Leveraging single-cell co-culture and gene expression profiling to identify tumor antigen-specific T cells in oral cancer

Oral squamous cell carcinoma (OSCC) is an aggressive malignancy with a 5-year survival rate below 50%. Despite the moderate successes of immune checkpoint blockade, durable control is rare for OSCC, underscoring the need for improve immunotherapeutic approaches. Critical to immunotherapy efficacy is the specific targeting of tumor antigens by T cell receptors (TCRs). However, methods to identify and expand tumor-infiltrating lymphocytes (TILs) specific for OSCC antigens have not been established. To address this, we have developed two innovations in OSCC. First, we have optimized a high-throughput, single-cell co- culture system employing the Bruker Lightning platform. By studying single tumor cell-single TIL interactions, we can isolate individual tumor-killing T cells and identify the TCRs they carry. Second, we have performed single-cell RNA and TCR sequencing studies of OSCC and have identified a cytotoxic TIL sub-cluster with conserved gene expression patterns across patients suggestive of tumor-specificity. This cluster, which we have termed TProlif_Tox, can be identified across OSCCs and is enriched in markers for proliferation, T cell differentiation, cytotoxicity, and immune checkpoint blockade. Aim 1 leverages our single-cell co-culture platform to identify tumor antigen-specific TCRs. Patient-derived specimens will undergo both single-cell RNA/TCR sequencing as well as tumor and TIL culture. Patient- matched tumor and TILs will then be used for single-cell co-culture with identification of tumor antigen-specific TILs, and subsequent single-cell TCR sequencing. Tumor antigen-specific TCRs will then be projected back onto each patient’s initial single-cell RNA/TCR sequencing profile to identify in vivo gene expression profiles of tumor antigen-specific TILs. We hypothesize that these tumor antigen-specific TILs will be consistently identified within the TProlif_Tox cluster across OSCC patients. Aim 2 will translate tumor antigen-specific TIL identification into anti-tumor efficacy by engineering T cells with TCRs identified from the TProlif_Tox cluster (neoTCRs). We will then test the functional capabilities of these neoTCR T cells against autologous OSCC both in vitro and in vivo. Using Crispr-Cas9 editing, the endogenous T cell receptor (TRAC/TRBC1/TRBC2) will be disrupted in patient-matched circulating T cells with transfection and integration of neoTCRs. T cells carrying neoTCRs from the TProlif_Tox cluster can then be assessed for anti- tumor functionality through in vitro (co-culture with autologous malignant OSCC lines) and in vivo (humanized patient-derived xenograft) models. We anticipate that neoTCR-engineered T cells will demonstrate superior anti-tumor activity over control T cells, confirming the tumor antigen-specificity of the TProlif_Tox cluster. By refining the identification of tumor antigen-specific TILs and their TCRs, our study is poised to improve the precision and potency of immunotherapy for OSCC. Project Number: 5R01DE034360-02 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of Dental and Craniofacial Research (NIDCR) | Principal Investigator: Joseph Zenga (+1 co-PI) | Institution: MEDICAL COLLEGE OF WISCONSIN, MILWAUKEE, WI | Award Amount: $390,000 | Activity Code: R01 | Study Section: Therapeutic Immune Regulation Study Section[TIR] View on NIH RePORTER: https://reporter.nih.gov/project-details/11309552