Abstract
Chimeric antigen receptor (CAR) modified T-cell therapy consists of ex-vivo genetic manipulation of autologous lymphocytes in order to establish robust T-cell mediated anti-tumor immunity. Our group was the first to design and evaluate a CAR targeted toward the B cell antigen CD19 in mice. Currently we utilize a second generation CAR comprised of a single-chain variable fragment (scFv) derived from an antibody against CD19 fused to the CD3 ζ chain and the CD28 intracellular signaling domain (19-28z) to provide the necessary signal 1 and signal 2 for enhanced T-cell activation and persistence. We have gone on to test the safety and efficacy of 19-28z CAR T-cells in patients with chronic lymphocytic leukemia (CLL) and acute lymphoblastic leukemia (ALL). We observed rapid complete molecular remissions in the first 14/16 patients treated with relapsed/refractory ALL. We hypothesize that contributing to the enhanced efficacy seen in ALL, when compared to solid tumors or extra-medullary CLL, is the fact that ALL is a bone marrow predominant disease, which may provide a microenvironment more amenable to T-cell therapy.
Waldenström’s Macroglobulinemia (WM) is an ideal disease to test 19-28z CAR-modified T-cell therapy, as it is often bone marrow predominant, and WM cells from patient samples typically uniformly express high levels of CD19. Furthermore, despite recent progress made with novel BCR-directed therapy, complete eradication of the WM clone from the bone marrow niche remains elusive, therefore providing an ideal clinical scenario for treatment consolidation via alternative cytotoxic methods such as cellular immunotherapy.
Using the human WM cell line, BCWM.1, we evaluated the in vitro efficacy of 19-28z CAR-modified T-cells when compared to mock transduced T cells or T cells transduced with an irrelevant second generation CAR directed towards the ovarian antigen MUC16. In a 4 hour co-culture assay, we observed significant cytotoxicity, even at low effector:target ratios (52% lysis at 1:1; 92% lysis at 10:1; p<0.01). This corresponded to increased secretion of INFγ and IL-2, markers of T-cell activation (p<0.01).
We then conducted in vivo studies using sublethally irradiated SCID/beige mice to generate a systemic model of WM via tail vein injection of 1x106 luciferase transduced BCWM.1 cells. This model is characterized by tumor growth in the bone marrow followed by rapid spread to the liver, lungs, kidney, and CNS. Mice were monitored by weekly bioluminescent imaging (BLI) and ultimately were sacrificed when they developed hind leg paralysis. 19-28z CAR modified T-cells administered at day 7, after tumor establishment, when compared to non-treated and irrelevant CAR-modified T cell controls, delayed the progression of disease and doubled the median survival time of the mice after treatment (p=0.001).
Taken together, the pre-clinical efficacy demonstrated in this abstract and the clinical features of WM, listed above, provide the rational for testing 19-28z CAR modified T cells clinically for WM. We have now opened a clinical trial for patients with relapsed or refractory WM, in which chemotherapy preconditioning is followed by a single dose of 19-28z CAR modified autologous T-cells (NCT00466531).
Disclosures
Brentjens: Juno Therapeutics: Consultancy, Scientific co-founder and Stock holder Other.
Optimizing Manufacturing Protocols of Chimeric Antigen Receptor T Cells for Improved Anticancer Immunotherapy
