Chimeric antigen receptor-modified iNKT cell therapy for CD7+ malignancies

PROJECT SUMMARY/ABSTRACT The major hurdle in developing chimeric antigen receptor (CAR)-T therapy for T-cell malignancies is CAR-T cell fratricide by self-killing, which leads to insufficient numbers of CAR-T cells for infusion. CRISPR- mediated genomic editing provides an alternative approach to enabling expansion of CAR transduced T cells from allogeneic healthy donors. However, the safety and clinical implications of CRISPR-based gene editing remain lingering. The objective of this SBIR Phase I project is to develop a non-edited, CD7 CAR-modified allogeneic invariant Natural Killer T (iNKT) cell product for the treatment of relapsed and refractory patients with T-cell acute lymphoblastic leukemia (T-ALL) and acute myeloid leukemia (AML). The rationale for this study is based on our strong preliminary data. We propose two Specific Aims. Aim 1: To optimize the iNKT cell primary stimulation for maximal proliferation and CAR transduction. We plan to test major commercially available T-cell activation reagents, i.e., T-Cell Activation/Expansion kit (CD2/CD3/CD28 microbeads), T-Cell TransAct (CD3/CD28 microbeads), and CD3/CD28 Dynabeads in primary stimulation and transduce them with lentivirus CD7 CAR. CD7 CAR-modified iNKT, control CD19 CAR-modified iNKT or mock cells will be evaluated for cytotoxicity and IFN-? production against CD7+ T-ALL and AML, CD19+ B-cell tumor cells, and ?GalCer pulsed CD1d+ target cells. In addition, expansion rate, CAR expression, and expression of CD3 and invariant T-cell receptor (iTCR) will also be assessed. CD7 CAR-modified iNKT cells with a highest expansion and CAR transduction while maintaining iNKT cell function and phenotype in primary culture will be examined for expansion in secondary stimulation. Expanded final cell products will be characterized for their expansion rate, cytotoxicity, cytokine profiling, phenotype, and expression of checkpoint receptors. Aim 2: To determine the effect of IL-7 co-expression on enhancing CD7 CAR-modified iNKT cell survival and efficacy. To test our hypothesis that IL-7 co-expression in CD7 CAR-modified iNKT cells can enhance their survival and anti-tumor efficacy, we will initially examine IL-7 and/or IL-15 co-expression in CD7 CAR-modified iNKT cells in response to CD7+ or CD7- tumor cells and characterize 1) secreted IL-7 and/or IL-15 levels, 2) STAT5 phosphorylation to reflect IL-7 and IL-15 signaling, 3) cytotoxicity and IFN-? release, 4) fold expansion, 5) quantification of antiapoptotic factors (Bcl-2, Bcl-xL, MCI-1), 6) serial tumor challenge assay to assess enhanced survival, 7) frequency of naïve, central and effector memory T cell subsets, and 8) expression of checkpoint receptors (PD-1, CTLA4, TIM3, LAG3). We will then evaluate their anti-tumor efficacy in our established aggressive T-ALL xenograft model as assessed by reduced tumor mass via bioluminescence imaging and prolonged survival. iNKT cell survival will also be assessed by flow cytometry.