Novel Antigen-Specific Expansion of T Cells Transduced with a CD19 Chimeric Antigen Receptor

Abstract 3262

Relapsed acute lymphoblastic leukemia (ALL) remains a difficult challenge for both pediatric and adult patients. Chimeric antigen receptors (CARs) are genetically engineered molecules expressed in transduced T lymphocytes. CARs express both a target binding motif and TCRzeta signals needed for T cell activation, with or without other costimulatory domains. CARs that target CD19 (expressed on most ALL blasts) are being studied in several clinical trials in adults and are planned for pediatric relapsed/refractory ALL. To date, CARs have been expanded ex vivo using antigen-independent techniques. This project sought to develop antigen specific expansion of cells transduced with a CD19-specific CAR by employing artificial APCs (aAPCs) and to explore whether the method of expansion impacted functionality of CD19-CAR T cells. aAPCs used in these studies express the high affinity Fc receptor (CD64) and the costimulatory molecule CD137L (aAPC-41BBL). We created an Fc-CD19 fusion protein that when loaded onto the aAPCs, engages the chimeric antigen receptor and induces antigen specific activation. Antigen-specific vs. non-specific approaches to activation and expansion of CAR expressing T cells were compared using three different expansion protocols (EPs). CD19-CAR transduced T cells were expanded with A) irradiated aAPC-41BBL loaded with anti-OKT3, B) irradiated aAPC-41BBL loaded with rFc-CD19 or C) irradiated allogeneic PBMC feeder cells with anti-OKT3. Briefly, OKT3 and IL-2 activated T cells were transduced with CD19CAR, control CAR (Her2-specific), or non-transduced (Mock). All cultures were maintained in IL-2. Functionality was assessed in a 4-hour ⁵¹Cr release assay against 4 distinct CD19+ ALL cell lines, K562 cells, and K562 stably transfected with CD19 (K562-CD19) or NGFR (K562-NGFR). Results demonstrate similar significant levels of CD19-specific cytotoxicity in the 4h assay at E:T ratios as low as 2.5:1, regardless of the expansion protocol used (20-40% lysis of all CD19+ targets when expanded by OKT3/aAPC, 40–60% lysis with rFc-CD19/aAPC, and 35–45% lysis with allogeneic feeders and OKT3). To evaluate cytotoxicity in long term culture, CD19-CAR T cells expanded using the three protocols were co-cultured with ALL cell targets for 4 days, then flow cytometry was performed to enumerate surviving ALL cells as determined by CD22+ staining. NALM6 and K562-CD19 cells were entirely eliminated even at an E:T ratio of 2.5:1 by CD19-CAR T cells, regardless of expansion protocol, and not by any of the 3 Mock EPs. Therefore, these results demonstrate that the cytolytic potential of CAR transduced T cells is similar, regardless of whether expansion occurs via CD3 signaling or via the chimeric receptor itself. Interestingly, we did observe substantial NK mediated killing in these assays, which correlated with CD56+ cell content and was eliminated by cold target inhibition using K562 cells. Studies are underway to determine whether differences in NK killing varies with expansion protocol. In summary, Fc-bearing artificial antigen presenting cells combined with CAR specific Fc fusion proteins provide a potential off-the-shelf reagent for antigen specific expansion of T cells with chimeric antigen receptors. This could overcome variable transduction efficiencies and allow administration of a more homogenous population of CAR specific T cells.