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423 Single cell RNA sequencing reveals functionally validated signatures of cytotoxicity in anti-FRα CoStimulatory Antigen Receptor (CoStARTM) activated CD4+ T cells
  1. Katrina Sherbina Baklan1,
  2. Martina Sykorova1,
  3. Leyuan Bao1,
  4. Owen Moon2,
  5. Robert E Hawkins1,
  6. Mark Dudley3,
  7. Gray Kueberuwa1,
  8. Jinzhou Yuan1 and
  9. John S Bridgeman2
  1. 1Instil Bio, Dallas, TX, USA
  2. 2Instil Bio, Macclesfield, UK
  3. 3Instil Bio, Washington, DC, USA
  • Journal for ImmunoTherapy of Cancer (JITC) preprint. The copyright holder for this preprint are the authors/funders, who have granted JITC permission to display the preprint. All rights reserved. No reuse allowed without permission.


Background ITIL-306 is a genetically engineered autologous TIL cell therapy that amplifies TCR-specific antigen recognition signals (Signal 1) with an FRα-specific CoStimulatory Antigen Receptor (CoStAR; Signal 2).1 However, the transcriptional effects which underlie this amplification are as yet unclear. This study set out to examine the gene expression profile of activation of T cells through CoStAR in a comprehensive manner.

Methods T cells from three healthy donors were engineered to express a high affinity HLA-A*02/CEA specific TCR and/or a FRα-specific CoStAR and subsequently enriched for expression. T cells were cocultured with target cell lines presenting the TCR-specific peptide plus CoStAR target antigen (H508-FRα) before processing of samples through 10X genomics 5’ GEX scRNA-seq workflow. Bioinformatic analysis was performed to compare TCR and/or CoStAR+ populations, as well as CD4+ v CD8+. Functional validation of gene sigantures was performed using cytokine and chemokine analysis by bead arrays and multiplexed analyte analysis (Mesoscale Discovery), as well as flow cytometric staining and xCELLigence cytotoxicity assays.

Results A custom gene reference was created and could successfully quantify scRNA-seq reads of anti-FRα-CoStAR, as well as the recombinant TCR introduced into the T cells. TCR+CoStAR signaling resulted in increased expression of genes associated with activation and a cytotoxic phenotype in CD4+ cells compared to TCR alone signaling. TCR+CoStAR signaling resulted in increased expression of genes associated with a less differentiated phenotype and cytotoxicity and decreased expression of genes associated with exhaustion in CD8+ cells compared to TCR alone signaling. A handful of genes – predominantly costimulatory receptors – were upregulated following CoStAR signalling alone in both CD4+ and CD8+ T cells.

The cytotoxicity signature in CD4+ T cells was confirmed using xCELLigence cytotoxicity assay, with TCR+CoStAR signaling associated with enhanced cytotoxicity compared to TCR signaling alone. Cytokine and chemokine data for several key effector molecules was also concordant with gene expression data, with flow cytometry confirming upregulation of costimulatory receptors in CD4+ T cells upon CoStAR engagement alone.

Conclusions CoStAR provides functional benefit to both CD4+ and CD8+ T cells in overlapping and distinct ways. Enhancement of CD4+ cytotoxicity by CoStAR was an unexpected, yet intriguing observation which strengthens the rationale for CoStAR engineering approaches for TIL therapy, where CD4+ T cell-mediated anti-tumour responses have been identified as potential drivers of cancer regression in patients.


  1. 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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