BCMA- and CST6-specific CAR T cells lyse multiple myeloma cells and suppress murine osteolytic lesions
Fumou Sun, … , John D. Shaughnessy Jr., Fenghuang Zhan
Fumou Sun, … , John D. Shaughnessy Jr., Fenghuang Zhan
Published October 26, 2023
Citation Information: J Clin Invest. 2024;134(1):e171396. https://doi.org/10.1172/JCI171396.
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BCMA- and CST6-specific CAR T cells lyse multiple myeloma cells and suppress murine osteolytic lesions

Abstract

We have previously demonstrated that cystatin E/M (CST6), which is elevated in a subset of patients with multiple myeloma (MM) lacking osteolytic lesions (OLs), suppresses MM bone disease by blocking osteoclast differentiation and function. CST6 is a secreted type 2 cystatin, a cysteine protease inhibitor that regulates lysosomal cysteine proteases and the asparaginyl endopeptidase legumain. Here, we developed B cell maturation antigen (BCMA) CST6 chimeric antigen receptor T cells (CAR-T cells), which lysed MM cells and released CST6 proteins. Our in vitro studies show that these CAR-T cells suppressed the differentiation and formation of tartrate-resistant acid phosphatase–positive (TRAP+) osteoclasts. Using xenografted MM mice, bioluminescence images showed that both BCMA–CAR-T and BCMA–CST6–CAR-T cells inhibited MM growth to a similar extent. Reconstructed micro–computed tomography images revealed that BCMA–CST6–CAR-T cells, but not BCMA–CAR-T cells, prevented MM-induced bone damage and decreased osteoclast numbers. Our results provide a CAR-T strategy that targets tumor cells directly and delivers an inhibitor of bone resorption.

Authors

Fumou Sun, Yan Cheng, Jin-Ran Chen, Visanu Wanchai, David E. Mery, Hongwei Xu, Dongzheng Gai, Samer Al Hadidi, Carolina Schinke, Sharmilan Thanendrarajan, Maurizio Zangari, Frits van Rhee, Guido Tricot, John D. Shaughnessy Jr., Fenghuang Zhan

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Figure 1

Construction and characteristics of BCMA–CST6–CAR-T cells.

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Construction and characteristics of BCMA–CST6–CAR-T cells. (A) Generatio...
(A) Generation of BCMA-CST6-CAR constructs. The BCMA-CST6-CAR vector was constructed by BCMA-specific scFvs, a safety switch (RQR8) in the hinge region, and a 4-1BB coactivation domain with CD3ζ. A P2A self-cleaving peptide was inserted between the CAR vectors and CST6. (B) The BCMA-CAR vector contained BCMA-scFv, a safety switch, and a 4-1BB coactivation domain with CD3ζ. (C) The MOCK-CAR vector only contained a safety switch and a 4-1BB coactivation domain with CD3ζ. All vectors contained SFFV promoters to drive expression of targeted gene(s) in CAR T cells. (D) Schematic representation of the BCMA–CST6–CAR-T structure. (E) Lentiviruses combined with the RetroNectin transfection technology were engineered with CD3+ T cells derived from healthy donor PBMCs. CAR-T cells were detected with RQR8-specific anti-CD34 antibodies by flow cytometry. CD34+ rates were 29.6% for BCMA–CST6–CAR-T cells. CD34+ cells were enriched to 75.6% for BCMA–CST6–CAR-T cells sorted with anti-CD34 microbeads. CD34+ cells were enriched to 76.9% for sorted BCMA–CAR-T cells. (F) Flow cytometric analyses revealed CD4+/CD8+ ratios of MOCK–CAR-T cells, BCMA–CAR-T cells, and BCMA–CST6–CAR-T cells (n = 3, representative result from 3 independent experiments). (G) Bar plots represent the frequency of CAR-T cells gated on CD4+ and CD8+ T cells (n = 3). Data represent the mean ± SD. One-way ANOVA was used for the statistical analysis; NS = P > 0.05. LTR, long terminal repeat.