Background: It is challenging to choose subsequent treatment option in patients with Osimertinib resistance in epidermal growth factor receptor (EGFR)-mutated non-small cell lung cancer (NSCLC). Meanwhile, antitumor effect of the antibody-dependent cellular cytotoxicity of natural killer (NK) cells mediated by cetuximab –anti-EGFR monoclonal antibody– has been reported. Therefore, we aimed to evaluate the safety and efficacy of SNK01 (non-genetically modified autologous NK cell) in combination with cytotoxic chemotherapy including cetuximab in NSCLC after failure to prior tyrosine kinase inhibitor (TKI) in a preclinical humanized cell line-derived xenograft (CDX) mouse model and phase I/IIa clinical study. Methods: We established a humanized CDX mouse model using Osimertinib-resistant lung cancer cell line. Reconstruction of the humanized mouse, CDX-related skills, and analytic data were made according to C&SR Inc. manufacturing technique & SOP protocol. The mice were divided into 4 groups based on treatment (no treatment [n = 2]; Cetuximab only [n = 3]; SNK01 only [n = 4]; SNK01 plus Cetuximab [n = 4]) and treated weekly for 5 weeks (SNK01, 1x10⁷ cells/dose; Cetuximab, 20 ug/dose). In the clinical study, 12 patients with EGFR-mutated NSCLC who failed prior TKI treatment were finally enrolled. They received weekly SNK01 in combination with Gemcitabine/Carboplatin (n = 6) or Gemcitabine/Carboplatin/Cetuximab (n = 6), and dose escalation of SNK01 following “3+3” design (4×10⁹ cells/dose or 6×10⁹ cells/dose). The primary endpoint was safety, and secondary endpoint was efficacy. Results: In the preclinical study, flow cytometry analysis showed that NK cells (CD45+/CD56+/CD3-) were significantly increased in the groups administrated SNK01. The volume of tumor extracted after completion of treatment was the smallest in SNK01 plus cetuximab group. In the clinical study, the median age of patients was 61 years, 33.3% were male, and all patients had adenocarcinoma. The enrolled patients received weekly infusions of SNK01 for 7 to 8 weeks (4×10⁹ cells/dose [n = 6]; 6×10⁹ cells/dose [n = 6]). Since dose limiting toxicity was not observed, maximum tolerated dose of SNK01 was determined to be 6×10⁹ cells/dose. SNK01-related adverse event ≥grade 3 was also not observed. In the efficacy analysis, objective response rate was 25%, disease control rate was 100% (partial response, n = 3/12; stable disease, n = 9/12), and median progression free survival (PFS) was 143 days. PFS will be updated as some patients are still being followed. Conclusions: SNK01 in combination with cytotoxic chemotherapy, including cetuximab in EGFR-mutated NSCLC with resistance to TKI was safe and showed a potential antitumor effect in this preclinical study and early phase I/IIa clinical trial. Clinical trial information: NCT04872634.