Discovery of Tumor-Specific T Cell Receptors Through Single-Cell Sequencing of Tumor-Infiltrating Lymphocytes in Melanoma

Çelik E., Çakıcı B., Uysak B. N., Karaçay M., Yağcıoğlu B., Özarı S., ...More

6th International Molecular Immunology & Immunogenetics Congress (MIMIC-VI), İstanbul, Turkey, 27 - 30 April 2025, pp.15, (Summary Text)

  • Publication Type: Conference Paper / Summary Text
  • City: İstanbul
  • Country: Turkey
  • Page Numbers: pp.15
  • Bursa Uludag University Affiliated: Yes

Abstract

Objective: T cell receptor (TCR)-based immunotherapies have emerged as a promising strategy for

cancer treatment, particularly in melanoma, where tumor-infiltrating lymphocytes (TILs) play a

crucial role in antitumor immunity. TIL therapy has recently received FDA approval for treating

advanced melanoma; however, its clinical application remains practically challenging.

Our study aims to identify tumor-specific TCRs by performing single-cell RNA (scRNA-seq) and TCR

sequencing on melanoma-derived TILs. By characterizing TCR repertoires and their antigen

specificity, we seek to develop a framework for enhancing personalized TCR-based therapies,

including TCR-NK cell therapies, to improve immunotherapy in melanoma.

Materials-Methods: Melanoma tumor samples were obtained from eleven patients undergoing

surgical resection. TILs from two patients were isolated and expanded for scRNA-seq and paired

TCRα/β sequencing to identify clonally expanded T cells with tumor-reactive properties.

Additionally, CD8⁺ T cells isolated from peripheral blood were co-cultured with tumor cells to grow

tumor-reactive clones and subjected to scRNA-seq. Computational analysis was performed to

classify TCR clonotypes based on frequency and their potential tumor reactivity.

Results: scRNA-seq revealed a clonally expanded TCR repertoire with potential effector signatures.

For the dominant clonotypes, a detailed analysis of TCR VDJ sequences was carried out and paired

TCRα/β sequences including CDR and constant region sequences were extracted. A 3rd generation

lentiviral vector was designed for the expression of novel TCRs, incorporating a 2A-peptide based

design for the paired expression of TCRα/β sequences.

Conclusion: Our study successfully identified tumor-specific TCRs from melanoma-derived TILs and

peripheral CD8+ T cells using scRNAseq. Functional validation of discovered sequences using TCRNK

technology is ongoing. Our approach seems to be feasible in establishing a functional pipeline

for discovery of novel tumor-specific TCRs. These findings provide a foundation for TCR-based

immunotherapies, potentially leading to novel personalized treatments such as TCR-NK for

melanoma patients.