IDENTIFICATION AND UTILIZATION OF TCRS AGAINST RECURRENT MUTATIONS IN UVEAL MELANOMA

​

Ortal Ariel-Cohen (1),  Dragoslav Zikich (1), Ofer Dotan (1), Abraham Nissani (1), Li-at Zeltzer (1), Shai Killim (1), Adva Kubi (1), Amihai Lieberman (2,3), Anat Shemer (2), Neta Zukerman (4), Gal Cafri (2), Ronnie Shapira-Frommer (1), Orit Itzhaki (1) and Yochai Wolf (1,5)  

 

(1) Ella Lemelbaum Institute for Immuno-Oncology, Sheba Medical Center, Tel-Hashomer, Israel (2) Immunotherapy and Genetic Engineering Lab, Cancer Research Center, Sheba Medical Center, Tel-Hashomer, Israel
(3) BMT Processing laboratory, Sheba Medical Center, Tel-Hashomer, Israel.
(4) Central Viral laboratory, Ministry of Health, Sheba Medical Center, Tel-Hashomer, Israel
(5) Department of Pathology, Faculty of Medicine, Tel Aviv University, Tel-Hashomer, Israel 

 

Background: Uveal melanoma (UM) is a rare cancer arising from uveal melanocytes. Unlike cutaneous melanoma, UM exhibits a limited response to immune checkpoint blockade (ICB). Over 95% of UM cases harbor recurrent mutations in GNAQ or GNA11, particularly at codon Q209. Despite their prevalence, immunogenic neoantigens derived from these mutations have not yet been identified.

 

Objectives: Identify short peptides from GNAQ/GNA11 Q209 mutations as potential neoantigens.
Isolate and validate neoantigen-reactive T-cell receptors (TCRs) from tumor-infiltrating lymphocytes (TILs).
Clone the identified TCRs into naïve T cells and assess their reactivity.

 

Methods: TILs from uveal melanoma patients were co-cultured overnight with autologous melanoma cells and analyzed by flow cytometry for activation markers (CD4, CD8, 4-1BB, OX40). Activated TILs were sorted for RNA extraction and TCR library preparation using the SMARTer Human TCR α/β Profiling Kit, followed by Illumina MiSeq sequencing. Cytokine levels in co-culture supernatants were measured using an MSD immunoassay. A 9-mer peptide library encompassing GNAQ/GNA11 Q209 mutations was synthesized. Peptide-HLA binding was predicted by NetMHCpan 4.1. U-Load dextramers were loaded with MHCI-peptide monomers and used to stain TILs to identify antigen-specific TCRs.

 

Results: TILs co-cultured with autologous melanoma cells showed upregulation of the activation markers 4-1BB and OX40, along with increased cytokine secretion. TCR sequencing of activated TILs identified dominant TCR clones in all five patients. One mutated 9-mer peptide demonstrated strong binding to HLA-C*06:02 using Immudex U-Load easYmers, indicating potential neoantigen specificity. Furthermore, dextramer staining with this peptide revealed positive staining in one TIL sample, suggesting the presence of neoantigen-specific T cells.

 

Conclusions: These findings support the feasibility of identifying immunogenic neoantigens and reactive TCRs in UM using autologous TILs and tumor cells, as well as the U-Load dextramer strategy. This approach may enable future TCR-based immunotherapies targeting GNAQ/GNA11 mutations in UM.