Chimeric Antigen Receptors Based on Low Affinity Mutants of FcεRI Re-direct T Cell Specificity to Cells Expressing Membrane IgE

IgE is the key mediator of allergic responses. Omalizumab, an IgE-specific monoclonal antibody that depletes IgE, is effective for treating severe allergic asthma. The need for frequent administration of the expensive drug, however, limits its applications. Taking advantage of T cell memory, adoptive T cell therapy (ACT) targeting IgE-producing cells has the potential to achieve long-term suppression of IgE and relief of symptoms for severe allergic diseases. The transmembrane form of IgE (mIgE), which is present on all IgE-producing cells, serves as an excellent molecular target for ACT that employs chimeric antigen receptors (CARs). Here, we designed and tested CARs that use the extracellular domain of high affinity IgE receptor, FcεRIα, for mIgE recognition. When expressed on Jurkat T cells, FcεRIα-based CARs mediated robust responses in terms of CD69 upregulation to U266 myeloma cells expressing low levels of mIgE. FcεRIα-based CARs specifically recognized cells expressing mIgE, but not cells with secreted IgE captured through Fcε receptors. CAR+ Jurkat cells did not respond to LAD2 mast cells with secreted IgE bound through FcεRI or Ramos cells with secreted IgE bound through FcεRII. Co-culture of CAR+ Jurkat cells and LAD2 mast cells with IgE bound did not trigger LAD2 cell degranulation. The activity of CAR using wild type FcεRIα for mIgE binding was inhibited by the presence secreted IgE, which likely blocked CAR-mIgE interaction. The activities of CARs using low affinity mutants of FcεRIα, however, tolerated secreted IgE at relatively high concentrations. Moreover, primary human CD8+ T cells expressing a low affinity mutant CAR responded to U266 cells with INFγ production and cytotoxicity despite the presence of secreted IgE. The potency, specificity, and robustness of our CAR design, combined with repaid advances in the safety of ACT, hold promise for novel and highly effective cell-based therapies against severe allergic diseases

Redirecting T cell specificity to IgE-expressing B cell for the management of severe allergic asthma

Cooper, Carlton R.Ma, ZhengyuThe prevalence of allergic diseases has dramatically increased in recent decades, affecting people of all ages. Severe allergic asthma affects a substantial proportion of the population and cannot be controlled by available medications. Patients often require large doses of corticosteroids in combination with other potentially toxic medications and still, may suffer from poorly controlled symptoms and frequent life-threatening asthma attacks and thus significantly impacting on their quality of life. ☐ Allergic immune responses trigger the disease in two-thirds of patients with asthma and up to 50% of patients with severe asthma. IgE plays a central role in allergic immune response, therefore, targeting the source of IgE, the IgE-expressing B cells represents an attractive therapeutic approach. Adoptive cell immunotherapy using T cells engrafted with IgE-specific chimeric antigen receptors (CARs) has the potential to achieve long-term suppression of IgE through specific killing of IgE-expressing B cells. ☐ Previously, first-generation FcεRIα-based CARs were constructed and efficiently expressed on the Jurkat T cell line. The wildtype (WT) and 4 low affinity mutant CARs expressed on Jurkat cells were able to bind IgE, and mediated potent and specific responses to target cells. However, the first-generation CARs showed limited persistence due to the lack of costimulatory signaling domains. ☐ To improve CAR T cell persistence, we have constructed 2nd and 3rd generation FcεRIα-based CARs with costimulatory domains, expressed them on primary human CD8+ T cells through lentiviral transduction, tested their in vitro persistence and cytotoxicity towards IgE-expressing B cells. We show that the 2nd generation CARs were robustly expressed on primary human CD8+ T cells and that these CARs mediated potent killing of target cells. CAR T cell in vitro persistence, however, was not improved by addition of co-stimulatory domains alone. After excluding the roles of IgE binding in culture system and excessive stimulation of T cells from the CD3/CD28 activation beads, we concluded that artificially high levels of CAR expression may lead to spontaneous signaling that is toxic to T cells. In support of this, modulating the expression level of CARs by using a lower expression platform increased CAR T cell in vitro persistence. ☐ Taken together, these results demonstrate the effectiveness of the FcεRIα-based CARs in targeting IgE-expressing B cells in severe allergic asthma and addressed the issue of CAR expression persistence. Through detailed experimental analysis, we have advanced the understanding of factors critical for CAR T cell survival. We believe future studies building on the results of current research will lead to the successful development of adoptive T cell therapy for severe allergic diseases using FcεRIα-based CARs.University of Delaware, Department of Biological SciencesM.S