Background ImmTAC molecules are TCR-anti-CD3 bispecific T cell engagers that redirect and activate polyclonal T cells against tumor cells. Brenetafusp, a PRAME-targeting ImmTAC, showed robust disease control and tumor reduction in a Phase1 study (NCT04262466;^{1 2})in HLA-A*02:01+ patients. A blood T cell fitness signature (TCF) was associated with clinical benefit from brenetafusp.² Here we explored TCF at the single cell level to identify its biological attributes.

Methods Bulk and single cell RNAseq were conducted on blood and PBMC respectively prior to brenetafusp treatment (NCT04262466) (n=16 patients). TCF was determined from bulk RNAseq based on mean expression of TESPA1, CD28 and GRP183 in blood at baseline. Patients were stratified by median TCF. Seurat R package was used for scRNAseq analysis. Mann Whitney U and ANOVA tests were used to compare 2 or multiple groups respectively.

Results Presented here are limited to those with p<0.05. 106178 cells were analyzed by scRNAseq. Clustering analysis identified 24 distinct subsets including CD4 and CD8 T cell populations of naïve (3.2%, 1.4% of total cells respectively), stem cell memory (SCM, 11%, 1.6%), central memory (CM, 3.5%, 0.7%), effector memory (EM, 20.6%, 3.1%) and terminal effector CD8 T cells (TE, 9.9%). TCF was primarily driven by naïve and memory CD4 and CD8 T cells but not TE T cells. Patients with high TCF exhibited CD4 T cells with higher levels of the co-stimulatory molecules CD2, CD28, and CD40L (17%, 22%, 32% higher) compared to patients with low TCF. CD40 gene expression by B cells was 17% higher in TCF high patients compared to TCF low patients. Memory CD8 T cells from TCF high patients exhibited 68% higher expression of CXCR3, a chemokine receptor that enables T cell migration into tumors in response to inflammatory chemokines. Conversely, naïve and SCM CD8 T cells from TCF low patients expressed 90% higher levels of CISH, a negative regulator of TCR signaling.

Conclusions A TCF gene signature, which was associated with clinical benefit from ImmTAC T cell engagers, reflects T cells with high stemness and self-renewal capacity combined with increased co-stimulatory receptors and higher tumor homing potential. An active CD40/CD40L axis was associated with high TCF which is consistent with the hypothesis that epitope spread contributes to long term benefit from ImmTACs.

References

1. Hamid O, et al. Results from phase I dose escalation of IMC-F106C, the first PRAME × CD3 ImmTAC bispecific protein in solid tumors. Annals of Oncology. 2022;33(suppl_7):S331-S355.

2. Hamid O, et al. Phase 1 safety and efficacy of IMC-F106C, a PRAME × CD3 ImmTAC bispecific, in post-checkpoint cutaneous melanoma (CM). Journal of Clinical Oncology. 2024;42(suppl 16):9507.