Background: Immunotherapy has provided treatment options for cancers that are otherwise refractory to standard approaches. One such technique is to use adoptive transfer of engineered autologous T cells expressing a chimeric antigen receptor (CAR) directed against a tumor antigen. The efficacy of CAR T cells directed against hematological malignancies, particularly CD19-expressing B cell leukemia and lymphomas, has been demonstrated in multiple clinical studies. The success of this approach has prompted development of CAR T cells directed to different tumor antigens for other tumor types. To ensure the selectivity and specificity of the CAR T cells against their intended target, screening methods need to be employed. Multiple myeloma is an incurable malignancy of plasma cells. B-cell maturation antigen (BCMA), also known as tumor necrosis factor superfamily member 17 (TNFRSF17) is nearly ubiquitously expressed on multiple myeloma cells, plasma cells and subsets of mature B cells.
Methods: In order to screen for the specificity of novel CAR T cells directed against BCMA, we utilized a cell microarray platform developed by Retrogenix. In this screen, approximately 4500 human plasma membrane proteins (representing up to 75% of the human plasma membrane proteome) are individually expressed in human HEK293 cells. Fluorescently labeled CAR T cells, which showed cytolytic activity against MM cell lines expressing BCMA, were applied to the cell microarray and specific binding of the CAR T cells to target cells was determined.
Results: Primary hits were sequenced to confirm identity and secondary specificity screens were performed on the identified hits. Specific binding of both mock transduced and BCMA CAR transduced T cells were confirmed for different plasma membrane proteins expressed from the HEK293 cells. These included known T cell interactors, such as ICOSLG, CD244 and CD86, where binding is proposed to be independent of CAR expression. Subtracting the hits of the mock transduced T cells from the BCMA CAR T cells demonstrated specific binding of the CAR T cells to BCMA. Utilizing the fully human IgGs directed against BCMA from which the single-chain variable fragments (scFvs) of the CARs were derived, we further confirmed specific binding to BCMA in additional secondary screens. Additionally, a lack of off-target binding of the fully human IgGs to normal tissue was demonstrated in a tissue cross reactivity screen.
Conclusions: These studies highlight the tractability of this cell microarray approach for determining the specificity of novel CAR constructs expressed in T cell. Demonstrating the selectivity and specificity of anti-BMCA CAR T cells further supports the progression of KITE-585 towards Phase 1 clinical studies in MM patients.
Citation Format: Gregor B. Adams, Jun Feng, Atefeh Ghogha, Armen Mardiros, Ruben Rodriguez, Tassja J. Spindler, Jed Wiltzius, Tony Polverino. Selectivity and specificity of engineered T cells expressing KITE-585, a chimeric antigen receptor targeting B-cell maturation antigen (BCMA) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2135. doi:10.1158/1538-7445.AM2017-2135