Recurrent and metastatic head and neck squamous cell carcinoma (R/M HNSCC) remains associated with limited therapeutic options. Amivantamab, Petosemtamab, Ivonescimab, and Ficerafusp alfa are emerging targeted bispecific/bifunctional antibodies under investigation. These agents aim to overcome resistance to current standards by targeting EGFR, MET, PD-1/PD-L1, VEGF, or TGF-β pathways. Early clinical studies suggest potential activity and manageable safety profiles. This study aimed to provide a comprehensive biological rationale for the clinical implementation of bispecific and bifunctional antibodies in R/M HNSCC.

Gene expression (GE) data were retrieved from 13 studies processed by microarray and 7 studies using RNA sequencing (RNAseq), analyzed as independent testing set. In addition, single-cell (scRNA-seq) data were obtained from the public dataset GSE191818, including 20 HNSCC patients, 13 HPV-negative (HPV-) and 7 HPV-positive (HPV+) with complete clinical annotation. Seurat R package and UMAP were applied to plot the expression of antibodies’ targets on HPV- and HPV+ cases. Statistical analyses (Chi-square and Cramér’s V) were used to assess differences in cell distribution.

Bulk analyses showed significantly higher expression of EGFR, MET, CD274, and VEGFA in tumors compared with normal tissues, whereas TGFBR2 was reduced and LGR5 showed low, non-differential expression. EGFR and MET were positively correlated, supporting coordinated activation of proliferative signaling. Single-cell analysis revealed marked cellular heterogeneity. In HPV-negative tumors (13,496 cells), EGFR (1,658 cells) and MET (934) were predominantly expressed in malignant cells. VEGFA was the most widely expressed gene (2,450 cells), enriched in both tumor cells and macrophages. TGFBR2 (2,074 cells) was broadly distributed across stromal and immune compartments, while LGR5 was rare (69 cells). HPV-positive tumors (9,577 cells) showed lower overall positivity, including EGFR (1,212 cells), MET (473), and VEGFA (1,481). Cluster analysis identified oncogenic and angiogenic programs concentrated in specific epithelial states, with EGFR and MET enriched in dominant malignant clusters. Co-expression patterns were significantly more frequent in HPV-negative disease, including EGFR/MET double-positive cells (630 vs 185 cells), indicating stronger pathway convergence and greater biological complexity.

This study provides biological validation of bispecific and bifunctional antibody use in R/M HNSCC setting. In particular, we identified that the therapeutic benefit is correlated to HPV status. In the future, survival analysis will be integrated in order to validate this data.

This work was supported by the Associazione Italiana per la Ricerca sul Cancro (AIRC) (IG23573 to LDC).