Anti-IFNG monoclonal antibody, clone YNH2.3 [Brilliant Violet 421]

BACKGROUND: Treatment with PD-1 inhibitors can be hampered by severe auto-immune-related toxicities. Our objective was to identify single-nucleotide polymorphisms (SNPs) in genes previously associated with auto-immunity, which are associated with toxicities in nivolumab-treated NSCLC patients. This was in order to identify patients prone to develop severe toxicities and to gain more insight into the underlying pathobiology. METHODS: We analysed 322 nivolumab-treated patients and assessed the association with toxicities for seven SNPs in four genes, which are considered contributors to PD-1-directed T-cell responses, i.e., PDCD1, PTPN11, ZAP70 and IFNG. Every SNP was tested for its association with toxicity endpoints. Significant associations were tested in a validation cohort. RESULTS: A multivariable analysis in the exploration cohort showed that homozygous variant patients for PDCD1 804C>T (rs2227981) had decreased odds for any grade treatment-related toxicities (n = 96; OR 0.4; 95% CI 0.2-1.0; p = 0.039). However, this result could not be validated (n = 85; OR 0.9; 95% CI 0.4-1.9; p = NS). CONCLUSIONS: Our results show that it is unlikely that the investigated SNPs have a clinical implication in predicting toxicity. A finding, even though negative, that is considered timely and instructive towards further research in biomarker development for checkpoint inhibitor treatments.

Both adjuvants and focal ablation can alter the local innate immune system and trigger a highly effective systemic response. Our goal is to determine the impact of these treatments on directly treated and distant disease and the mechanisms for the enhanced response obtained by combinatorial treatments. Methods: We combined RNA-sequencing, flow cytometry and TCR-sequencing to dissect the impact of immunotherapy and of immunotherapy combined with ablation on local and systemic immune components. Results: With administration of a toll-like receptor agonist agonist (CpG) alone or CpG combined with same-site ablation, we found dramatic differences between the local and distant tumor environments, where the directly treated tumors were skewed to high expression of F4/80, Cd11b and Tnf and the distant tumors to enhanced Cd11c, Cd3 and Ifng. When ablation was added to immunotherapy, 100% (n=20/20) of directly treated tumors and 90% (n=18/20) of distant tumors were responsive. Comparing the combined ablation-immunotherapy treatment to immunotherapy alone, we find three major mechanistic differences. First, while ablation alone enhanced intratumoral antigen cross-presentation (up to -8% of CD45(+) cells), systemic cross-presentation of tumor antigen remained low. Combining same-site ablation with CpG amplified cross-presentation in the draining lymph node (similar to 16% of CD45(+) cells) compared to the ablation-only (-0.1% of CD45(+) cells) and immunotherapy-only cohorts (similar to 10% of CD45(+) cells). Macrophages and DCs process and present this antigen to CD8(+) T-cells, increasing the number of unique T-cell receptor rearrangements in distant tumors. Second, type I interferon (IFN) release from tumor cells increased with the ablation-immunotherapy treatment as compared with ablation or immunotherapy alone. Type I IFN release is synergistic with toll-like receptor activation in enhancing cytokine and chemokine expression. Expression of genes associated with T-cell activation and stimulation (Eomes, Prf1 and !cos) was 27, 56 and 89-fold higher with ablation-immunotherapy treatment as compared to the no-treatment controls (and 12, 32 and 60-fold higher for immunotherapy-only treatment as compared to the no-treatment controls). Third, we found that the ablation-immunotherapy treatment polarized macrophages and dendritic cells towards a CD169 subset systemically, where CD169(+) macrophages are an IFN-enhanced subpopulation associated with dead-cell antigen presentation. Conclusion: While the local and distant responses are distinct, CpG combined with ablative focal therapy drives a highly effective systemic immune response.