Background ITIL–306 is a genetically engineered autologous TIL cell therapy that amplifies TCR–specific antigen recognition (Signal 1) with an FRα-specific CoStimulatory Antigen Receptor (CoStAR; Signal 2).1 T-cell activation through the endogenous TCR is dependent on the concentration of cognate peptide-MHC antigen. This study examined T-cell activation across a range of physiologically relevant FRα expression levels and characterized whether functional T-cell avidity (response to cognate antigen concentration) was impacted with CoStAR engagement.
Methods In vitro cocultures were used to determine T-cell response to variations in strength of Signal 1 and Signal 2. To evaluate the role of FRα on amplification of T-cell responses, stable cell lines expressing membrane-anchored OKT3 and different FRα levels were established. Healthy donor (HD) T cells transduced with anti–FRα CoStAR or non-transduced (control) were used as effector cells. Cytolytic activity and cytokine levels (IL-2, IFN-γ, TNF-α) were assessed.
To assess the effect of CoStAR on TCR functional avidity, HD T cells were non-transduced or transduced with a defined TCR recognizing HLA-A*02/MART-1 antigen, anti–FRα CoStAR, or both. Parental T2 or FRα-transduced T2 cells were used as targets. Target cells were pulsed with titrated concentrations of 4 different MART-1–altered peptide ligands of varying antigenicity. Cytokine secretion after coculture was measured, and antigen half-maximal effective concentration (EC50) was calculated.
Results CoStAR amplified T-cell responses at all FRα expression levels. IL-2 secretion was significantly higher at any FRα expression level versus no FRα (P<.0001). CoStAR-transduced and non-transduced T cells were not activated in coculture with cells expressing any level of FRα alone. Kinetic activation studies demonstrated that engaging CoStAR (Signal 2) followed by TCR activation (Signal 1) at a later time resulted in amplified T-cell activity.
Cytokine secretion was increased from MART-1−TCR+CoStAR T cells versus MART-1−TCR T cells when cocultured with T2-FRα cells pulsed with titrated concentrations of all cognate peptides evaluated. EC50 was not impacted by CoStAR for cognate peptides with EC50 between 10-10 to 10-7 M.
Conclusions CoStAR augmented T-cell function across a range of physiologically relevant FRa expression levels and TCR/cognate peptide affinities. TCR/cognate antigen affinity (EC50) was unchanged by CoStAR, suggesting that CoStAR TIL will have identical specificity as unmodified TIL. Further, CoStAR improved T-cell function at low FRa expression levels, supporting the evaluation of ITIL-306 activity across multiple tumors, including those with low FRα expression. These results are being explored in a first-in-human clinical study with ITIL-306 (NCT05397093).
Acknowledgements The authors would like to thank Akshata Udyavar, MS, PhD; Sujita Sukumaran, PhD; Clare Yarka, MS; Stella Ouyang, MS; Owen Moon, PhD, BSc Hons; and Michael King, PhD, for help with experimental execution and abstract writing. Medical writing support was provided by Nexus Global Group Science, with funding from Instil Bio, Inc.
Sukumaran S, Kalaitsidou M, Mojadidi M, et al. Costimulatory antigen receptor (CoStAR): a novel platform that enhances the activity of tumor infiltrating lymphocytes (TILs). J Immunother Cancer . 2021;9(Suppl 2):198.
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