Abstract
Abstract BACKGROUND The CD20 antigen is an attractive immunotherapy target for B cell non-Hodgkin lymphomas, and adoptive transfer of T cells genetically modified to express a chimeric antigen receptor (CAR) targeting CD20 is a promising strategy. A theoretical limitation of this approach is that residual serum rituximab from prior chemoimmunotherapy regimens might block CAR binding to CD20 and prevent T cell mediated anti-lymphoma responses. However, previous data from our group and others have suggested that CD20 CAR+ T cell function is only partially blocked by anti-CD20 antibody (Ab), and T cell function in the setting of anti-CD3 x anti-CD20 bispecific Ab is not blocked by rituximab levels of up to 100 μg/ml. We have further tested the impact of various concentrations of rituximab on CD20-CAR T cell activity in vitro and in vivo. METHODS CD3+ T cells (proliferation and cytokine assays) or CD8+ selected T cells (cytotoxicity assays) were isolated from healthy donors, activated with anti-CD3/CD28 beads, and transduced with epHIV7 lentiviral vectors encoding 2nd or 3rd generation anti-CD20 CAR constructs (Leu16-28-ζ, Leu16-28-BB-ζ, or fully human 1-5-3-NQ-28-BB-ζ). Functional assays, performed using target cells pre-incubated for 30 min. with varying concentrations of rituximab, included a CFSE assay to assess CAR T cell proliferation, Luminex assays for cytokine secretion, and a 5-hour standard 51 chromium release assay for cytotoxicity. Target cells included K562 cells transduced to express CD80 with or without CD20 (denoted "K80" and "K80-20"), Raji, Daudi, Granta, Rec-1, and FL-18 lymphoma cells. K80-20 cell lines expressing low, medium, and high CD20 were established by limiting dilution cloning. For in vivo experiments, NOD/SCID/γ-/- (NSG) mice were inoculated i.v. with rituximab-resistant Raji-ffLuc lymphoma cells. After 5 days, rituximab was administered i.p. at 25 μg/ml or 200 μg/ml, and then at 24 hours after rituximab administration CAR+ central memory T cells expressing the 1-5-3-NQ-28-BB-ζ vector were injected i.v. Tumor growth was measured with bioluminescence imaging twice weekly and mice were followed for survival. RESULTS The availability of CD20 binding sites on Ramos lymphoma cells pre-incubated with various concentrations of rituximab was assessed with flow cytometry, and as expected, we found a dose-dependent blockade of CD20, with complete blockade at 50 μg/ml rituximab at 4°C. However, when anti-CD20-PE was incubated at 37°C, low-level CD20 binding could occur even at 200 μg/ml of rituximab. Despite the low number of available CD20 binding sites after rituximab, proliferation of CFSE-labeled CAR+ T cells was largely unimpaired in rituximab concentrations up to 200 μg/ml. In contrast, cytokine secretion was impaired in a dose-dependent manner, although even at 100 μg/ml of rituximab, interferon-γ, interleukin-2, and tumor necrosis factor a were still produced at 34-51%, 70-92%, and 79-108% of baseline levels, respectively. Cytotoxicity also decreased with increasing rituximab concentration but >75% of baseline cytolytic activity was retained at 100 μg/ml. We hypothesized that the level of CD20 expression on target cell lines might impact sensitivity to rituximab blockade. Using K80-20 cells with low, medium, or high CD20 expression we found that cytokine secretion and cytotoxicity (but not proliferation) were highly impaired upon stimulation with CD20low target cells, whereas T cell function remained completely intact when CD20high cells were used as targets. In vivo, mice bearing rituximab-refractory Raji-ffLuc tumors experienced only slight delay of tumor growth when treated with either low or high doses of rituximab, and mice treated with T cells alone had significant clearance of tumor. In mice that received low or high-dose rituximab prior to T cell infusions, tumor rejection and survival prolongation were equivalent to or better than that observed with mice receiving T cells alone (see figure). CONCLUSION We have shown that the in vitro and in vivo activity of CD20-targeted CAR T cells is minimally impacted after rituximab, despite a low number of available CD20 binding sites. These data suggest that residual serum rituximab levels will not present a significant impediment to CD20-targeted CAR therapy in patients who have received rituximab-containing chemotherapy regimens. Download : Download high-res image (96KB) Download : Download full-size image Figure 1 . Disclosures Jensen: Juno Therapeutics: Equity Ownership, Patents & Royalties, Research Funding. Gopal: Merck: Research Funding; BioMarin: Research Funding; Seattle Genetics: Consultancy, Honoraria; Gilead: Consultancy, Research Funding; Spectrum: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Piramal: Research Funding; Millenium: Honoraria, Research Funding; BMS: Research Funding; Janssen: Consultancy; Emergent/Abbott: Research Funding; Sanofi-Aventis: Honoraria. Riddell: Juno Therapeutics: Equity Ownership, Patents & Royalties, Research Funding; Cell Medica: Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Consultancy. Till: Pfizer: Research Funding; Roche/Genentech: Research Funding.