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. 2018 Sep 11;10(9):323.
doi: 10.3390/cancers10090323.

CAR-T Cells Based on Novel BCMA Monoclonal Antibody Block Multiple Myeloma Cell Growth

Robert Berahovich  1 , Hua Zhou  2 , Shirley Xu  3 , Yuehua Wei  4 , Jasper Guan  5 , Jian Guan  6 , Hizkia Harto  7 , Shuxiang Fu  8 , Kaihuai Yang  9 , Shuying Zhu  10 , Le Li  11   12 , Lijun Wu  13 , Vita Golubovskaya  14
Affiliations

CAR-T Cells Based on Novel BCMA Monoclonal Antibody Block Multiple Myeloma Cell Growth

Robert Berahovich et al. Cancers (Basel). .

Abstract

The cell-surface protein B cell maturation antigen (BCMA, CD269) has emerged as a promising target for CAR-T cell therapy for multiple myeloma. In order to create a novel BCMA CAR, we generated a new BCMA monoclonal antibody, clone 4C8A. This antibody exhibited strong and selective binding to human BCMA. BCMA CAR-T cells containing the 4C8A scFv were readily detected with recombinant BCMA protein by flow cytometry. The cells were cytolytic for RPMI8226, H929, and MM1S multiple myeloma cells and secreted high levels of IFN-γ in vitro. BCMA-dependent cytotoxicity and IFN-γ secretion were also observed in response to CHO (Chinese Hamster Ovary)-BCMA cells but not to parental CHO cells. In a mouse subcutaneous tumor model, BCMA CAR-T cells significantly blocked RPMI8226 tumor formation. When BCMA CAR-T cells were given to mice with established RPMI8226 tumors, the tumors experienced significant shrinkage due to CAR-T cell activity and tumor cell apoptosis. The same effect was observed with 3 humanized BCMA-CAR-T cells in vivo. These data indicate that novel CAR-T cells utilizing the BCMA 4C8A scFv are effective against multiple myeloma and warrant future clinical development.

Keywords: CAR-T cells; cancer; cell therapy; chimeric antigen receptor; immunotherapy; multiple myeloma; tumor antigen; xenograft.

PubMed Disclaimer

Conflict of interest statement

L. Wu is CEO of Promab Biotechnologies and the co-authors are employees of Promab Biotechnologies and Forevetek Biotechnology. The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
BCMA mAb 4C8A binds BCMA protein. (A) Binding of BCMA mAb 4C8A using Forte Bio Blitz system. BCMA mAb 4C8A was loaded onto a Blitz mouse Fc capture sensor at different concentrations. The Kd of binding was detected with Blitz software. (B) Binding of BCMA mAb to BCMA protein by ELISA. BCMA mAb 4C8A was incubated in ELISA plates coated with BCMA protein, BCMA protein with a C-terminal deletion of 37 residues and the first two N-terminal residues; or negative control protein, CD363 protein. * p < 0.0001 for BCMA protein versus BCMA and control. (C) Dose-dependent binding of 4C8A mAb to BCMA protein. Dilutions of BCMA mAb 4C8A were incubated in ELISA plates coated with BCMA protein or CD363 negative control protein. * p < 0.0001 for BCMA protein versus control. (D) BCMA binding to BCMA protein in 293 cells by immunofluorescent staining (IF). BCMA mAb 4C8A was incubated with HEK293 cells, HEK293 cells expressing BCMA, or HEK293 cells expressing negative control protein, CD18. Binding of BCMA mAb 4C8A was detected with Alexa Fluor 488-conjugated anti-mouse IgG. (E) Binding of BCMA monoclonal antibody to BCMA in multiple myeloma cells. BCMA mAb 4C8A, BCMA mAb 19F2 and a mouse IgG1 isotype control mAb were incubated with myeloma lines RPMI8226, H929, and MM1S, as well as Burkitt’s lymphoma line Raji and the BCMA-negative cell line K562. Binding of the antibodies to the cells was detected by flow cytometry with PE-conjugated anti-mouse IgG. (F) Quantification of binding shown in Figure 1E. To quantitate the binding in panel E, the mean fluorescence intensity (MFI) of each BCMA mAb was divided by the MFI of the isotype control mAb. * p < 0.05 for BCMA mAb 4C8A versus BCMA mAb 19F2 (MM1S and Raji only). (G) BCMA mAb 4C8A binds BCMA in CHO-BCMA cells. BCMA mAb 4C8A, BCMA mAb 19F2, and a mouse IgG1 isotype control mAb were incubated with CHO (Chinese Hamster Ovary) cells stably expressing human BCMA, and binding of the antibodies was detected by flow cytometry with PE-conjugated anti-mouse IgG.
Figure 2
Figure 2
Immunohistochemical staining of normal human tissues by BCMA 4C8A mAb. (A) BCMA 4C8A but not the isotype control mAb stained (brown color) RPMI8226 myeloma cells and normal human liver. (B) BCMA 4C8A did not stain any other normal human tissues. Blue color: nucleus counterstain. Original magnification 400×.
Figure 3
Figure 3
Characterization of BCMA 4C8A CAR-T cells in vitro. (A) Diagram of the BCMA and mock CARs, with the following regions (from N-terminus to C-terminus): scFv, CD8 hinge, CD28 transmembrane domain, CD28 costimulatory domain, CD3 zeta activation domain. (B) FACS analysis with BCMA protein detects BCMA-CAR expression. BCMA CAR-T cells, mock CAR-T cells, and non-transduced T cells were analyzed by flow cytometry, using an APC-conjugated anti-CD3 mAb (Y-axis) and biotinylated BCMA protein (X-axis). The percentage of cells binding to both the CD3 mAb and the BCMA protein (i.e., BCMA CAR-T cells) is shown in the upper right quadrant. (C) BCMA-CAR-T cells lyse RPMI8226 multiple myeloma cells by LDH assay. BCMA CAR-T cells, mock CAR-T cells, and non-transduced T cells were incubated with RPMI8226 myeloma cells, and the levels of LDH released into the medium was measured by a colorimetric enzyme assay as described in Materials and Methods. The LDH assay demonstrates specific LDH release by target cells. * p < 0.0001 for BCMA CAR-T cells versus mock CAR-T cells and non-transduced T cells. (D) BCMA-CAR-T cells secrete IFN-γ against multiple myeloma cells. BCMA CAR-T cells, mock CAR-T cells and non-transduced T cells were incubated with myeloma lines: RPMI8226, H929, and MM1S, as well as the BCMA-negative cell line K562. The levels of IFN-γ released into the medium was measured by ELISA; * p < 0.0001 for BCMA CAR-T cells versus mock CAR-T cells and non-transduced T cells. (E) BCMA-CAR-T cells killed CHO-BCMA cells by real-time cytotoxicity assay (RTCA). BCMA CAR-T cells, mock CAR-T cells, and non-transduced T cells were added to monolayers of CHO cells (top row) and CHO-BCMA cells (bottom row), and the impedance proportional to cell number of the monolayers was monitored over time. The average of three replicates is shown. (F) Quantitation of cytotoxicity at the end of the RTCA assay in panel E; * p < 0.0001 for BCMA CAR-T cells versus mock CAR-T cells and non-transduced T cells. (G) BCMA-CAR-T cells significantly increased IFN-gamma secretion against CHO-BCMA cells. The levels of IFN-gamma released into the RTCA medium was measured by ELISA; * p < 0.0001 for BCMA CAR-T cells versus mock CAR-T cells and non-transduced T cells.
Figure 3
Figure 3
Characterization of BCMA 4C8A CAR-T cells in vitro. (A) Diagram of the BCMA and mock CARs, with the following regions (from N-terminus to C-terminus): scFv, CD8 hinge, CD28 transmembrane domain, CD28 costimulatory domain, CD3 zeta activation domain. (B) FACS analysis with BCMA protein detects BCMA-CAR expression. BCMA CAR-T cells, mock CAR-T cells, and non-transduced T cells were analyzed by flow cytometry, using an APC-conjugated anti-CD3 mAb (Y-axis) and biotinylated BCMA protein (X-axis). The percentage of cells binding to both the CD3 mAb and the BCMA protein (i.e., BCMA CAR-T cells) is shown in the upper right quadrant. (C) BCMA-CAR-T cells lyse RPMI8226 multiple myeloma cells by LDH assay. BCMA CAR-T cells, mock CAR-T cells, and non-transduced T cells were incubated with RPMI8226 myeloma cells, and the levels of LDH released into the medium was measured by a colorimetric enzyme assay as described in Materials and Methods. The LDH assay demonstrates specific LDH release by target cells. * p < 0.0001 for BCMA CAR-T cells versus mock CAR-T cells and non-transduced T cells. (D) BCMA-CAR-T cells secrete IFN-γ against multiple myeloma cells. BCMA CAR-T cells, mock CAR-T cells and non-transduced T cells were incubated with myeloma lines: RPMI8226, H929, and MM1S, as well as the BCMA-negative cell line K562. The levels of IFN-γ released into the medium was measured by ELISA; * p < 0.0001 for BCMA CAR-T cells versus mock CAR-T cells and non-transduced T cells. (E) BCMA-CAR-T cells killed CHO-BCMA cells by real-time cytotoxicity assay (RTCA). BCMA CAR-T cells, mock CAR-T cells, and non-transduced T cells were added to monolayers of CHO cells (top row) and CHO-BCMA cells (bottom row), and the impedance proportional to cell number of the monolayers was monitored over time. The average of three replicates is shown. (F) Quantitation of cytotoxicity at the end of the RTCA assay in panel E; * p < 0.0001 for BCMA CAR-T cells versus mock CAR-T cells and non-transduced T cells. (G) BCMA-CAR-T cells significantly increased IFN-gamma secretion against CHO-BCMA cells. The levels of IFN-gamma released into the RTCA medium was measured by ELISA; * p < 0.0001 for BCMA CAR-T cells versus mock CAR-T cells and non-transduced T cells.
Figure 4
Figure 4
BCMA 4C8A CAR-T cells block RPMI 8226 xenograft tumor growth. (A) NSG (NOD Scid gamma) mice were injected subcutaneously with RPMI8226 myeloma cells and tumor size was measured bi-weekly with calipers. On days 16 and 24 (arrows), the mice received BCMA CAR-T cells, mock CAR-T cells, or PBS intravenously; * p < 0.01, ** p < 0.0001 for BCMA CAR-T cells versus mock CAR-T cells and PBS. (B) The tumors were excised and photographed. (C) The weight of the tumors significantly decreased in BCMA-treated mice. * p < 0.05 for BCMA CAR-T cells versus mock CAR-T cells and PBS. (D) BCMA-CAR-T cells did not change mice weight. (E) BCMA-treated mice had significantly increased level of IFN-gamma in the blood plasma. Human IFN-γ levels were measured in the plasma by ELISA at the end of the study; * p < 0.0001 for BCMA CAR-T cells versus mock CAR-T cells and PBS. (F) The level of human T cells and CAR-T cells in the blood of BCMA-treated mice was significantly increased versus control treated mice. The peripheral blood cells were analyzed by flow cytometry at the end of the study for binding to human BCMA protein and an antibody specific for human CD3. The percentage of cells binding to the CD3 mAb is shown on the left, and the percentage of those human T cells that also bound to the BCMA protein is shown on the right; * p < 0.0001 for BCMA CAR-T cells versus mock CAR-T cells.
Figure 5
Figure 5
BCMA-CAR-T cells significantly block growth of subcutaneous established (150 mm3) RPMI8226 xenograft tumors. (A) NSG mice were injected subcutaneously with RPMI8226 myeloma cells and tumor size was measured bi-weekly with calipers. On days 18 and 24 (arrows), the mice received PBS, BCMA CAR-T cells or mock CAR-T cells intravenously; * p < 0.0001 for BCMA CAR-T cells versus PBS and mock CAR-T cells. (B) The tumors were excised and photographed. (C) The excised tumors were weighed; * p < 0.0001 for BCMA CAR-T cells versus mock CAR-T cells. (D) The mice were weighed weekly during the study. (E) The peripheral blood cells were analyzed by flow cytometry at the end of the study for binding to human BCMA protein and an antibody specific for human CD3. The percentage of cells binding to the CD3ζmAb is shown on the left, and the percentage of those human T cells that also bound to the BCMA protein is shown on the right; * p < 0.05 for Human T cells (BCMA versus mock and PBS), p < 0.0001 for CAR-T cells (BCMA versus mock).
Figure 6
Figure 6
BCMA-CAR-T cells significantly block growth of subcutaneous large size established (500 mm3). RPMI8226 xenograft tumors. (A) NSG mice were injected subcutaneously with RPMI8226 myeloma cells and tumor size was measured bi-weekly with calipers. On days 27 and 31 (arrows), the mice received BCMA CAR-T cells or mock CAR-T cells intravenously; * p < 0.0001 for BCMA CAR-T cells versus mock CAR-T cells. (B) The tumors were excised and photographed. (C) The excised tumors were weighed; * p < 0.05 for BCMA CAR-T cells versus mock CAR-T cells. (D) The mice were weighed weekly during the study. (E) Sections of the tumors were stained immunohistochemically (brown color) with antibodies specific for human CD3ζ, Ki-67 and cleaved caspase-3. Top row: tumors from mice receiving mock CAR-T cells. Bottom: tumors from mice receiving BCMA CAR-T cells. Blue color: nucleus counterstain. Original magnification 400×.
Figure 7
Figure 7
Characterization of humanized BCMA 4C8A CAR-T cells. (A) Three humanized clones were evaluated for cytolytic activity by RTCA on CHO and CHO-BCMA target cells at an E:T ratio of 10:1. The quantitation of cytotoxicity at the end of the assay is shown on the right; * p < 0.0001 for all 3 clones versus mock and non-transduced T cells. (B) The levels of IFN-γ produced in the RTCA assay were quantitated by ELISA; * p < 0.0001 for all 3 clones versus mock and non-transduced T cells. (C) The humanized BCMA 4C8A CAR-T cells were cultured overnight with BCMA-positive RPMI8226 myeloma cells and BCMA-negative K562 cells, then the levels of IFN-γ produced in the culture were quantitated by ELISA; * p < 0.0001 for all 3 clones versus mock and non-transduced T cells. (D) The humanized BCMA 4C8A CAR-T cells were tested in the RPMI8226 tumor model for inhibition of tumor growth; * p = 0.043 and ** p ≤ 0.005 for all 3 clones versus mock. (E) The peripheral blood cells were analyzed by flow cytometry at the end of the study for binding to human BCMA protein and antibodies specific for human T (CD4+/CD8+) cells. The percentage of cells binding to the CD4 mAb is shown on the left, and the percentage of those human T cells that also bound to the BCMA protein is shown on the right; * p < 0.05 for Human T and BCMA-CAR-T cells (BCMA versus Mock).
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