TCR-Engineered Lymphocytes Targeting NY-ESO-1: In Vitro Assessment of Cytotoxicity against Tumors

Abstract

Adoptive T-cell therapies tailored for the treatment of solid tumors encounter intricate challenges, necessitating the meticulous selection of specific target antigens and the engineering of highly specific T-cell receptors (TCRs). This study delves into the cytotoxicity and functional characteristics of in vitro-cultured T-lymphocytes, equipped with a TCR designed to precisely target the cancer-testis antigen NY-ESO-1. Flow cytometry analysis unveiled a notable increase in the population of cells expressing activation markers upon encountering the NY-ESO-1-positive tumor cell line, SK-Mel-37. Employing the NanoString platform, immune transcriptome profiling revealed the upregulation of genes enriched in Gene Ontology Biological Processes associated with the IFN-γ signaling pathway, regulation of T-cell activation, and proliferation. Furthermore, the modified T cells exhibited robust cytotoxicity in an antigen-dependent manner, as confirmed by the LDH assay results. Multiplex immunoassays, including LEGENDplex™, additionally demonstrated the elevated production of cytotoxicity-associated cytokines driven by granzymes and soluble Fas ligand (sFasL). Our findings underscore the specific targeting potential of engineered TCR T cells against NY-ESO-1-positive tumors. Further comprehensive in vivo investigations are essential to thoroughly validate these results and effectively harness the intrinsic potential of genetically engineered T cells for combating cancer.

Keywords: NY-ESO-1; T-cell activation; TCR; TCR-T cells; cellular cytotoxicity.

Figure 1

The scheme of the gamma retroviral vector (MMLV) encoding a T-cell receptor specific to the NY-ESO-1 epitope. The vector incorporates key components, including long terminal repeat (LTR), TCRβ (T cell receptor chain beta) ①, TCRα (T cell receptor chain alpha) ②, and P2A (self-cleaving peptide sequence).

Figure 2

Retroviral transduction estimation of the anti-CD3 primed PBMCs by flow cytometry.

Figure 3

HSNE plots of the transduced T cells, after the co-cultivation with tumor cell lines, with a flow cytometry data markers’ overlay: clusters are color-labeled in accordance with the heatmap.

Figure 4

Box plot analysis of transduced T cell population clusters following NY-ESO-1-expressing SK-MEL-37 cell line cultivation.

Figure 5

Volcano plot of differentially expressed genes. Orange dots depict upregulated genes, purple dots depict downregulated genes. Black dots represent non-significantly differentially expressed genes.

Figure 6

Ring plot of the gene set enrichment analysis of the upregulated genes.

Figure 7

Analysis of LDH-measured cytotoxicity in the SK-Mel-37, NW-Mel-38, and HCT-116 cell lines, *** stands for q-value < 0.0005, ** stands for q-value < 0.001, (+) stands for the presence of the NY-ESO-1 expression, (−) stands for the absence of the NY-ESO-1 expression.

Figure 8

Bar plot of the cytokines secreted by transduced T cells following co-cultivation with tumor cell lines expressing the target antigen (SK-Mel-37). Each bar’s height represents the median levels of cytokines, and the error bars indicate the interquartile range. Significance levels are indicated by asterisks, with *** denoting a p-value < 0.001, ** representing a p-value < 0.01, and * indicating a p-value < 0.05.