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Abstract Checkpoint inhibitors represent promising cancer therapeutics as they have the potential to reignite T cell activity and reverse T cell exhaustion, which is a critical mechanism of immune evasion by tumors. Hematopoietic progenitor kinase 1 (HPK1) is a negative regulator of T-cell signaling that has recently emerged as a novel target for cancer immunotherapy. Inhibition of HPK1 can restore T cell function and anti-tumor immune responses, and several HPK1 inhibitors are currently under pre-clinical development, or clinical evaluation. Identifying clinically useful pharmacodynamic (PD) biomarkers in response to HPK1 inhibition is critical to characterize exposure-response relationship in early-phase trials and inform dose selection for later-phase studies. However, development and implementation of clinical PD assays are challenging because high sensitivity and specificity are required. Here, we developed a multi-biomarker strategy that combines a proximal biomarker (SLP-76 (Ser376) phosphorylation, a specific substrate of HPK1) and distal biomarkers (cytokine production) to evaluate target engagement (TE) and establish proof of mechanism (POM) of HPK1 inhibitors to determine their comprehensive PD profile. These PD biomarker assays were developed using peripheral blood as a surrogate tissue to assess HPK1 inhibition-induced pSLP-76 level decrease and cytokine levels increase, thus reflecting drug inhibitory effects and T-cell activity recovery. However, the lack of basal levels of these biomarkers presented a challenge for clinical workflow, as ex-vivo T-cell receptor stimulation of the clinical specimen is required after collection. Phosphoflow cytometry was prioritized over several technology platforms evaluated for quantifying blood pSLP-76 levels because of superior assay performance and signal stability under the developed clinical workflow. Blood cytokines upregulated upon HPK1 inhibition were first identified using targeted proteomics. IFNγ, IL2, TNFα, and IL17A were next confirmed and validated using quantitative immunoassays. Both pSLP-76 and whole blood cytokine workflows were thoroughly optimized for every step of sample handling from specimen processing at clinical sites to sample analysis at testing lab to ensure standardized, precise, and robust assay performance for clinical application. In addition, consistent HPK1 inhibition potency values were achieved using blood from cancer patients, demonstrating that the established PD biomarker assays apply to the clinical target population. In conclusion, robust peripheral TE and POM biomarkers were successfully developed to support future phase I/II studies with HPK1 inhibitors. These are expected to enable characterization of the exposure-response relationships and help define the therapeutic window and optimal dose. Citation Format: Lin Jia, Yasmin Marikar-Coplin, Jeff Chen, Kevin Xu, Deanna A. Mele, Nadia Luheshi, Sonia Iyer, Anton I. Rosenbaum, Sonia Terrillon. Development and validation of clinical pharmacodynamic biomarkers to assess treatment response to HPK1 inhibition and guide dosing decisions abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts) ; 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84 (6Suppl): Abstract nr 7177.
Jia et al. (Fri,) studied this question.