In Vitro: ZYF0033 (100 nM; 24 h) reduces phosphorylation of SLP76 (serine 376), a key biomarker of T cell and HPK1 activation. ZYF0033 causes HPK1 inhibition, increased proliferation of CD4+ and CD8+ T cells, and IFN-γ secretion.
In Vivo: The maximum tolerated dose of ZYF0033 exceeds 50 mg/kg (daily; po) and exceeds 120 mg/kg in a 6-day toxicity study. In the 4T-1 syngeneic mouse model, ZYF0033 regulates immune cell subsets, increases DC and NK cell infiltration, and promotes anti-cancer immune response.