Poster Presentation 36th Lorne Cancer Conference 2024

Engineered receptors T cell fusion constructs (TRuC) and peptide-MHC-I targeting chimeric antigen receptor (CAR) on T cells form native T cell receptor-like immune synapse morphology and cytotoxicity. (#142)

Shiqi Wang 1 2 , Kylie Luong 2 , Hannah Huckstep 1 3 , Jian Ding 4 , Melinda Iliopoulos 2 , Kathy Watson 2 , Brad McColl 5 , Shereen Jabar 5 , Fiona Gracey 5 , Robert Tighe 4 , Ryan Cross 2 , Robert Hofmeister 4 , Alexandra Garnham 3 , Misty Jenkins 2 6
  1. Murdoch's Childrens' Research Institute, Parkville, VIC, Australia
  2. Immunology, Walter Eliza Hall Institute of Medical Research, Melbourne, VIC, Australia
  3. Bioinformatics, Walter Eliza Hall Institute of Medical Research, Melbourne, VIC, Australia
  4. TCR2 Therapeutics, Boston, Massachusetts , United States of America
  5. Myrio Therapeutics, Scoresby, VIC, Australia
  6. Biochemistry and Chemistry, La Trobe Institute of Molecular Science, Melbourne, VIC, Australia

Synthetic receptors such as Chimeric antigen receptors (CARs) and T cell fusion constructs (TRuCs), engineered into T cells to harness the cytotoxic functions of T cells towards to any tumour antigen of choice. There has been increasing interest in understanding how the receptor design could influence killing kinetics, persistence, and efficacy to improve and develop novel immunotherapies.

To evaluate the effectiveness our CARs and TRuCs, we studied the morphology of the immune synapse, the junction between the T cell and tumour cell, which consists of highly organised positioning of cytoskeletal and signalling components required for effective T cell cytotoxicity. Using laser scanning confocal microscopy and lattice lightsheet microscopy, we were able to visualise and interrogate the morphology of the T cell immune synapse and perform time lapse studies into T cell killing kinetics, measuring the time between T cell contact, signalling, degranulation, and target cell death.

We generated synthetic receptors against known tumour antigens in various formats, including surface antigen-targeting TRuCs and second-generation peptide-MHC-I (pMHC)-targeting and surface antigen-targeting CARs, to compare the morphology and functionality of CARs and TRuCs against the native T cell receptor (TCR).

We found that both pMHC-targeting CARs (Wang et al, 2021, Biomedicines) and TRuCs form a morphologically TCR-like immune synapse and TCR-like cytotoxicity, contrasting with CARs targeting surface antigen HER2 (Davenport et al, 2016, PNAS). Interestingly, despite other groups reporting transcriptional and functional differences between receptor design and choice of co-stimulation (Boroughs et al, 2020), mesothelin-specific CAR T cells with either 41-BB or CD28 co-stimulation domains both showed similar TCR-like immune synapse morphology and TCR-like killing kinetics, both effectively killing antigen-expressing tumour cells.

Our findings imply that both the synthetic receptor design and the tumour antigen play a role in the formation of the immune synapse, which directly contributes to T cell cytotoxicity. Further work is required to understand how the combination of the receptor and tumour antigen influences immune synapse formation and T cell killing kinetics, so that we could further optimise and improve our synthetic receptor designs to create more effective immunotherapies.

  1. Wang and Luong et al, A Novel Peptide-MHC Targeted Chimeric Antigen Receptor T Cell Forms a T Cell-like Immune Synapse, 2021, Biomedicines
  2. Davenport AJ et al, Chimeric antigen receptor T cells form nonclassical and potent immune synapses driving rapid cytotoxicity, 2016, PNAS