Pheral CD8+ T cells making them poised for exhaustion even before TCR binding. These findings suggest that IR blockade also plays a substantial part in reversing immune tolerance outside of the TME and cytokine blockade could play a function in reversing PD1 blockade resistance. Ethics Approval The study was approved by the University of Pittsburgh’s IRB and Ethics Board, approval quantity: PRO16070383. P557 Overcoming genetically-based resistance mechanisms to PD-1 blockade Davis Torrejon, MD1, Gabriel Abril-Rodriguez, MS2, Jennifer Tsoi2, Ameya Champhekar2, Giulia Parisi2, Gardenia Cheung-Lau2, Tom Wohlwender2, Mykola Onyshchenko2, Beata Berent-maoz2, Catherine Grasso2, Bego Comin- Anduix, PhD2, Siwen Hu-Lieskovan, MD, PhD2, Antoni Ribas, MD, PhD2 1 UCLA Hematology-Oncology, Los Angeles, CA, USA; 2UCLA, Los Angeles, CA, USA Correspondence: Antoni Ribas ([email protected]) Journal for ImmunoTherapy of Cancer 2018, 6(Suppl 1):P557 Background We studied loss of function (LOF) mutations inside the interferon (IFN) pathway (JAK1 or JAK2) and in the antigen presentation pathway (beta-2-microglobulin-B2M) discovered in biopsies from patients who are resistance to anti-PD-1 therapy, and tested approaches to overcome the resistance. Solutions Applying CRISPR/Cas9 genome editing we generated JAK1, JAK2 and B2M knockout (KO) sublines of your murine MC38 carcinoma, a model of higher mutational load cancer that responds effectively to anti-PD-1, too as of human MART-1-positive melanoma cell lines, tested working with Ubiquitin-Specific Peptidase 34 Proteins manufacturer in-vitro T cell co-culture systems. We analyzed signaling modifications in human cell lines (parental and KOs) exposed to c-Jun N-terminal kinase 2 (JNK2) Proteins Recombinant Proteins IFN-gamma employing RNAseq. Furthermore, we performed in-vivo antitumor activity inside the MC38 variants applying mass cytometry (CyTOF) to characterize the tumor microenvironment. Ultimately, we tested approaches to overcome resistance mechanisms with SD-101 (TLR-9-agonist) and NKTR-214 (CD-122 biased agonist).Results The JAK1-KO sublines lost sensitivity to IFN-alpha, IFN-beta and IFNgamma, even though the JAK2-KO cell line was insensitive only to IFN-gamma induced signaling (PD-L1, MHC class I) and growth arrest (p0.001 compared with IFN-alpha or beta). There was no difference inside the in-vitro cytotoxicity by MART-1 distinct T-cells against JAK1/2- KO-MART-1+ melanoma cells compared to the parental (94 , 95 vs 90 cytotoxicity at ten:1 E:T ratio, pNS). On the other hand, B2M-KO was resistant to killing by MART-1 specific T-cells (2 vs 90 cytotoxicity at ten:1 E:T ratio, p0.0001). RNAseq differential gene expression evaluation showed that the IFN-gamma-induced improved expression of antigen presenting machinery, IFN-gamma signaling and chemokines (CXCL9, CXCL10) were not expressed by JAK1/2-KOs. Inside the MC38 model, the substantial antitumor activity of anti-PD-1 against the parental was lost in JAK1/2 and B2M KOs (Table1); in these KO sublines, CyTOF analysis revealed that anti-PD-1 therapy was unable to transform tumor CD8 T-cell infiltration. Employing JAK1/2-KOs cell lines we showed that intratumoral administration of your TLR-9 agonist SD-101 was in a position to overcome neighborhood resistance to anti-PD-1 even in abscopal websites, along with the NKTR-214 overcame resistance to anti-PD-1 inside the B2M-KO tumor development and significantly increased survival. Conclusions JAK1/2 LOF mutant tumors lead to loss of sensitivity to IFN induced antitumor effects but usually do not impair T cell recognition and cytotoxicity, although B2M LOF outcomes in lack of antigen presentation to T cells and loss of antitumor activity. B.