Background Neoplasm of IgE-producing B lineage cells gives rise to IgE myeloma, chronic B cell leukemia and Hodgkin’s lymphoma. Although several chimeric antigen receptor (CAR) T-cell therapies have been approved for treating B cell malignancies by targeting the B cell lineage marker CD19 or B cell maturation antigen (BCMA), these approaches indiscriminately eliminate both cancerous B cells and most normal B cells. This leads to general B cell and plasma aplasia and compromised humoral immune responses.
Methods To specifically target IgE-producing cancer cells, we developed a CAR that targets the extracellular membrane-proximal domain (EMPD) of membrane IgE (mIgE) expressed on the surfaces of IgE-producing cells. EMPD is a 52-residue peptide that exists only on mIgE but not on secreted IgE. Specifically, we generated a human EMPD-specific hybridoma 2E3E10, cloned the heavy and light chain variable regions, and constructed an EMPD-specific second-generation CAR with intracellular 4-1BB and CD3ζ signaling domains. To determine the activity of the CAR, we used a human myeloma cell line U-266 expressing low levels of mIgE as target. In addition, to minimize alloreactive CD8+ T-cell-mediated background killings, we eliminated HLA class I expression on U266 cells by knocking out (KO) beta-2-microglobulin (β2m) using CRISPR/Cas9. We also modified the U-266 cells to express firefly luciferase, enabling a bioluminescence-based killing assay.
Results Through lentiviral transduction, the EMPD-specific CAR was successfully expressed on more than 50% of primary human CD8+ and CD4+ T cells stimulated with anti-CD3/CD28 beads. The expression persisted for more than three weeks in cultures. Coculturing engineered U-266-b2mKO-luci target cells with primary human T cells that expressed the EMPD-specific CAR led to significant T cell cytokine production and target cell killing.
Conclusions The EMPD-specific CAR is capable of mediating primary human T cell activation and cytotoxicity through recognition of the mIgE on IgE-expressing cancer cells. The results demonstrate the proof-of-concept for using EMPD-specific CAR T cells to treat IgE-producing B cell malignancies. Although the CAR T cell eliminates both IgE-producing cancerous and normal B cells, since the vast majority of B cells produce IgG, this approach should not cause general B cell aplasia.
Acknowledgements The work is supported by NIH grants R21AI149243 and R03TR004206 and the Nemours Foundation.
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