Flash Talk and Poster Presentation 36th Lorne Cancer Conference 2024

High affinity T-cell receptors against HLA-presented cancer antigens using single-cell RNA sequencing for precision T-cell receptor therapy. (#104)

Gwo Yaw Ho 1 2 , Peter Eggenhuizen 2 , Anh Doan 2 , Anusha Yellapragada 2 , Ting Yi Tian 2 , Qi Xiong 2 , Tracy Putoczki 3 , Belinda Lee 3 4 , Janet Chang 2 , Jessica Wu 2 , Holly Barker 3 , Jason Steen 2 5 , Justin Bedo 3 6 , Sophia Frentzas 1 2 , Cassandra Vandenberg 3 , Hanim Abd Halim 2 , Kylie Loh 2 , Paul Hertzog 7 , Sean Grimmond 4 8 , Tony Papenfuss 3 , Clare Scott AM 3 4 , Eva Segelov 2 9 , Tu Nguyen-Dumont 5 8 , Pouya Faridi 2 , Joshua Ooi 2
  1. Monash Health, Clayton, VIC, Australia
  2. School of Clinical Sciences, Monash University, Clayton , Victoria
  3. Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia
  4. Peter MacCallum Cancer Centre, Melbourne, VIC, Australia
  5. Central Clinical School, Monash University, Melbourne, Australia
  6. Department of Computing and Information Systems, University of Melbourne, Parkville
  7. Hudson Institute of Medical Research, Clayton, Victoria, Australia
  8. Department of Clinical Pathology, University of Melbourne, Parkville
  9. Faculty of Medicine University of Bern and Department of Radiation Oncology, Bern University Hospital, University of Bern, Switzerland

T-cell receptor therapy (TCR-T) is a cell-based immunotherapy that targets human leukocyte antigen (HLA)-presented neoantigens using naturally occurring TCRs, enabling targeted immune-activation at very low-antigen density [1]. This ability to target intracellular-antigens and activate immune responses at low-antigen levels gives TCR-T an advantage over chimeric antigen receptor therapy (CAR-T), especially in treating solid-tumours and cancers with limited surface antigens [2]. TCR-T requires identification of cancer-specific HLA-presented tumour neoantigens (TNA) and corresponding high-affinity TCRs against these HLA-presented TNAs. Our team has developed an integrated multi-omics TNA discovery platform, combining immunopeptidomics, genomics, and immunologic techniques. We successfully identified an immunogenic mutant HLA-presented TNA in aggressive immunologically "cold" ovarian cancer with low tumour mutational burden: neuropeptide W E100Q (NPW_E100Q), restricted to HLA-A*33:03.

Using single-cell RNA sequencing (scRNAseq), we discovered three high-affinity TCRs against NPW_E100Q in context of HLA-A*33:03. First, we optimised the antigen presenting cell (dendritic cell; DC) and CD8+ T-cell co-culture conditions (14-day culture with IL-7/IL-15/IL-21) to selectively expand NPW_E100Q-specific CD8+ T-cells, utilising HLA-matched healthy donor blood. We demonstrated that CD8+ T-cells expanded in vitro in the presence of DC pulsed with NPW_E100Q peptide showed increased NPW_E100Q-specific CD8+ T-cell proliferation, identified by the presence of NPW_E100Q-specific dextramer staining compared to wild-type NPW dextramer. The proliferated CD8+ T-cells were fluorescence-activated cell sorted. Subsequent scRNAseq revealed 1,023 paired α/β-TCR clonotypes from the CD8+ T-cells co-cultured with NPW_E100Q peptide-pulsed DCs; showing high specificity with <28 TCRs shared between NPW_E100Q and wild-type NPW stimulations. High-affinity TCRs were identified as the α/β-TCR pairs of the most abundant clonotypes. TCRs with cross-reactivity against wild-type NPW and expression of TCR gamma chains were excluded. Lastly, we confirmed the specificity of T-cell responses by AIM (Antigen Identification by MHC) analysis. We are now in the process of engineering precision TCR-T onto donor CD8+ T-cells for in vivo cytocidal testing.

In conclusion, we confirmed our ability to identify immunogenic HLA-presented TNAs and their corresponding high-affinity TCRs. We plan to expand this study into a cohort of well-curated pancreatic biospecimens with matched genomic and clinical data. This work has the potential to revolutionise the solid tumour treatment landscape with precision cell-based immunotherapy.

  1. Estelle Baulu et al., TCR-engineered T cell therapy in solid tumors: State of the art and perspectives.Sci. Adv. 9, eadf3700(2023).DOI:10.1126/sciadv.adf3700
  2. Tsimberidou, AM., Van Morris, K., Vo, H.H. et al. T-cell receptor-based therapy: an innovative therapeutic approach for solid tumors. J Hematol Oncol 14, 102 (2021). https://doi.org/10.1186/s13045-021-01115-0