Background Immune mobilising TCRs against cancer (ImmTAC) are a novel class of soluble bispecifics which bind both CD3 on T cells and specific peptide/MHC antigen on tumour cells.¹ Four ImmTAC molecules are currently in clinical trials, one of these being tebentafusp which targets a gp100-derived HLA-A*02:01 peptide. Tebentafusp has demonstrated monotherapy activity in cutaneous and uveal melanoma.^{2 3} Here, we explored approaches to enhance ImmTAC-mediated T cell activation and anti-tumour activity. We hypothesised that the cytokine IL-2, which is approved for treatment of cutaneous melanoma and renal cell cancer, might augment ImmTAC-mediated T cell responses.⁴

Methods Tumour cell killing was determined by IncuCyte assay. Cell lines, healthy donor PBMC, tumour cell populations and cytokines including IL-2 (Proleukin, Novartis) were sourced commercially. ImmTAC molecules recognising peptides from either gp100 or the tumour antigen PRAME⁵ were used for in vitro assays. Surface marker expression was determined by flow cytometry.

Results Clinically relevant IL-2 levels (60 to 600U/ml) improved both the speed (maximum killing after ~15 hours following 60 U/ml IL-2 pre-treatment versus ~30 hours without IL-2) and potency (mean EC50 13.6pM with IL-2 versus 81.2pM in absence of supplementation) of antigen-specific ImmTAC-dependent tumour killing in vitro by either PBMC or purified T cell populations. Addition of IL-2 was sufficient to improve killing responses when either T cell numbers or target peptide:HLA levels were limiting, and IL-2-cultured TILs exhibited ImmTAC-dependent killing. Dose-dependent cytokine enhancement of ImmTAC-mediated killing response was also consistent with increased surface CD3 and CD69 expression on T cell populations. Notably, weak PBMC donor populations with minimal intrinsic killing activity could be induced to provide robust ImmTAC-dependent tumour cell killing responses by physiological IL-2 treatment.

Conclusions These results suggest that the anti-tumour activity of ImmTAC molecules could be enhanced by combination treatment with the cytokine IL-2.

Ethics Approval The study was approved by the South Central - Oxford A Research Ethics Committee (UK), REC reference 13/SC/0226

References

1. Lowe, et al. Cancer Treat Rev 2019. Vol.77, p35–432.

2. Middleton, et al. ( 2019) J. Clin. Oncol. 37, no. 15_suppl (May 20, 2019).

3. Shoustari, et al. SITC poster 2019, J. Immunother Cancer 2019;7.

4. Tang and Harding Cytokine: X 2019

5. Moureau, et al. ( 2020) AACR poster