Targeting T-Cell Malignancies with Chimeric Antigen Receptors Using Two Structurally Different Antigen Binding Domains Against CD5

Patients with relapsed T-cell acute lymphoblastic leukemia (T-ALL) have extremely poor survival when treated with chemotherapy alone. Allogeneic hematopoietic stem cell transplantation (HSCT) offers a chance of cure for these patients, but attaining clinical remission in order to undergo HSCT remains the biggest therapeutic challenge. Chimeric antigen receptor (CAR) T-cell therapy has been used with great success in relapsed/refractory B-ALL patients. The same approach, however, is difficult in targeting T-cell disease given the lack of a T-lymphoblast specific surface antigen. The use of natural killer (NK) cells as the effector cell provides a potential solution. We are testing two different CAR structures directed against CD5, a pan T-cell marker; one consisting of a more traditional immunoglobulin (Ig) based single chain variable fragment (scFv), while the other comprising of a variable lymphocyte receptor (VLR) as the antigen recognition domain. VLRs are single chain crescent shaped proteins that represent the functional unit of the adaptive immune system in jawless vertebrates. Our previous work has shown that a VLR can be used as an alternative for CAR-based antigen recognition. Here, we directly compare a VLR to its scFv counterpart in a CAR setting. The single chain structure of a VLR allows for the rapid creation of novel CARs, compared to the corresponding use of a scFv-CAR, in which the variable heavy and light sequences need further engineering for adapting to CAR technologies. Thus, our hypothesis was that natural killer cells engineered to express an anti-CD5 CAR can specifically be used to target T-cell malignancies, with the VLR-CAR being equal or superior to scFv-based cassettes. We constructed two second generation CARs using the VLR or scFv sequence targeting CD5 as our antigen recognition domain. Both CD5-CAR constructs showed similar activity when tested in CD5-positive Jurkat cells. For our cytotoxicity studies, we used the IL-2 dependent natural killer cell line NK-92. To generate uniform populations of CAR-expressing NK cells we transduced NK-92 cells with a dual lentiviral construct expressing the CD5-CAR and green fluorescent protein (GFP), and then selected for the positively transduced cells using flow sorting for GFP. In vitro cytotoxicity studies showed significantly increased killing using the CD5-CAR expressing NK-92 cells compared to unmodified NK-92 cells (p<0.05), for both VLR and scFv modified cells. We then tested the two CD5-CARs in a T-cell leukemia mouse model. NOD scid gamma (NSG) mice were intravenously injected with luciferase expressing Jurkat leukemia cells to establish the T-cell leukemia xenograft model. Four doses of NK-92 cells were injected over a two week period starting on day 7 post-tumor injection. Bioluminescence imaging was used to monitor tumor burden. The CD5-scFv-CAR NK-92 treated mice group had a significant survival advantage over the other groups (p<0.01). Interestingly, the CD5-VLR-CAR NK-92 treated mice did not have a significant advantage over naïve NK-92 or saline treated groups. Given the superior in vivo cytotoxicity demonstrated in our studies, we plan to use the CD5-scFV-CAR going forward as we transition to primary cells. Overall, these studies provide the foundation to move these studies into preclinical testing as a treatment for relapsed T-ALL.