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Background HPK1 (MAP4K1), a serine/threonine protein kinase predominately expressed in the immune compartment, negatively regulates T-cell receptor signaling and immune activation. HPK1 is a promising therapeutic target for enhancing the antitumor immune response, and several small-molecule HPK1 inhibitors are in clinical trials. HPK1 also has kinase-independent regulatory roles in several innate immune cells, eg, dendritic and natural killer (NK) cells, potentially not addressed by small-molecule kinase inhibition. We characterized PROteolysis TArgeting Chimera (PROTAC) HPK1 degraders, which induce ubiquitination of HPK1 and its subsequent degradation by the proteasome.Methods PROTAC HPK1 degraders were characterized by biochemical, proteomic, and cell-based assays. Mechanistic studies were conducted in human peripheral blood mononuclear cells and various immune cells. Pharmacological efficacy and pharmacokinetic/pharmacodynamic/pathway engagement relationship were assessed in syngeneic tumor models as single agents or combined with an anti-PD-1 antibody. Multicolor, flow cytometry tumor-infiltrating lymphocyte analysis and RNAseq in syngeneic melanoma tumor-implanted mice provided mechanistic differentiation of the tumor immune landscape after PROTAC HPK1 degrader treatment.Results PROTAC HPK1 degraders exhibited half-maximal degradation concentrations of 1-20 nM, potent cellular pathway engagement (pSLP76 half-maximal inhibitory concentrations: 2-25 nM), and robust T-cell activity (interleukin-2 release half-maximal effective concentrations: 2-20 nM). PROTAC HPK1 degraders demonstrated the ability to reverse T-cell exhaustion, block suppressive effects of tumor-mediated metabolites, eg, prostaglandin E 2 and adenosine (return of interleukin-2 secretion to control levels), induce T cell and NK cell-mediated tumor cell death, and activate dendritic cells. PROTAC HPK1 degraders achieved robust HPK1 degradation (>80%) and efficient pathway engagement (>70% pSLP76 inhibition) at a single 30 mg/kg oral dose in naïve BALB/c mice. In a colonic syngeneic tumor model, PROTAC HPK1 degraders (30 mg/kg) showed greater tumor growth inhibition (TGI) compared with an anti-PD-1 antibody (>80% vs ~50%); combination of a PROTAC HPK1 degrader with an anti-PD-1 antibody resulted in 50% complete responders (5/10 mice). In a low-immunogenic, immune checkpoint blockade-resistant melanoma model, PROTAC HPK1 degraders elicited superior TGI (79%) vs an anti-PD-1 antibody (0) or HPK1 inhibitor (13%). PROTAC HPK1 degraders transcriptionally reprogrammed immune cells in the melanoma tumor microenvironment, with significantly increased (~2-fold) inflammatory monocytes and activated NK cells, and decreased infiltrating neutrophils compared with an anti-PD-1 antibody or HPK1 inhibitor.Conclusions We demonstrated pharmacological degradation of HPK1 has greater antitumor activity than HPK1 inhibitors or immune checkpoint blockade in preclinical models. These results support future investigation of PROTAC HPK1 degraders alone or combined with other agents in patients with certain cancers.Ethics Approval Animals were maintained in accordance with the guidelines of the Guide for the Care and Use of Laboratory Animals. All animal research protocol procedures were reviewed and approved by the Arvinas Animal Care and Use Committee.