SMARCA4 deletion enhances CAR T cell cytotoxicity and persistence against cancer Free

Chimeric antigen receptor (CAR) T therapy is now used in second and later lines of treatment in patients with B-cell lymphoma and leukemias, and multiple myeloma. However, patient response rates remain imperfect, with over 60% of patients failing to respond long term. Several preclinical and clinical studies have shown that enrichment of less-differentiated T cells in the CAR T cell infusion product is correlated with prolonged CAR T cell persistence and better patient outcomes. A major challenge facing autologous CAR T cell therapy is the lack of early-differentiated (i.e., stem-like and memory) T cell phenotypes in baseline patient apheresis material that further worsens after CAR T manufacturing and infusion. Therefore, inhibiting drivers of T cell differentiation could improve CAR T cell products and long-term anti-tumor activity.

T cell differentiation is regulated in part by the SWItch/Sucrose-Non-Fermentable (SWI/SNF) family of chromatin remodeling complexes. Within SWI/SNF complexes, the ATPase module SMARCA4 slides and evicts nucleosomes to increase chromatin accessibility at target loci, thereby promoting transcriptional changes and enabling differentiation. We therefore hypothesize that deletion of SMARCA4 will delay differentiation of CAR T cells, resulting in greater long-term anti-tumor efficacy.

To test this hypothesis, we knocked out SMARCA4 in healthy donor T cells using CRISPR/Cas9 prior to activation and manufacture of CAR19-BBz CAR T cells (CART19). SMARCA4 knockout (KO) and wildtype (WT) CART19 were subsequently expanded for 17 days, and their differentiation was characterized by flow cytometry. During manufacturing, SMARCA4 KO CART19 cells had a higher proportion of CD8⁺ T cells compared to WT CART19 (p=0.0078, paired t-Test), despite both groups starting with a 1:1 mixture of CD8⁺:CD4⁺ T cells. Moreover, we observed that the SMARCA4 KO CD8⁺ CART19 cells had increased co-expression of the stem-cell-memory-like markers CD45RA, CCR7, CD27, and CD95 (p=0.0016, paired t-Test), as well as the transcription factor TCF1 (p=0.0120, paired t-Test).

After manufacturing, we challenged CART19 cells with coculture in vitro against the OCI-Ly18 cell line, a model of diffuse large B-cell lymphoma (DLBCL), stably expressing GFP and luciferase. In a transient tumor challenge, SMARCA4 KO CART19 cells demonstrated improved 72-hour cytotoxicity at both 0.125:1 and 0.0625:1 Effector:Target ratios (p=0.0131 and p=0.0348, paired t-Test), as measured by tumor luciferase activity. Additionally, SMARCA4 KO CART19 cells achieved a higher frequency of IFNγ production after PMA-ionomycin stimulation, with a 1.5-fold increase in the percentage of CART19 cells able to express IFNγ compared to WT CART19 (p=0.0001, paired t-Test). We then tested the SMARCA4 KO CART19 cells against a repeat OCI-Ly18 coculture, where fresh tumor cells were added every 72 hours to mimic chronic antigen stimulation. Despite no difference in killing after seven rounds of repeat OCI-Ly18 coculture, the SMARCA4 KO CART19 cells maintained a 1.9-fold higher frequency of IFNγ expression over WT (p=0.0058, paired t-Test).

Additionally, we found that SMARCA4 KO CART19 cells improved survival in vivo against the OCI-Ly18 xenograft model of DLBCL. Immunodeficient mice were implanted subcutaneously with 5 million OCI-Ly18 tumor cells. After seven days of engraftment, mice were treated with 4 million CART19 cells. The SMARCA4 KO CART19-treated mice demonstrated improved survival (6/6 OS) over those treated with WT CART19 (2/6 OS) (p=0.0179, log-rank test, holm corrected).

In summary, we found that SMARCA4 deletion resulted in less differentiation during CART19 manufacturing, more robust anti-tumor cytotoxicity in vitro, and long-term tumor control in vivo. These results highlight the emerging role of chromatin remodelers in modulating T cell effector functions and the potential for targeting SMARCA4 to improve CAR T cell therapy.