(P 247) SINGLE-CELL RNA SEQUENCING OF AFAMITRESGENE AUTOLEUCEL INDICATES MECHANISMS OF PERSISTENCE AND ANTI-TUMOR ACTIVITY

Objective: Afamitresgene autoleucel (afami-cel) is an engineered T-cell receptor (TCR) T-cell therapy targeting tumors expressing melanoma-associated antigen (MAGE) A4 and is the first such therapy with advanced FDA approval for a solid tumor. In the Phase 2 SPEARHEAD-1 trial in patients with advanced synovial sarcoma or myxoid (with/without round cell) liposarcoma, greater long-term afami-cel pharmacokinetic exposure was associated with clinical benefit (D’Angelo S, et al. Lancet 2024). We explored gene expression profiles and functional characteristics of afami-cel manufactured product (MP) associated with long-term persistence.

Methods: Afami-cel MP were flow sorted into transduced (t)CD4+ and tCD8+ subpopulations. Single-cell RNA sequencing was performed on afami-cel tCD4+ and tCD8+ MP subpopulations from 22 and 23 patients, respectively, using 10X Genomics single-cell 5’ technology. In vitro cytotoxicity assays measured killing of a target HLA-A2/MAGE-A4–expressing tumor cell line over 125 hours co-culture with afami-cel MP subpopulations. qPCR specific to the engineered TCR measured afami-cel persistence in patient peripheral blood, with pharmacokinetic exposure calculated as area under the curve over 0–6 months after infusion.

Results: We saw distinct gene expression profiles associated with greater pharmacokinetic exposure in patients. In tCD8+ MP samples, this was characterized by an effector function and long-lived memory formation expression profile, whereas expression of genes involved in T-cell dysfunction was associated with lower pharmacokinetic exposure. In tCD4+ MP samples, higher exposure was associated with expression of T-cell activation genes and lower exposure was associated with pro-apoptotic, anti-proliferative gene expression. Afami-cel tCD8+ MP samples demonstrated strong cytotoxicity in vitro and sustained cytotoxic activity was associated with greater long-term pharmacokinetic exposure. Loss of initial cytotoxic activity was associated with a higher percentage of cells expressing a T-cell dysfunction–like gene profile. Greater in vitro cytotoxicity of afami-cel tCD4+ T-cells was associated with CD4+ T-cell activation and effector gene expression.

Conclusion: We hypothesize that MP with T-cell subsets poised for sustained effector function and long-lived memory formation are capable of longer post-infusion persistence, sustaining higher afami-cel pharmacokinetic exposure, and will discuss the gene expression programs underlying these different cellular potentials.