Abstract 560: Generation of tertiary lymphoid structures and CD3+ CD8+ CD56+ NKG2D+ CAR TaNK cells following subcutaneous injection of CD3-directed lentiviral-loaded PBMCs

Background: We have previously established the ability of CD3-directed lentiviral vectors encoding for CD19 or CD22 CARs to mediate robust anti-tumor immunity in humanized lymphoreplete mouse models. We now present additional mechanistic data for this novel subcutaneous (SC) CAR-T approach. Methods: Human PBMCs loaded with a self-inactivating lentiviral vector (LV) encoding CD19 CAR with a synthetic driver element were injected SC into autologous PBMC humanized NSG MHC I/II double knock out (DKO) mice. The LV was packaged with a modified envelope with the ability to target and activate CD3+ T cells. To first track the site of CAR formation in vivo following SC injection, PBMCs were loaded with an LV encoding a CD19 CAR and luciferase, and bioluminescence imaging (BLI) was performed. Additionally, histopathology of the site of injection and distal organs were examined. Evaluation for CAR+ cells was performed through immunohistochemistry and PCR detection. To examine the tropism of the CD3-directed CD19 CAR LV and characterize CAR+ cells, the phenotype of in vivo expanded CAR+ cells was evaluated. Further characterization of CAR+ cells was performed with in vitro studies. Results: Following SC injection of LV-loaded PBMCs, the first evidence of transgene expression utilizing BLI for luciferase was detected four to five days following SC injection. At the same time point, histologic examination of the SC site of injection revealed the formation of tertiary lymphoid structures (TLS) consisting of human CD8+ and CD4+ T cells, CD68+ macrophages, CD68+ dendritic cells, and a few CD20+ B cells. On day 13 post-SC injection, BLI detected the presence of CAR+ cells systemically beyond the site of injection and within subcutaneous Raji tumors implanted on the contralateral side. On day 14 post-SC injection, histologic examination showed sustained TLS within the SC tissue without signs of dermal acute inflammation or ulceration and evidence of CAR+ cells appearing in the spleen. CAR+ cells exhibited robust anti-tumor immunity with expansion into peripheral blood. CAR+ cells consisted of a distinct population of CD8+ T cells with NK-like features (TaNKs) and a CD3+ CD8+ CD56+ NKG2D+ cell phenotype. In vitro transduction of CD56 NK cell-depleted PBMC with the CD3-directed LV also led to CAR-TaNK formation. Conclusion: The subcutaneous injection of CD3-directed LV-loaded PBMCs leads to the formation of tertiary lymphoid structures at the site of injection and the developm