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Long Read Isoform Sequencing as High-Throughput Approach in Advancing Neoantigen Discovery for Gastric Cancer Immunotherapy

Authors
Benedict LIAN Shi Xiang1, HUANG Kye Kiong1,3, Patrick TAN1,2,3,4

Affiliations

  1. Cancer and Stem Cell Biology Program, Duke-NUS Medical School, Singapore.
  2. Singapore Gastric Cancer Consortium, Singapore.
  3. Cancer Science Institute of Singapore, National University of Singapore, Singapore.
  4. Genome Institute of Singapore, Agency for Science, Technology and Research, Singapore.

Abstract
Immunotherapy-based immune checkpoint inhibitors (ICI) have become the mainstream treatment for cancer patients owing to their fantastic treatment outcomes. However, immunotherapy becomes ineffective for several reasons, including tumour heterogeneity and the immune-suppressive microenvironment in tumours. Neoantigen-based adoptive T-cell therapy has high specificity and could overcome the immune evasion mechanism in the tumour microenvironment. Unfortunately, the targetable neoantigens for various cancers are still very limited. Hence, high efficacy and scalable neoantigens are needed to improve the effectiveness of neoantigen-based immunotherapies and treatment resistance in cancer patients.

Previously, our group performed neoantigen prediction using the PacBio long-read Isoform sequencing (Iso-seq) on ten gastric cancer cell lines (Huang et al.,2021)*. The long-read Iso-seq method can define the full-length structure of a transcript, which could not be achieved by conventional short-read RNA sequencing. We have identified numerous novel transcript isoforms highly expressed in gastric cancer cell lines using the long-read Iso-seq database and mass spectrometry proteomics database for protein expression validation. The identified novel transcript with a high affinity to major histocompatibility complex (MHC) binding sites will be considered neoantigen candidates and screened by T-cell-based immunogenicity assay.

The immunogenicity of cytotoxic CD8⁺ T-cells is evaluated from the expression of cytokines that can kill cancer cells, such as IFN-γ and TNF-α. Several qualitative and quantitative methods will detect and quantify these cytokines, including flow cytometry assay, enzyme-linked immunosorbent assay (ELISA), and enzyme-linked immunosorbent spot (ELISpot). Lastly, the immunoreactive neoantigens and respective TCR repertoires are used to explore the adoptive cellular therapeutic strategy for gastric cancer patients. This research study will demonstrate the feasibility of a high-throughput sequencing approach to neoantigen discovery for gastric cancer, potentially accelerating the development of neoantigen-based immunotherapy for gastric cancer patients.

References
*Huang, K.K., Huang, J., Wu, J.K.L., Lee, M., Tay, S.T., Kumar, V., Ramnarayanan, K., Padmanabhan, N., Xu, C., Tan, A.L.K., Chan, C., Kappei, D., Göke, J., Tan, P., Long-read transcriptome sequencing reveals abundant promoter diversity in distinct molecular subtypes of gastric cancer, Genome Biology. 2021 Jan; 22(1):44. [PMID: 33482911]