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442-K A CAR T cell approach for treating B cell lineage cancers expressing membrane IgE
  1. Shenyu Zhang1,
  2. Brittany Fay2 and
  3. Zhengyu Ma2
  1. 1University of Delaware, Wilmington, DE, USA
  2. 2Nemours Children’s Hospital, Wilmington, DE, USA


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.

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