Background Hematopoietic Progenitor Kinase 1 (HPK1/MAP4K1) has been demonstrated to restrain T cell activation through phosphorylating SLP-76 at Serine 376 during the T-cell activation process. Given its irreplaceable negative regulation role in TCR pathway, HPK1 is considered as a promising target in immuno-oncology. Recently, HPK1 has been shown to function as scaffolding protein and bind to adaptor proteins to promote JNK signaling and actin cytoskeleton rearrangement involved in lymphocyte adhesion. Additionally, the turnover rate of HPK1 is low. These evidences suggest that HPK1 is a suitable target to develop protein-degrader. Herein, we described our state-of-the-art work in discovery and identification of HPK1 degrader HZ-S109. This degrader exhibits improved pharmacokinetic properties and stronger engagement with the HPK1 target, leading to the restoration of suppressed T-cell function and inhibition of tumor growth.

Methods HZ-S109 was identified based on our in-house unique DaTProD^® platform. Western blot analysis was utilized to assess the levels of HPK1 protein, while experiments were carried out to study T cell activation and reinvigoration under different conditions. Oral-bioavailability were profiled in three species. In-vivo anti-tumor efficacy was assessed in C57BL/c mice which were engrafted with CT-26 cell line.

Results HZ-S109 exhibits potent HPK1 degradation with DC50 values lower than 10 nM across various cell types. It has minimal impact on the downregulation of proteins other than HPK1 downstream targets. HZ-S109 can significantly enhance the activation of primary T cell at nano-molar level concentrations and the maximum activation fold can be more than 15 folds. Furthermore, HZ-S109 restores T cell function in the presence of muiltiple immune-suppressed conditions including NECA/PGE2/TGF-b or their combination. Despite its higher molecular weight, HZ-S109 boasts superior oral bioavailability. In in-vivo anti-tumor efficacy evaluation, HZ-S109 robustly suppressed the tumor growth as a single agent in CT26 and A20 xenograft model. Moreover HZ-S109 demonstrated remarkable combination effect with immune checkpoint inhibitors (ICI), with TGI more than 90%.

Conclusions In summary, HZ-S109 stands out as a potent and distinct HPK1 modulator, exhibiting enhanced potency and oral bioavailability. At present, HZ-S109 is in the IND-enabling study stage.

Ethics Approval All animal studies were performed in strict accordance with the institutional guidelines as defined by the Institutional Animal Care and Use Committee(IACUC), approved by the Animal Care and Use Committee, Hangzhou Medical College Animal Center (Hangzhou, China), approval ID: 2023-030. All participants gave informed consent before taking part.