Development and Preclinical Evaluation of Tetraspecific Antibodies in Combination with Ex Vivo Expanded NK Cells Infusion for Improved Control of Esophageal Cancer

Background: Esophageal squamous cell carcinoma (ESCC) is highly heterogeneous, leading to limited drug effectiveness and resistance. Antibody therapies offer limited benefits due to low response rates. multispecific antibodies, targeting multiple tumor antigens and NK cell receptors, may overcome these challenges, but their effectiveness and mechanisms in ESCC therapy are not yet understood. Methods: Whole transcriptome sequencing was performed on tumor tissues from 32 ESCC patients to identify tumor antigens. Tetraspecific antibodies, referred as NK cell engagers (NKCEs), were designed and constructed to target two ESCC antigens and two surface markers of NK cells. AlphaFold was used to predict the spatial structure of tetraspecific NKCEs and its binding properties with antigens. Fast protein liquid chromatography and surface plasmon resonance were applied for quality control of NKCEs. Using multiple human ESCC cell lines, chemoresistant ESCC cell lines and NK cells from different donors, we carried out flow cytometry to assess NK cell cytotoxicity and activation. Immunofluorescence assays were used to confirm NKCEs binding and colocalization. Transcriptome sequencing, flow cytometry and western blot were performed to explore molecular mechanisms. Subcutaneous and metastatic xenograft tumor models were established in nude mice to evaluate the in vivo anti-tumor efficacy of NKCEs combined with adoptive transfer of NK cells. Results: We found that EGFR was consistently highly expressed in all ESCC samples while MET was specifically upregulated in recurrent ESCC. Tetraspcific NKCEs were successfully engineered and could specifically bind to EGFR, MET, CD16, NKp30 or NKp46. The tetraspecific NKCEs significantly enhanced the cytotoxicity of NK cells against ESCC cell lines as well as paclitaxel-resistant ESCC cell lines in vitro, compared with the commercial antibody Amivantamab. Transcriptomic analysis followed by functional validation revealed that tetraspecific NKCEs triggered immune activation pathways and increased the expression of TNF-α, IFN-γ, and granzyme B in NK cells, but also induced NK cell exhaustion. In vivo studies demonstrated that tetraspecific NKCEs combined with NK cell infusions exerted better antitumor efficacy against esophageal tumors in different stages. The combination therapy was well tolerated, with enhanced NK cell infiltration in tumor tissues. Conclusions: We developed tetraspecific NKCEs targeting two tumor antigens (EGFR, MET) and two NK-cell activating receptors (CD16, NKp30 or NKp46) to effectively bridge ESCC cells and NK cells. The tetraspecific NKCEs exert the potential to enhance antitumor activity of NK cells against ESCC. This study supports the clinical translation of multispecific NKCEs for NK cell-based immunotherapy for ESCC.