Background
IgE-producing multiple myeloma, non-Hodgkin’s lymphoma and chronic B cell leukemia are caused by neoplasm of IgE-producing B cells. Several chimeric antigen receptor (CAR) T-cell therapies have been approved for treating B cell malignancies, but they target B cell linage markers CD19 or BCMA and indiscriminately eliminate both malignant B cells and 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 specifically recognizes the transmembrane form of IgE (mIgE). The second-generation CAR employs a single chain variable fragment (scFv) 2E3E10 that recognizes the extracellular membrane-proximal domain (EMPD), which is found only on mIgE and not on secreted IgE. To determine the activity of EMPD-specific CAR, U-266 myeloma cells expressing low levels of mIgE and Daudi lymphoma cells expressing high levels of mIgE were used as target cells. To minimize T cell receptor (TCR)-mediated alloreactive killing that is independent of CAR activity, beta-2-microglobulin (β2m) expression on U-266 cells were knocked out using CRISPR/Cas9 to abrogate HLA class I expression. In addition, U-266 and Daudi cells were modified to express firefly luciferase for monitoring target cell killing in vitro and in vivo.
Results
Primary human CD8+ and CD4+ T cells transduced using lentiviral vectors encoding the CAR showed high levels of CAR expression on around 50% of cells for more than three weeks in culture. Coculturing U-266-b2mKO-luci or Daudi-mIgE-luci target cells with the CAR T cells led to significant T cell cytokine production and target cell killing, demonstrating the ability of EMPD-specific CAR to mediate primary human T cell activation and cytotoxicity. To assess CAR T cell activity in vivo, myeloma and lymphoma mouse models were established through intravenous injection of U-266-b2mKO-luci and Daudi-mIgE-luci cells, respectively, into immunocompromised NSG mice. Administration of EMPD-specific CAR T cells three days later led to complete elimination of cancer cells in both xenograft mouse models. EMPD-specific CAR-T cells were detected in tail vein blood after 15 weeks and displayed stem and memory-like phenotypes.
Conclusions
Our results indicate that EMPD-CAR T cells are capable of mediating primary human T cell activation and cytotoxicity through recognition of the mIgE on IgE-expressing cancer cells both in vitro and in vivo, demonstrating the proof-of-concept for using EMPD-CAR T cells to treat IgE-producing blood cancers without the side effect of causing B cell aplasia.