OBJECTIVES/GOALS: Smoking is a well-established risk factor for lung cancer, but never smokers account for up to 25% of lung cancer cases. There is mounting evidence that lung cancer in never smokers is biologically distinct. We aim to characterize the genomic and immunologic features of lung adenocarcinoma in never smokers versus smokers. METHODS/STUDY POPULATION: We examined clinical, genomic, and bulk-RNA sequencing data from 499 patients in the TCGA lung adenocarcinoma cohort. Tumor mutation burden was analyzed using maftools (R package). Tumor immune characterization was completed using CIBERSORTx, a digital cytometry tool that uses single cell reference profiles to determine immune cell type frequencies from bulk-RNA sequencing data. Single cell reference profiles for 19 different immune cell types were constructed from sequencing of freshly resected lung tumor tissue from UCSF patients. Partitioning Around Medoids (PAM; R package) was used to identify distinct immune phenotypes based on immune cell composition. Fisher’s exact test was used to evaluate for associations between immune phenotypes and smoking status. RESULTS/ANTICIPATED RESULTS: Of the 499 TCGA lung adenocarcinoma patients, 75 were never smokers, 269 were female, and 246 were over the age of 65. Never smokers had lower tumor mutation burden and lower predicted neoantigen burden compared to smokers (p < 0.001). There was no difference in total tumor immune cell infiltration between never smokers and smokers. PAM yielded 2 distinct clusters/immune phenotypes. The first was enriched in M1 Macrophages, cytotoxic T Cells, helper T Cells, regulatory T Cells, and Plasma Cells. The second was enriched in plasmacytoid Dendritic Cells, M2 Macrophages, and exhausted cytotoxic T Cells. Never smoking status was associated with an increased odds of having the first immune phenotype (OR 1.95, 95% CI: 1.15 - 3.35) and this association was statistically significant (p = 0.0086). DISCUSSION/SIGNIFICANCE: Our findings suggest that never smokers have an immune phenotype that is distinct from that observed in smokers. The distinct immune characteristics we observed could explain clinical trial data suggesting immune checkpoint inhibitors are less effective in never smokers and hold implications for tailoring therapy.