Improved Anti-Tumor Response of Chimeric Antigen Receptor T Cell (CART) Therapy after GM-CSF Inhibition Is Mechanistically Supported By a Novel Direct Interaction of GM-CSF with Activated Carts

Abstract

<h3>Introduction</h3> Despite the remarkable activity of CD19 directed chimeric antigen receptor T cell (CART19) therapy in the treatment of B cell malignancies, limitations include neurotoxicity (NT) and cytokine release syndrome (CRS). Emerging literature suggests that inhibitory myeloid cells and their cytokines induce CAR-T cell toxicities and inhibit effector functions. GM-CSF was specifically identified as a critical cytokine in the development of NT and CRS. Neutralization of GM-CSF in preclinical models has been shown to prevent CRS, enhance CART anti-tumor activity, and reduce the presence of tumor associated macrophages. In addition, there appears to be a direct effect on CART19 cells. <h3>Objectives</h3> In this study, we aimed to evaluate the direct effect of GM-CSF on CART cells. <h3>Methods</h3> We used a guide RNA (gRNA) targeting exon 3 of GM-CSF in a CRISPR-Cas9 lentiviral vector to knock out GM-CSF during CART cell manufacturing. Edited and wildtype T cells and CART cells were sent for whole exome sequencing (WES) and RNA sequencing. We also used flow cytometry to assess the expression of GM-CSF receptors on CART cells, activated using CD3/CD28 beads or irradiated NALM6 cells. <h3>Results</h3> WES analysis revealed no significant difference in the single nucleotide variants or indel counts between GM-CSF^k/o and GM-CSF^wt CART19 (Fig 1A). WES was significant for only two alterations in the gene targeted by the gRNA (Fig 1B). This high efficiency and accuracy indicated that the improvement in CART function is unlikely related to an off-target effect of the gRNA and suggests a direct interaction between GM-CSF and CART cells as a potential mechanism behind the improved anti-tumor activity. Further investigation robustly indicates that while resting CART cells do not express any GM-CSF receptors, activated CART cells significantly upregulate both α and β subunits of the GM-CSF receptor. This finding was significant both when CART cells were activated through their T cell receptor (Fig 1C) or CAR (Fig 1D). This was also seen in GM-CSF^k/o CART19 cells, indicating this is specifically induced by T cell stimulation. These data suggest a direct interaction between GM-CSF and upregulated GM-CSFR on activated CART cells. Finally, we sought to uncover the downstream changes resulting from this effect. Transcriptome interrogation of GM-CSF^k/o CART19 revealed a distinct signature, including significant inhibition of the Fas death pathway (Fig 1E). This suggests a potential mechanism for enhanced CART19 activity following GM-CSF depletion. <h3>Conclusion</h3> Our results strongly indicate that CART cells increase expression of GM-CSF receptor subunits when activated, resulting in modulation of CART cell functions. Furthermore, GM-CSF^k/o CART19 revealed a distinct transcriptome signature compared to GM-CSF^wt CART19. These results illuminate a novel mechanism for a direct modulatory effect of GM-CSF on activated CART cells.