Low-Dose Radiation Improves Anti-CD19 CAR T-Cells Efficacy in Diffuse Large B-Cell Lymphoma through Fas/FasL Pathway

Multiple anti-CD19 chimeric antigen receptor T-cell (CART19) products were approved to treat relapsed/refractory diffuse large B-cell lymphoma (R/R DLBCL). However, a significant subset of patients fails to respond or relapses after CART19 therapy. While most of the pivotal trials did not allow bridging therapy, many patients required radiation therapy to maintain disease control and palliate symptoms awaiting CART19 infusion. Retrospective reports showed the feasibility of this approach. However, it remains unknown if radiation therapy improves the efficacy of CART19 in DLBCL.

We sought to determine the impact of DLBCL low dose radiation (LDR) on cytotoxicity of CART19 cells, and potential mechanism of action.

We first assessed the direct effect of LDR on DLBCL cell lines, OCI-Ly10 (ABC-DLBCL) and SUDHL-4 (GCB-DLBCL) using 1Gy and 2Gy, respectively. We found no significant decrease in viability 24 hours after radiation assessed by flow cytometry (FC) when compared to non-irradiated control (mean ± SEM: OCI-Ly10: 85 ± 2% vs 96 ± 1%, SUDHL-4: 91 ± 0.6% vs 94 ± 1%, n=4). Because LDR did not affect cell viability, we chose these doses for our studies.

To evaluate LDR impact on CART19 cells cytotoxicity, we obtained healthy donor peripheral blood mononuclear cells and transduced purified T cells with a CD19.4-1Bb.3z lentiviral vector to obtain CART19 cells. We then CFSE-labeled OCI-Ly10 and SUDHL-4 cells (target cells). Then, irradiated the target cells at 1Gy and 2Gy, respectively, to later co-culture with CART19 cells or control T cells from the same donor (effector cells) at different effector-target ratios (5:1, 2.5:1 and 1:1) to assess cytotoxicity. The viability of the CFSE⁺ target cells was evaluated 16 hours later by FC using DAPI stain. We observed significant improvement (p <0.05) in CART19 cells cytotoxicity against irradiated target cells compared to non-irradiated cells (Figure 1). These results were confirmed in an OCI-LY10 xenograft model.

To elucidate the mechanism by which LDR enhanced CART19 cytotoxicity, we assessed the role of Fas/FasL signaling apoptosis pathway as activated T-lymphocytes, including CART cells, express FasL, which bind to the tumor-expressed Fas prompting apoptosis of the malignant cells. Thus, we measured changes of Fas expression in response to LDR in DLBCL cell lines. We irradiated multiple ABC-DLBCL cell lines (OCI-Ly10, SUDHL-2 and RC-K8) and GCB-DLBCL cell lines (SUDHL-4, SUDHL-5, SUDHL-6, OCI-LY1 and OCI-Ly4) at 1Gy/2Gy and 24 hours later we measured Fas expression by FC and RT-qPCR. We observed consistent upregulation of Fas in all irradiated DLBCL cell lines compared to non-irradiated controls (i.e., mean Fas fold change 0 to 1Gy; OCI-Ly10: FC 1.87, RT-qPCR 3.76; SUDHL-4: FC 1.19, RT-qPCR 1.77, n=3).

To better understand the dynamic change of Fas expression, OCI-Ly10 were irradiated at 1 Gy, Fas was measured by FC over multiple time points. We observed Fas upregulation started 3 hours after irradiation, peaked at 6 hours, and continued to be at same level after 24 hours.

To establish the link between Fas upregulation in irradiated DLBCL cell lines and enhanced CART19 cytotoxicity, we performed a cytotoxicity assay with OCI-Ly10 and Fas blocking antibody. The cells were stained with CFSE and incubated with Fas blocking antibody (2 μg/ml) for 1 hour before irradiation at 1Gy. OCI-Ly10 then co-cultured with CART19 for 16 hours. The CFSE⁺ OCI-Ly10 viability was then measured by FC. We observed that CART19 cytotoxicity was significantly impaired when Fas blocking antibody added to irradiated OCI-Ly10 (mean ± SEM: E:T 2.5:1, 47± 0.2% vs 54± 0.6%, p= 0.009, n=2). This highlights the critical role that Fas/FasL pathway plays in improving CART19 cytotoxicity after DLBCL cell lines radiation.

We describe (1) DLBCL low dose radiation improved the efficacy of CART19, (2) ABC/GCB DLBCL cell lines upregulated Fas expression in response to LDR, (3) pivotal role for Fas/FasL signaling pathway in boosting CART19 cytotoxicity against irradiated DLBCL cell lines.

These results provide proof of principle for potential use of LDR in the context of CART19 treatment for DLBCL. We are setting up a first-in-human phase I clinical trial to combine CART19 with LDR for R/R DLBCL to determine the safety of this novel combination, establish recommended radiation doses for a phase II study and generate preliminary efficacy data.

Martinet:Grifols: Research Funding. Boyer:BMS: Consultancy, Honoraria; CSL Behring: Consultancy, Honoraria, Research Funding; UCB: Consultancy, Honoraria, Patents & Royalties, Research Funding; Argenx: Consultancy, Honoraria, Research Funding; Egle Tx: Consultancy, Honoraria; OGD2: Consultancy, Honoraria. Galluzzi:Onxeo: Consultancy, Honoraria, Research Funding; AstraZeneca: Consultancy, Honoraria; OmniSEQ: Consultancy, Honoraria; The Longevity Labs: Consultancy, Honoraria; Inzen: Consultancy, Honoraria; Sotio: Consultancy, Honoraria; Noxopharm: Consultancy, Honoraria; EduCom: Consultancy, Honoraria; Luke Heller TECPR2 Foundation: Consultancy, Honoraria; Promontory: Consultancy, Current holder of stock options in a privately-held company, Honoraria, Research Funding; Lytix Biopharma: Research Funding; Boehringer Ingelheim: Consultancy, Honoraria. Formenti:MedImmune: Honoraria; EMD/Serono: Honoraria; Accuray: Honoraria; Eisai: Honoraria, Research Funding; Elekta: Honoraria; Janssen: Honoraria; Merck: Consultancy, Honoraria, Research Funding; Roche-Genentech: Consultancy; Viewray: Consultancy, Honoraria; AstraZeneca UK: Consultancy, Honoraria; Eli-Lilly, Janssen: Research Funding; Varian: Honoraria, Research Funding; Bristol Myers Squibb: Honoraria, Research Funding; Regeneron: Honoraria, Research Funding; Boehringer Ingelheim: Consultancy, Honoraria; Accuray: Consultancy. Guzman:BridgeMedicines: Research Funding; Samus Therapeutics: Other: Inventor on licensed IP; Seq RX: Current holder of stock options in a privately-held company.