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Immunotherapy has fundamentally changed the landscape of cancer treatment. Despite the encouraging results with the checkpoint modulators, response rates vary widely across tumor types, with a majority of patients exhibiting either primary resistance without a significant initial response to treatment or acquired resistance with subsequent disease progression. Hematopoietic progenitor kinase 1 (HPK1) is predominantly expressed in hematopoietic cell linages and serves as a negative regulator in T cells and dendritic cells (DC). While HPK1 gene knockout (KO) studies suggest its role in anti-tumor immune responses, the involvement of kinase activity and thereof its therapeutic potential remain unknown. To investigate the potential of pharmacological intervention using inhibitors of HPK1, we generated HPK1 kinase dead (KD) mice which carry a single loss-of—function point mutation in the kinase domain and interrogated the role of kinase activity in immune cells in the context of suppressive factors or the tumor microenvironment (TME). Our data provide novel findings that HKP1 kinase activity is critical in conferring suppressive functions of HPK1 in a wide range of immune cells including CD4+, CD8+, DC, NK to Tregs, and inactivation of kinase domain was sufficient to elicit robust anti-tumor immune responses. These data support the concept that an HPK1 small molecule kinase inhibitor could serve as a novel agent to provide additional benefit in combination with existing immunotherapies, particularly to overcome resistance to current treatment regimens.

Introduced in the late 1980s as a reducing reagent, Tris (2-carboxyethyl) phosphine (TCEP) has now become one of the most widely used protein reductants. To date, only a few studies on its side reactions have been published. We report the observation of a side reaction that cleaves protein backbones under mild conditions by fracturing the cysteine residues, thus generating heterogeneous peptides containing different moieties from the fractured cysteine. The peptide products were analyzed by high performance liquid chromatography and tandem mass spectrometry (LC/MS/MS). Peptides with a primary amine and a carboxylic acid as termini were observed, and others were found to contain amidated or formamidated carboxy termini, or formylated or glyoxylic amino termini. Formamidation of the carboxy terminus and the formation of glyoxylic amino terminus were unexpected reactions since both involve breaking of carbon-carbon bonds in cysteine.

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Clinical validation of S1P receptor modulation therapy was achieved with the approval of fingolimod (Gilenya, 1) as the first oral therapy for relapsing remitting multiple sclerosis. However, 1 causes a dose-dependent reduction in the heart rate (bradycardia), which occurs within hours after first dose. We disclose the identification of clinical compound BMS-986104 (3d), a novel S1P 1 receptor modulator, which demonstrates ligand-biased signaling and differentiates from 1 in terms of cardiovascular and pulmonary safety based on preclinical pharmacology while showing equivalent efficacy in a T-cell transfer colitis model. KEYWORDS: GPCR, S1P1, S1P3, biased signaling L ymphocyte infiltration from blood into sites of inflammation is critical to the pathogenesis of autoimmune diseases and allograft rejection. Gilenya (FTY720, 1) blocks lymphocyte migration through sequestration of lymphocytes in the thymus and secondary lymphoid organs, leading to a marked lymphopenia. 1 Compound 1 is a pro-drug; its phosphorylated form, FTY-P (1-P), binds four out of the five S1P receptors (S1P-1, 3, 4, 5) and elicits a full agonist response in functional assays such as GTP-S binding, ERK phosphorylation, cAMP, and calcium mobilization. Among these four receptors, S1P 1 has been shown to be critically involved in lymphocyte trafficking and agonism of this receptor is responsible for the peripheral blood lymphopenia believed to be key to the efficacy seen with 1. 2,3 Clinical studies have demonstrated a side effect profile of 1 that includes cardiovascular effects (transient bradycardia, sustained blood pressure elevation) as well as a decline in pulmonary function. 4 In rodent studies, S1P 3 activity was shown to play a role in some of the observed acute toxicity of nonselective S1P receptor agonists, including bradycardia, hypertension, and bronchoconstriction. 5,6 As agonism of S1P 3 does not appear to contribute to efficacy, the identification of S1P 1 agonists sparing of S1P 3 has been a primary emphasis of many research programs in this area. 7 However, clinical studies with S1P agonists with selectivity for S1P 1 over S1P 3 have suggested that in humans the heart rate reduction effects are controlled at least in part through agonism of S1P 1 . 8 Additionally, through the course of our own studies it was discovered that simply abolishing S1P 3 agonism was not sufficient to eliminate the acute and chronic pulmonary toxicity elicited in rodents by 1 or by selective S1P 1 full agonists, findings that led us to discontinue our efforts related to S1P 1 full agonists and seek alternative profiles that could overcome these liabilities. 9 In this letter we describe the identification of a differentiated S1P 1 receptor modulator, BMS-986104 (3d), which distinguishes itself from 1 in terms of cardiovascular and pulmonary safety based on preclinical pharmacology while showing equivalent efficacy in a T-cell transfer colitis model.In our search for S1P 1 agonists that could further dissociate efficacy from toxicity, we evaluated...