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Immune check point blockade (ICB) has recently transformed the treatment of non-small cell lung (NSCLC). However, the majority of patients do not respond to ICB. For ICB to be effective, tumor neoantigens need to be presented in a human leukocyte antigen (HLA) class I-restricted manner. Recent studies by our group and others have shown that HLA-B44 supertype is associated with extended survival in patients treated with ICB. Herein, we hypothesize that HLA-B44-specific motif neoepitopes generate an active tumor immune microenvironment and a favorable ICB response in NSCLC. Utilizing the TCGA database, we found that nearly half of the NSCLC patients have at least one HLA-B44 allele, of whom 36.8% and 37.3% of patients harbor at least one B44 motif neoepitope in lung adenocarcinoma (LUAD) and lung squamous carcinoma (LUSC), respectively. In a pooled analysis of patients with LUAD and LUSC, gene expression of PD-L1, CTLA-4, LAG-3, CXCL9, CXCL10, CXCL13, CD8A and cytolytic gene signature (includes GZMA and PRF1) is elevated in patients with B44 motif neoepitopes compared to those without motif neoepitopes. Immune cell component analysis showed increased infiltration of Th1, Th2 and CD8 T cells in LUAD and regulatory and CD8 T cells in LUSC. Although a linear regression model revealed a positive correlation between tumor mutational burden (TMB) and number of B44 motif neoepitopes, pooled analysis of LUAD and LUSC with similar TMB showed significant elevation of CTLA-4, LAG3, CXCL10 and a trend of increased expression of CXCL9 in motif patients. Upon investigation of possible immune editing, we found a significantly higher proportion of genes containing B44 neoepitope downregulated at the RNA level both in LUAD and LUSC. Lastly, we showed that combining B44 motif with either PD-L1 or TMB improved prediction of progression-free survival in NSCLC patients treated with ICB. In summary, we demonstrate that HLA-B44 motif neoepitopes are associated with a favorable tumor immune microenvironment and can serve as a potential biomarker for ICB benefit in NSCLC patients. Citation Format: Rui Li, Charlene M. Fares, Tristan R. Grogan, Tianhao Zhang, Debory Li, Matthew Theisen, Maria A. Velez, Paige M. Brodrick, Jackson P. Lind-Lebuffe, Yazeed Radwan, Gregory A. Ayzenberg, David Elashoff, Bin Liu, Aaron E. Lisberg, Amy L. Cumming, Edward B. Garon. HLA-B44 motif neoepitope is associated with a favorable tumor immune microenvironment and predicts ICB response in NSCLC. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4340.

Purpose: Immune checkpoint blockade (ICB) has revolutionized treatment of melanoma and lung cancer. Recent evidence suggests that human leukocyte antigen (HLA) B supertype influences efficacy of ICB. HLA-B27 and B44 supertypes have electronegative and electropositive binding pockets, respectively, and preferentially bind and display oppositely charged neoepitopes. We sought to evaluate the relationship between baseline immune activation and charged neoepitopes in the context of oppositely charged HLA-B binding pockets. Methods: Datasets from a total of 466 MEL and 513 LUAD patients included in the TCGA were used. HLA type was obtained with OptiType. Nonsynonymous mutations were annotated with SnpEff, Ensembl VEP, and VAtools. Only transcribed variants were filtered for analysis. pVAC-Seq using NetMHCpan algorithm predicted nonamer neoepitopes. Favorable B27 neoepitopes were defined as having new positively charged amino acid (AA) substitutions (H/K/R) from oppositely charged or uncharged wildtype AA, while neoepitopes with new negatively charged AAs (D/E) were considered favorable for B44. Three gene signatures were evaluated with normalized RNA expression values from 14 antigen presentation, 15 immunostimulator, and 11 immunoinhibitor genes. Linear regression tests were performed between gene expression values and fraction of nonsynonymous mutations resulting in neoepitopes with new positively and negatively charged AAs. Paired t-tests were performed between the mean of Pearson's r for fraction of new positively versus negatively charged AAs compared to gene expression values, and subsetted to B27/B44. Heterozygotes for B27/B44 were excluded from analysis to avoid competing charges. Results: 26.4% and 48.7% of MEL patients had B27 and B44, respectively. In LUAD, 28.6% had B27 and 47.2% had B44. Of new charged AAs resulting from nonsynonymous mutations, 72.6% in MEL and 75.0% in LUAD were positively charged (p=0.0053). In MEL, difference in Pearson's r values between fraction of new positively and negatively charged AAs as compared to immunostimulator (B27 p=.016; B44 p Conclusion: Immune gene signatures showed association with neoepitopes that have oppositely charged AAs than their HLA-B binding pocket in both MEL and LUAD, indicating that presentation of favorable neoepitopes stimulates an immune response. The association was limited to B27 in LUAD and strongest for B44 in MEL. Citation Format: Charlene M. Fares, Amy L. Cummings, Matthew K. Theisen, Nicholas Hornstein, Jaklin Gukasyan, Wisdom Akingbemi, Debory Li, Paige Brodrick, Aaron Lisberg, Edward B. Garon. Association between immune gene signatures and neoepitopes with electrostatic charge opposite to their HLA-B binding pocket in melanoma (MEL) and lung adenocarcinoma (LUAD) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6614.