207. Variable Lymphocyte Receptors Enable Development of Chimeric Antigen Receptors for the Treatment of T-Cell Malignancies
Authors: Sunil S. Raikar, Daniel Tylawsky, Robert Moot, Lauren Fleischer, David McCarty, Christopher Doering, H. Trent Spencer
Journal: Molecular Therapy (2016)

Relapsed T-cell leukemia has a poor prognosis. Chimeric antigen receptor (CAR) therapy could be an effective treatment modality, although targeting T-cell disease without a T-lymphoblast specific antigen is difficult. The use of natural killer (NK) cells as the effector cell and CD5 as the target antigen provides a potential solution. While traditionally CARs use an immunoglobulin (Ig) based single chain variable fragment (scFV) to target tumor cells, we designed a novel CAR structure using a variable lymphocyte receptor (VLR) as our antigen recognition sequence. VLRs represent the functional unit of the adaptive immune system in jawless vertebrates (lamprey and hagfish) and are analogous but not homologous to immunoglobulins. VLRs have a fundamentally different structure and geometry compared to Ig-based antibodies while still demonstrating high degrees of specificity and avidity. Additionally, VLRs exist naturally as single chain structures that allows for rapid creation of CAR cassettes. In the current study our objective was to develop a VLR-CAR-NK cell against T-cell leukemia using an anti-CD5 VLR sequence. We constructed a second generation CAR using a VLR sequence targeting CD5 as our antigen recognition sequence. The CAR structure consisted of the anti-CD5 VLR sequence, the CD28 co-stimulatory transmembrane domain, and the intracellular CD3ζ signaling domain. Using this construct, high titer CD5 VLR CAR self-inactivating lentivirus was produced at titers >10^8 TU/ml. To test the functionality of the CAR construct, CD5 expressing Jurkat cells were transduced with various doses of lentivirus, and activation was measured by expression of CD69. For cytotoxicity studies, NK-92 cells were used as the effector cell with CCRF-CEM cells being the target cell. Cytotoxicity was measured at different effector: target ratios using a flow cytometry based assay. Activation positively correlated with lentiviral copy numbers and CAR expression in Jurkat cells. Both copy number and activation decreased over time, which was anticipated as we show that highly activated cells had a growth disadvantage. NK-92 cells expressing the CD5 VLR CAR showed approximately two fold increase in cytotoxicity towards CCRF-CEM cells when compared with naïve NK-92 cells; however, low transduction efficiency and copy numbers (<0.5) were problematic, similar to previous data showing that NK-92 cells are resistant to lentiviral transduction. In order to improve upon these results, we created a bicistrionic vector co-expressing GFP and the CD5-VLR-CAR. Jurkat cells genetically modified to express the new construct showed a direct correlation between GFP expression and activation, thus confirming dual expression and function of both proteins. Transduced NK-92 cells were sorted and expanded. These cells expand robustly, demonstrate CAR expression and have the functional characteristics required for targeting T-cell disease. Our studies show the utility of VLR-derived CARs and provide the foundation to progress these studies into preclinical testing as a treatment for relapsed T-cell leukemia.