of the same length in the query sequence when aligned with a word of length W in the database sequence, either matching or meeting some positive threshold score T. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds to initiate searches to find longer HSPs containing the seeds. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. The cumulative score is 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. The extension of the word hits in each direction is stopped when the cumulative alignment score falls from its maximum achieved value by an amount X; when the cumulative score falls below 0 due to the accumulation of one or more negative-scoring residue alignments; or when 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 wordlength (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 wordlength of 3, and an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915), alignments (B) of 50, 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 minimum 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 minimum sum probability is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001, the nucleic acid is 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 the 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 in 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.Another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequences.

[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 comparative 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 applicable in the art. For example, the comparative antibody can be aligned with the antibody provided herein for maximum sequence homology, and the position in the aligned comparative antibody that aligns with Kabat position 48 can be determined to correspond thereto. Alternatively, instead of (or in addition to) a primary sequence alignment as described above, a three-dimensional structural alignment can also be used, for example, when the structure of the comparative antibody is aligned with the antibody provided herein for maximum correspondence, and the overall structures are compared. In this case, an amino acid that occupies the same essential position as Kabat position 48 in the structural model can be said to correspond.

[0098] The term isolated, as applied to a protein, refers to a protein that is essentially free of other cellular components with which it is naturally associated. It can be 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 the preparation is substantially purified. The term isolated refers to a protein that gives rise to essentially one band in an electrophoretic gel. In particular, 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 presence of the protein 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] A cell, as used herein, 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 a particular dye, 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, refer to a protein inhibitor (e.g., a BAFF-R antibody provided herein) interaction, 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 a reduction in a disease or symptoms of a disease (e.g., cancer or an autoimmune disease). Thus, inhibition includes at least partially, partially or completely blocking stimulation, reducing, preventing, delaying activation, inactivating, desensitizing, or downregulating signaling or enzyme activity or the amount of a protein. Similarly, an inhibitor is a compound or protein that inhibits BAFF-R activity, for example, by binding, partially or completely blocking, reducing, preventing, delaying, inactivating, desensitizing, or downregulating activity (e.g., BAFF-R signaling activity).

[0102] The agents provided herein (e.g., antibodies) are often administered as pharmaceutical compositions that contain an active therapeutic agent and a variety of other pharma- ceutical 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 pharma- ceutically 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 combination. 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 may 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 may be administered depending on the dosage and frequency as required and can be tolerated by the patient. Patients or subjects include both humans and other animals, particularly 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, filler substances, binding substances, diluents, buffers, wetting agents, preservatives, flavoring agents, dyes, disintegrants, and pharma- ceutically 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 immunostimulants (i.e., adjuvants).

[0106] Suitable preparations for rectal administration include, for example, suppositories (comprising nucleic acid packaged with 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 base (including, for example, liquid triglycerides, polyethylene glycols and paraffin hydrocarbons) and the selected compound.

[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, bacteriostatic substances, 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). The 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 provided 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 active ingredients. 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 coadministration using separate formulations or a single pharmaceutical formulation, as well as sequential administration in any order, where preferably there is a period during which both (or all) active agents exert their biological activities simultaneously.

[0111] The effective dose of the compositions provided herein may vary depending on different factors, including the means of administration, the target site, the physiological condition of the patient, whether the patient is human or animal, other pharmaceutical agents administered, and whether the treatment is prophylactic or therapeutic. However, one of skill in the art will readily recognize appropriate and / or equivalent doses, taking into account the dosages of the 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 a health condition 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 cancer 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, carcinomas and sarcomas. Exemplary cancers that may 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), 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, endocrine system cancer, brain cancer, 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 insulinoma, linoma, malignant carcinoid, bladder cancer, premalignant skin lesions, testicular cancer, lymphoma, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary cancer, malignant hypercalcemia, endometrial cancer, adrenal cortical 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 characteristics of the disease (acute or chronic); (2) the type of cells involved; myeloid (myelogenous), lymphoid (lymphogenous), or monocytic; and (3) the increased or non-increased number of abnormal cells in the blood (leukemic or non-leukemic (subleukemic)). Exemplary leukemias that may 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), 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, lymphoblast ... lymphosarcoma cell leukemia, mast cell leukemia, megakaryocytic leukemia, small myeloblastic leukemia, monocytic leukemia, myeloblastic leukemia, myelogenous leukemia, myelogenous granulocytic leukemia, myelomonocytic leukemia, Naegeli leukemia, plasma cell leukemia, multiple myeloma, plasma cell leukemia, promyelocytic leukemia, Rieder cell leukemia, Schilling leukemia, stem cell leukemia, subleukemic leukemia, or anaplastic cell leukemia.

[0115] As used herein, the terms metastasis and metastatic cancer are used interchangeably and may refer to the spread of a proliferation or disease (e.g., cancer) from one organ or another non-adjacent organ or body part. Cancer begins at a site of origin (e.g., breast), which is referred to as 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 tissues in the local area and / or penetrate the walls of the circulating 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 referred to as a metastatic tumor or secondary tumor. When cancer cells metastasize, it is assumed that the metastatic tumor and its cells are similar to 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 referred to as a metastatic lung cancer. Thus, the phrase 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 phrase non-metastatic cancer, or the subject with non-metastatic cancer, refers to a disease in which the subject has a primary tumor but does not have one or more secondary tumors.For example, metastatic lung cancer refers to a disease in which the subject has or has a history of a primary lung tumor, and has one or more secondary tumors at a second location or multiple locations (e.g., 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 the 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) a symptom of the disease.

[0117] As used herein, autoimmune disease refers to a disease or disorder resulting from the alteration of the immune response by the immune system of a subject against, for example, material tissues and / or cells that are 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-Barre syndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis, ankylosing spondylitis, psoriasis, Sjogren's syndrome, vasculitis, glomerulonephritis, autoimmune thyroiditis, Behcet'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] A humanized antibody is a genetically engineered antibody in which at least one CDR (or a functional fragment or variant thereof) from a mouse antibody (the "donor antibody", which may be a 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 sequence of the acceptor antibody may be, for example, a mature human antibody sequence (or a fragment thereof), a consensus sequence of a human antibody sequence (or a fragment thereof), or a germline region sequence (or a fragment thereof). Thus, a humanized antibody may be an antibody having one or more CDRs from a donor antibody and a variable region framework (FR). The FR may form part of the constant and / or variable region in a human antibody. In addition, to retain high binding affinity, amino acids in the human acceptor sequence can be replaced by the corresponding amino acids 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, which provide detailed instructions for the construction of humanized antibodies (herein incorporated by reference). 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 sequences (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 mouse antibody (also referred to herein as mouse CDRs) and a human variable region framework; and (ii) a heavy chain variable region comprising at least one CDR (often three CDRs) from a mouse 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 (combined 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 light chain human constant region and a heavy chain human constant region.

[0121] Chimeric antibodies are antibodies in which the variable regions of a mouse (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. While such antibodies retain the binding specificity of the mouse antibody, they are approximately two-thirds human. The proportion of non-human sequences present in mouse, chimeric and humanized antibodies suggests that the immunogenicity of chimeric antibodies is intermediate between that of mouse and humanized antibodies. Other types of engineered antibodies that may have reduced immunogenicity relative to murine antibodies include human antibodies made 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 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, in conjunction with, in the preparation of, or are products of the disclosed methods and compositions. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed, specific references to the various individual and collective combinations and permutations of each 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 are specifically contemplated, unless specifically indicated to the contrary. Similarly, any subset or combination thereof is 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 to be 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. EXAMPLES

[0125] Example 1. Novel BAFF receptor antibodies to natively folded recombinant protein eradicate drug-resistant human B cell malignancies in vivo. Conventional 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 proteins. Furthermore, antibodies may be made against off-target domains, such as transmembrane or intracellular domains of the target protein. As described herein, a strategy was applied to generate mAbs against natively folded and glycosylated immunogens expressed on eukaryotic cells. In particular, human BAFF-R was expressed as a native protein on mouse fibroblasts and engineered cell clones were used as immunogens in mice. Described herein is 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 xenogeneic tumor models.

[0126] Materials and Methods Animals, cell lines, and primary human tumor samples. BALB / c mice and NOD scid gamma (NSG) breeding pairs for antibody development were purchased from Jackson Laboratory (Bar Harbor, ME). NSG breeding colonies were maintained by the Animal Resource Center at City of Hope. Mice were housed in a pathogen-free animal facility according to 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 Lymphoma Satellite Tissue Bank at MD Anderson Cancer Center 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 from human B cells and cloned in frame with the GFP gene on 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 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 and screened for antibodies by ELISA using plates coated with D2C or parental L cells. Immunization and hybridoma procedures were performed at the Antibody Core Facility at MD Anderson Cancer Center.

[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 co-transfected into the FreeStyle 293 expression system (Life Technologies, Carlsbad, CA) according to the manufacturer's instructions. Antibodies in the culture supernatants were purified by HiTrap Protein A affinity chromatography columns (GE Healthcare, Marlborough, MA) according to the manufacturer's instructions.

[0130] Cytotoxicity assays. Target cells (L cells, human tumor lines, 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 by 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, luciferase gene was introduced into the tumor lines by 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 × 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 min prior to imaging. Imaging was performed on an AmiX imaging system (Spectral Instruments Imaging, Tucson, AZ).

[0134] Antibody studies: Mice (n=5 per group) were tumor challenged IV 3 days prior to four treatments, one every 5 days. Treatments consisted of a 300 μL IV injection (200 μg therapeutic antibody, 10×10 6 of effector human NK-92-176V cells, and 5 × 10 4 IU of IL-2 (Prometheus Laboratories, San Diego, CA). Control groups received the same volume of injections with control antibody or without antibody and / or NK cells. Bioluminescence imaging was performed weekly until day 80. Survival was followed until 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 is 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 following 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). Supernatants of all four clones contained antibodies that bound to the BAFF-R expressing Mino cell line (MCL) in a dose-dependent manner. No antibody binding was 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 concentration (0.05μg / 10 6 In contrast, the antibody effectively bound JeKo-1 (MCL), SU-DHL-6 (DLBCL), Raji (Burkitt's lymphoma), and RL (FL) (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 respective chimeric mAbs containing human IgG1 constant regions (designated C55 and C90). The chimeric antibodies retained specific dose-dependent binding to BAFF-R-expressing L cells (Figure 1C). C55 and C90 were conjugated to Alexa Fluor 488 and showed direct binding to non-Hodgkin's lymphoma (NHL) lines JeKo-1, SU-DHL-6, Raji, and RL (Figure 1D). Importantly, the chimeric mAbs readily bound patient primary tumor samples of 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 and a BAFF-R negative human multiple myeloma line (U266) (Figure 2A and Figure 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, the addition of NK cells was required for cytotoxicity (Figure 2C and Figure 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 (Figure 14), suggesting a possible disruption of BAFF / BAFF-R survival signaling in tumor cells. Furthermore, C55 and C90 showed limited internalization upon binding to BAFF-R (Figure 15).

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

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

[0144] To create a drug-resistant lymphoma model, a stable CD20 knockout (KO) clone of JeKo-1 was generated 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). The 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 IV, 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 groups (Figure 6A). Tumor-free monitoring and long-term overall survival confirmed significant antitumor efficacy of both BAFF-R antibodies, but not rituximab (Figure 6C). Similarly, significant efficacy was observed following treatment of ibrutinib-resistant Z-138 tumor-bearing mice with either BAFF-R antibody compared to controls (PBS or NK only) (Figure 6B-C).

[0147] BAFF-R mAbs also bind 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 (Figure 17). The positive staining results were verified with purified B cells (Figure 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 induced robust in vivo antitumor effects as a single agent against multiple B cell tumor types, including NHL, CLL, and ALL. Moreover, the antibody eradicated established tumors, which led to long-term tumor-free survival in vivo.

[0150] The differential properties of the BAFF-R mAbs may be due to the approach used for their generation. The approach provided is to express human BAFFF-R as a native surface protein on mouse fibroblasts for immunization, increasing the likelihood of presenting a natively folded and glycosylated immunogen. It is therefore highly likely that the antibody binds an accessible human BAFF-R epitope that is different from the other antibodies described. 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 invention is ADCC, since 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 the 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 with a CRISPR-edited MCL line (JeKo-1, which lacks CD20). The significant in vivo antitumor effect of C55 or C90 against this line, as well as against naturally ibrutinib-resistant Z-138 MCL, but not with rituximab treatment, suggests efficacy against drug-resistant lymphomas (Figure 5). Taken together 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 to be comparable in binding affinity to the parent chimeric antibody (Table 1). [Table 1]

[0153] Nine candidate antibodies were further evaluated to determine the top candidate. The binding of the humanized antibodies was observed to have specificity for BAFF-R, with all having similar relative binding in a dose-dependent manner (Figure 20A). In addition, the ADCC efficacy of the humanized antibodies was assessed. Again, it was found that the humanized candidates maintained specific cytotoxicity and performed 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 with the humanized variants on normal PBMCs shows specificity in binding. When assessed for binding to granulocytes, monocytes, B cells, T cells, and NK cells in normal healthy PBMCs, the antibody binds only 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 incorporation, the JeKo-1, Z138 and RS4 lines were administered varying concentrations of antibodies. The cells and antibodies were incubated with effector NK cells. The supernatants were analyzed 6 hours after treatment (Figure 21A). The antibodies have a clear cytotoxic effect on the tumor lines, demonstrating a dose-dependency with 10-fold dilutions of each. The results are comparable to those of Rituximab, but can also be observed in RS4 acute lymphoblastic lymphoma, where Rituximab is inactive. Further assays with LY-10, MEC-2, RL and Raji (Figure 21B) were continued to demonstrate the efficacy of the humanized antibody treatment. All results found were comparable to those of the current conventional treatment 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 engineered into the T cell signaling domain (δ chain) with a 4-1BB motif. The engineered CAR gene was introduced into purified healthy donor-derived CD8+ T cells via lentivirus along with co-expressed GFP. 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 were monitored by bioluminescence imaging until a visible population of tumor cells was observed, which was approximately 9 days after tumor challenge. Mice received 5×10 6 Mice were treated with two doses of CAR-T cells (anti-BAFF-R and anti-CD19) from 100-mL immunized mice. Control groups received untreated T cells or saline (PBS). To evaluate the therapeutic antitumor effects of CAR-T therapy, mice were closely monitored and imaged every 3 days to follow tumor progression.

[0157] The humanized anti-BAFF-R mAbs were further assessed on primary patient tumor samples for their binding and cytotoxicity. 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 that are 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 the previously developed chimeric antibodies (Figure 22B). The cell type specificity of the humanized antibodies was determined by assessment of their binding to normal PBMCs. No appreciable binding was noted for the 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 the previously characterized chimeric antibodies.

[0158] Anti-BAFF-R mAb is further used to generate chimeric antigen receptor (CAR) T cells. The experiment utilized chimeric C55 variable regions engineered into single chain (sFv) format. Anti-BAFF-R C55 sFv was attached to a T cell receptor signaling domain containing a 4-1BB motif and successfully introduced into healthy normal human donor CD8+ T cells isolated from PBMC. CAR-T cells were administered to tumor-bearing mice with appreciable tumor burden (Figure 24). Mice treated with anti-BAFF-R CAR-T cells had significant tumor clearance when compared to either saline or unengineered T cell control groups. In addition, the anti-tumor efficacy of the CAR-T cells of the present invention is comparable to that of the anti-CD-19 CAR-T treatment group.

[0159] The chimeric anti-BAFF-R antibody C90 was humanized with multiple variants. The humanization process took into account the analysis of the variable regions and especially the CDRs of the chimeric antibody. From there, three variants for each heavy and light chain were developed with degrees of similarity to human 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.

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