750. Anti-Tumor Effects of Anti-CD20 Chimeric Antigen Receptor Therapy in Canine B Cell Lymphoma Patients

Abstract

Chimeric antigen receptor (CAR) T cell therapy has shown great promise in treating human leukemias. However, for CAR T technology to achieve its full potential against solid malignancies, better pre-clinical models are needed that recapitulate the immune suppressive tumor microenvironment and the distribution of target antigens. Pet dogs are an outbred population that have a close phylogenetic relationship with man and frequently develop naturally occurring, spontaneous malignancies. We explored the feasibility of evaluating CAR T cell technologies in dogs with spontaneous cancers by assessing CD20-targeting CAR T cells in dogs with relapsed CD20+ B cell lymphoma. We have developed standard procedures for optimal ex vivo canine T cell activation, expansion, and lentiviral transduction. Canine PBLs were activated in vitro using artificial antigen presenting cells (aAPCs) loaded with anti-canine CD3, in the presence of rhIL-2 and rhIL-21, and the time of peak T cell activation and disappearance of aAPCs from the culture were determined. Peak CD25 expression was observed 3-4 days post-stimulation, when less than 5% of aAPCs remained in culture, eliminating potential competitor cells for viral infection. Transduction of canine T cells, 4 days post-stimulation using VSVg-pseudotyped lentivirus encoding a second-generation CD20-targeting CAR (CD20-28-ζ) at an MOI of 20 resulted in up to 26% surface expression of the CAR on T cells grown from a healthy dog. In vitro co-culture of CD20-specific canine CAR T cells derived from a lymphoma patient with CD20+ target cells resulted in antigen-specific CAR T cell expansion and effective killing. To determine whether canine CD20 CAR T cells could survive, expand and exhibit cytotoxic activity in vivo, three pet dogs with relapsed CD20+B cell lymphoma were treated with autologous CD20-28-ζ CAR T cells in an IACUC-approved protocol. PBLs were transduced (1.5-6.5%), expanded (22-153 fold) and infused into the patients either IV or IV plus intra-nodal injection. A 52% decrease in tumor volume was observed in the first patient 4 days post-IV infusion, coinciding with an 82% increase in T cell frequency within the malignant lymph node. CAR T cells were administered via intra-nodal injection into the largest malignant node of the second dog, which stayed stable in volume over two weeks while all other non-injected malignant nodes doubled in size. The third dog received the largest number of CAR T cells administered IV. In this dog, CAR T cells have expanded and persisted for three weeks and malignant nodes have remained below the limit of accurate measurement. These data demonstrate the ability of canine CAR T cells to function in vitro and to halt and even temporarily reverse solid tumor progression in canine patients, mirroring what has been observed in human disease and treatment. This work shows the feasibility of this approach in dogs and offers a spontaneous cancer model for pre-clinical testing of novel CAR targets and next generation CAR design that aims to inform human clinical trials.