# ROR1 as an Immunotherapeutic Target for Inducing Antitumor Helper T Cell Responses Against Head and Neck Squamous Cell Carcinoma

## TL;DR

This study shows that ROR1 is a promising target for immunotherapy in head and neck cancer, as it can trigger T cell responses that kill cancer cells.

## Contribution

The study identifies a novel ROR1-derived epitope that elicits antitumor helper T cell responses in HNSCC.

## Key findings

- ROR1 is significantly overexpressed in HNSCC tissues compared to healthy tissues.
- ROR1-reactive helper T cells exhibit cytotoxic activity against ROR1+ HNSCC cells in an HLA-DR-restricted manner.
- Combining ROR1-targeted vaccines with PD-L1/PD-L2 inhibitors enhances T cell antitumor activity.

## Abstract

ROR1, a tumor-associated antigen (TAA), is widely expressed in various cancers. However, its expression in HNSCC remains poorly characterized. Given the demonstrated tolerability of ROR1-targeting therapies in clinical trials, ROR1 may represent a promising TAA for T cell-based peptide vaccine development. Here, we demonstrate that ROR1 is widely expressed in HNSCC tissue specimens and cell lines, with significantly higher expression in HNSCC than in healthy tissues. To develop an ROR1-targeted peptide vaccine, we identified a novel ROR1-derived epitope capable of eliciting antitumor T cell responses against HNSCC. ROR1-reactive helper T cell (HTL) lines secreted effector cytokines and exhibited direct cytotoxic activity against ROR1+ HNSCC cell lines in a human leukocyte antigen (HLA)-DR-restricted manner. Furthermore, the tumoricidal activity of T cells was enhanced by ICIs targeting the PD-L1/PD-1 and PD-L2/PD-1 axes. These findings suggest that ROR1 could serve as a promising immunotherapeutic target in patients with HNSCC.

Background/Objectives: Head and neck squamous cell carcinoma (HNSCC) is the seventh most common cancer, with limited responsiveness to immune checkpoint inhibitors (ICIs). Cancer vaccine therapy is a promising novel immunotherapeutic approach that stimulates tumor-specific T cells. Receptor tyrosine kinase-like orphan receptor 1 (ROR1), which is overexpressed in malignant tumors but minimally expressed in normal tissues, presents a promising target for immunotherapy. This study aimed to evaluate ROR1 as a target for helper T lymphocyte (HTL)-based peptide vaccine immunotherapy in HNSCC. Methods: ROR1 expression in HNSCC tissues was assessed by immunohistochemistry. A novel ROR1-derived epitope (ROR1403–417) was identified and used to generate ROR1-reactive HTLs. Functional assays measuring IFN-γ and granzyme B secretion, as well as direct cytotoxicity, were performed. The effects of ICIs on HTL activity were also examined. The presence of ROR1-reactive T cells in the peripheral blood of patients with HNSCC was evaluated. Results: ROR1 positivity rates in HNSCC tissues were significantly higher (80.0%) than those in healthy controls (16.7%), and high ROR1 expression correlated with advanced clinical stages. HTL lines recognized the ROR1403–417 peptide in a human leukocyte antigen (HLA)-DR-restricted manner, secreted effector cytokines, and exhibited direct cytotoxicity against ROR1+ tumor cells. Dual PD-L1/PD-L2 blockade further enhanced HTL responses. ROR1-reactive T cells were detected in the peripheral blood of patients with HNSCC. Conclusions: ROR1 represents a promising target for immunotherapy in HNSCC. The ROR1403–417 peptide can elicit ROR1-reactive HTLs that exhibit antitumor responses against HNSCC cell lines, which can be enhanced by ICIs. These findings support the potential of ROR1-targeted peptide vaccine therapy for HNSCC.