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854 HPK1 inhibition relieves suppression downstream of TCR activation to drive enhanced cytokine production and antigen-specific killing, an effect that is further enhanced by immune checkpoint blockade
  1. Rajesh Singh,
  2. Bindi Patel,
  3. Rameshwari Rayaji,
  4. Sachie Marubayashi,
  5. Sean Cho,
  6. Stefan Garrido-Shaqfeh,
  7. Joseph Kulusich,
  8. Cesar Meleza,
  9. Nidhi Tibrewal,
  10. Joice Thomas,
  11. Pradeep Nareddy,
  12. Ehesan Sharif,
  13. Sharon Zhao,
  14. Dave Green,
  15. Manmohan Leleti,
  16. Jay Powers,
  17. Matt Walters and
  18. Daniel DiRenzo
  1. Arcus Biosciences, Hayward, CA, United States


Background T cell activation is critical in the initiation and potentiation of anti-tumor immune responses. Hematopoietic Progenitor Kinase 1 (HPK1) is a member of the MAP4K family whose activity restrains T cell activation through phosphorylation of SLP-76 (pSLP-76) at Serine 376, leading to disassembly of the TCR complex. Mouse genetic deletion and kinase dead mutants of HPK1 have been shown to enhance T cell activity and combine with immune checkpoint inhibition. Therefore, we sought to demonstrate that inhibitors of HPK1 activity can increase T cell activation, drive antigen-specific cancer cell killing, and combine with immune checkpoint blockade to amplify anti-tumor T cell responses. Herein, we describe the characterization of novel inhibitors of HPK1 and assess their effects on T cell activation in combination with PD-1 blockade.

Methods Measurement of pSLP-76 using a flow cytometry-based assay was employed to assess HPK1 activity in human and mouse whole blood. Jurkat cells, human CD8+ T cells and PBMCs were activated using anti-CD3/CD28 stimulation, CEF peptide pools, or Staphylococcal enterotoxin A (superantigen), in the presence of HPK1 inhibitors; levels of IL-2, IFN-γ and TNF-α were assayed in supernatants by cytokine bead array. Mouse OT-1 splenocytes were stimulated with SIINFEKL peptide and co-cultured with E.G7-OVA cells to assess cytokine production and target cell killing.

Results HPK1 inhibitors exhibited a potent, concentration-dependent reduction in pSLP-76, with a concurrent increase in IL-2 secretion in both Jurkat and human CD8+ T cells. Human T cells also demonstrated increased IFN-γ and TNF-α secretion as well as a greater percentage of activated CD69+ cells with HPK1 inhibition. These increases in T cell activation were mirrored in antigen-specific OT-1 T cell assays, in which HPK1 inhibition enhanced IFN-γ production in response to OVA peptide and greater killing of E.G7-OVA cancer cells. Similarly, antigen-recall assays demonstrated that inhibition of HPK1 increased IFN-γ production from human PBMC stimulated with CEF peptide and combined with PD-1 blockade to further enhance cytokine production. Consistent with these results, HPK1 inhibition and PD-1 blockade increased cytokine secretion in superantigen-stimulated human PBMC individually and further enhanced cytokine production in combination. Taken together, these data demonstrate that the combined activity of HPK1 inhibition with checkpoint therapy may yield greater anti-tumor T cell activity.

Conclusions These data demonstrate that pharmacological inhibition of HPK1 amplifies antigen-specific T cell activation alone or in combination with immune checkpoint blockade and provide a strong mechanistic rationale for targeting HPK1 to amplify anti-tumor immune responses.

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