B-cell maturation antigen is a promising target for adoptive T-cell therapy of multiple myeloma

Abstract

Purpose: Multiple myeloma is a usually incurable malignancy of plasma cells. New therapies are urgently needed for multiple myeloma. Adoptive transfer of chimeric antigen receptor (CAR)-expressing T cells is a promising new therapy for hematologic malignancies, but an ideal target antigen for CAR-expressing T-cell therapies for multiple myeloma has not been identified. B-cell maturation antigen (BCMA) is a protein that has been reported to be selectively expressed by B-lineage cells including multiple myeloma cells. Our goal was to determine if BCMA is a suitable target for CAR-expressing T cells.

Experimental design: We conducted an assessment of BCMA expression in normal human tissues and multiple myeloma cells by flow cytometry, quantitative PCR, and immunohistochemistry. We designed and tested novel anti-BCMA CARs.

Results: BCMA had a restricted RNA expression pattern. Except for expression in plasma cells, BCMA protein was not detected in normal human tissues. BCMA was not detected on primary human CD34(+) hematopoietic cells. We detected uniform BCMA cell-surface expression on primary multiple myeloma cells from five of five patients. We designed the first anti-BCMA CARs to be reported and we transduced T cells with lentiviral vectors encoding these CARs. The CARs gave T cells the ability to specifically recognize BCMA. The anti-BCMA-CAR-transduced T cells exhibited BCMA-specific functions including cytokine production, proliferation, cytotoxicity, and in vivo tumor eradication. Importantly, anti-BCMA-CAR-transduced T cells recognized and killed primary multiple myeloma cells.

Conclusions: BCMA is a suitable target for CAR-expressing T cells, and adoptive transfer of anti-BCMA-CAR-expressing T cells is a promising new strategy for treating multiple myeloma.

Figure 1

BCMA had a restricted pattern of expression. A, Cell-surface BCMA was detected on multiple myeloma cell lines, but BCMA was not detected on other cell lines. For all plots, the solid line represents staining with anti-BCMA antibodies, and the dashed line represents staining with isotype-matched control antibodies. BCMA was expressed by BCMA-K562 cells but not by NGFR-K562 cells. Flow cytometry staining revealed BCMA on the cell surface of the multiple myeloma cell lines H929, U266, and RPMI8226. BCMA was not detected on the sarcoma cell line TC71, on the T cell leukemia line CCRF-CEM, or on the kidney cell line 293T-17. Primary CD34⁺ hematopoietic cells lacked cell-surface BCMA expression. All plots are gated on live cells. The primary CD34⁺ cells plot is also gated on CD34⁺ cells. B, BCMA cDNA copies were measured in samples of the indicated normal tissues by qPCR. In addition, qPCR was performed on cDNA from cells of a plasmacytoma from Myeloma Patient 1 as a positive control. Neoplastic plasma cells made up 93% of the total cells in the sample. Actin copies were also measured by qPCR in all of the samples, and the results were expressed as the number of BCMA cDNA copies per 10⁵ actin cDNA copies.

Figure 2

IHC staining revealed BCMA expression by normal plasma cells and MM cells but not gastrointestinal epithelial cells. A, and B, BCMA-K562 cells exhibited strong BCMA expression while NGFR-K562 cells did not express BCMA. Isotype-matched control antibodies did not stain either cell line. Staining of sections of C, normal stomach and D, normal rectum showed BCMA-expressing plasma cells in the lamina propria but no BCMA expression by other cells of these organs. IHC staining was performed with both anti-BCMA and isotype-matched control antibodies on consecutive sections. E, Double staining of normal duodenum with anti-BCMA (surface, brown) and anti-MUM1(nuclear, red) revealed BCMA⁺ plasma cells and plasmacytoid cells. BCMA was not expressed by other cells of the duodenum. 10X and 100X views are shown. F, G, H, Staining of myeloma tissue sections from Myeloma Patients 1 through 3 showed cell-surface BCMA expression on the majority of neoplastic plasma cells. IHC staining was performed with BCMA and isotype-matched control antibodies on consecutive sections.

Figure 3

Anti-BCMA CARs were expressed on the surface of T cells. A, A general diagram of anti-BCMA CAR fusion proteins is shown (LTR, long terminal repeat; MSCV, mouse stem cell virus; SIN, self-inactivating). From the N-terminus to the C-terminus, the anti-BCMA CARs all included an anti-BCMA scFv, the hinge and transmembrane (TM) regions of the CD8α molecule, the cytoplasmic portion of the CD28 molecule, and the cytoplasmic portion of the CD3ζ molecule. B, T cells from Donor B were transduced with lentiviral vectors encoding one of three CARs or left untransduced (UT). Expression of two anti-BCMA CARs, which were designated anti-bcma1 and anti-bcma2, was detected by flow cytometry 5 days after the transduction. Expression of the control CAR SP6 was also detected. As controls, isotype-matched antibody staining was included for each cell type. The plots are gated on CD3⁺ lymphocytes. The numbers on the plots are the percentages of cells in each quadrant. This is one of seven experiments with similar results. C, T cells were transduced with either the anti-BCMA CAR anti-bcma2 or the negative-control CAR SP6. The cells were cultured with either the BCMA-expressing cell line BCMA-K562 or the BCMA-negative cell line NGFR-K562 for 4 hours. After transduction with anti-bcma2, T cells degranulated in a BCMA-specific manner. SP6-transduced T cells from the same donors did not degranulate in a BCMA-specific manner. The plots are gated on live CD3⁺ lymphocytes. The numbers on the plots are the percentages of cells in each quadrant. This is one of three experiments with similar results.

Figure 4. T cells expressing anti-BCMA CARs produced cytokines, proliferated, and killed in a BCMA-specific manner. Anti-BCMA CAR-transduced T cells eradicated tumors in vivo

A, T cells from Myeloma Patient 4 were transduced with either anti-bcma2 or the negative-control CAR SP6. Four days later, the CAR-transduced T cells were cultured for 6 hours with either the BCMA-expressing cell line BCMA-K562 or the BCMA-negative cell line NGFR-K562. Large fractions of the T cells transduced with anti-bcma2 produced IL-2 when cultured with BCMA-K562. Only small numbers of anti-bcma2-transduced T cells produced IL-2 when they were cultured with NGFR-K562. Only small numbers of SP6-transduced T cells produced IL-2 when cultured with either BCMA-K562 or NGFR-K562. The plots are gated on CD3⁺ lymphocytes. The numbers on the plots are the percentages of cells in each quadrant. This is one of three experiments with similar results. B, T cells from Donor B were transduced with either anti-bcma2 or SP6. The T cells were then labeled with CFSE. The T cells were cultured with either irradiated BCMA-K562 cells or irradiated NGFR-K562 cells. IL-2 was not included in the cultures. Four days later, the T cells were analyzed by flow cytometry. The plots are gated on CAR-expressing T cells. The CFSE fluorescence was less intense in anti-bcma2-expressing T cells that were cultured with BCMA-K562 cells (solid) than in anti-bcma2-expressing T cells that were cultured with NGFR-K562 cells (dashed, open). This indicates that anti-bcma2-expressing T cells proliferated specifically in response to BCMA. For SP6-expressing T cells, CFSE fluorescence intensity was similar for T cells cultured with either BCMA-K562 or NGFR-K562. This is one of two experiments with similar results. Anti-bcma2-transduced T cells from Donor A specifically killed the multiple myeloma cell lines C, H929 and D, RPMI8226 in 4-hour cytotoxicity assays at various effector:target cell ratios. T cells transduced with the negative control CAR SP6 caused much lower levels of cytotoxicity at all effector:target ratios. For all effector:target ratios, the cytotoxicity was determined in duplicate, and the results are displayed as the mean +/− the standard error of the mean. E, Mice were injected intradermally with RPMI8226 cells and tumors were allowed to grow for 17 to 19 days. On day 0, the mice received intravenous infusions of 8×10⁶ T cells that were transduced with either anti-bcma2 or the negative control CAR SP6. Another group of mice was left untreated. The mice all had established tumors at the time of T-cell infusion. One-hundred percent of mice receiving infusions of anti-bcma2 had rapid and complete regressions of their tumors. In contrast, all mice receiving infusions of SP6-transduced T cells and all mice left untreated had progressive enlargement of their tumors. F, Tumors did not recur in mice receiving anti-bcma2-transduced T cells for the duration of the experiment. All mice receiving anti-bcma2-transduced T cells survived and were healthy for the duration of the experiment. All mice receiving SP6-transduced T cells or left untreated died with progressive tumors. The P<0.0001 refers to the comparison of anti-bcma2 and SP6. E and F show combined results of 3 experiments (anti-bcma2, n=10; SP6, n=10; untreated, n=7).

Figure 5. Anti-BCMA-CAR-transduced T cells specifically recognized and killed BCMA-expressing primary multiple myeloma cells

A, Flow cytometry staining for BCMA (solid line) and isotype-matched control staining (dashed line) revealed BCMA expression on the surface of primary bone marrow multiple myeloma cells from Myeloma Patient 5. The plot is gated on CD38^high CD56⁺ plasma cells, which made up 40% of the bone marrow cells. B, Unmanipulated myeloma-containing bone-marrow cells (MM) from Myeloma Patient 5 or PBMC from Myeloma Patient 5 were cocultured overnight with allogeneic T cells from Donor C. The T cells had been transduced with either anti-bcma2 or the negative control CAR SP6. After the coculture, an IFNγ ELISA was performed. C, Flow cytometry for BCMA (solid line) and isotype-matched control staining (dashed line) revealed BCMA expression on the surface of MM cells from a plasmacytoma of Myeloma Patient 1. The plot is gated on plasma cells, which made up 93% of the total plasmacytoma cells. D, Unmanipulated myeloma cells (MM) from a plasmacytoma of Myeloma Patient 1 or PBMC from Myeloma Patient 1 were cocultured overnight with allogeneic T cells from Myeloma Patient 4. The T cells were transduced with either anti-bcma2 or the negative control CAR SP6. The PBMC from Myeloma Patient 1 did not contain BCMA⁺ cells. After the coculture, an IFNγ ELISA was performed. E, Unmanipulated myeloma cells (MM) from a plasmacytoma of Myeloma Patient 1 or PBMC from Myeloma Patient 1 were cultured overnight with autologous anti-bcma2-transduced T cells or autologous SP6-transduced T cells, and an IFNγ ELISA was performed. F, Myeloma cells from a plasmacytoma of Myeloma Patient 1 were specifically killed by autologous anti-bcma2-transduced T cells at low effector:target ratios while autologous SP6-transduced T cells caused only a low level of cytotoxicity of the myeloma cells in a 4-hour cytotoxicity assay. For all effector:target ratios, the cytotoxicity was determined in duplicate, and the results are displayed as the mean +/− the standard error of the mean.