273P IL-12 synergistically enhances HPV E7 mRNA vaccine efficacy by promoting effector T cells and overcoming exhaustion: Impact of nucleotide modification

Background: Hepatocellular carcinoma (HCC) is a leading cause of cancer mortality.First-line therapy with anti-PD-L1 plus anti-VEGFA antibodies yields 30% response, but most patients remain resistant due to sustained PD-L1 expression and insufficient T cell receptor (TCR) signaling within the tumor microenvironment.We identified WNK lysine deficient protein kinase 3 (WNK3) as a positive regulator of PD-L1 in cancer cells and a negative regulator of T cell function.WNK3 is abundant in liver and brain but lower in kidney.Unlike ubiquitously expressed Wnk1, whose knockout is embryonic lethal, Wnk3 knockout mice are viable, suggesting WNK3 as a safer target.However, no WNK3-selective inhibitors exist because ATP-binding pockets are highly conserved across WNK kinases. Methods:We applied structure-based design approach integrating modeling, fragment merging, and scaffold hopping to generate WNK3-selective inhibitors.Lead compounds were screened for potency, selectivity, and cellular activity.Pharmacokinetic studies assessed standard PK parameters, oral bioavailability, tumor-toplasma (T/P) ratio, and blood brain barrier (BBB) penetration.Functional validation was performed in T cell co-culture and Hepa1-6 OVA syngeneic models.Results: CX003 showed single-digit to sub-nanomolar potency against WNK3 with >40-160-fold selectivity over WNK1.It was well tolerated up to 40 M in normal cells.CX003 displayed a favorable PK profile with high T/P ratio (>0.7), low BBB penetration (< 0.1).Mechanistically, CX003 boosted CD8 + and CD4 + T cell proliferation and effector function, increasing IFN-, GRANZYME B, TNF-, and IL-2, via activation of AKT, c-JUN, and ERK.In parallel, it suppressed PD-L1 in cancer cells by inhibiting JNK/c-JUN driven transcription.These dual mechanisms converged to amplify TCR signaling and improve tumor killing in Hepa1-6 OVA co-culture and in xenografts.It achieved a TGI 46% as monotherapy and 93% in combination with anti-PD-L1/anti-VEGFA therapy.Conclusions: CX003 is an orally bioavailable, first-in-class WNK3 inhibitor that enhances T cell activity and reduces tumor PD-L1, supporting synergy with immune checkpoint blockade in HCC.