Kenton L. Longenecker

Abstract Immune checkpoint blockade is effective for some patients with cancer, but most are refractory to current immunotherapies and new approaches are needed to overcome resistance 1,2 . The protein tyrosine phosphatases PTPN2 and PTPN1 are central regulators of inflammation, and their genetic deletion in either tumour cells or immune cells promotes anti-tumour immunity 3–6 . However, phosphatases are challenging drug targets; in particular, the active site has been considered undruggable. Here we present the discovery and characterization of ABBV-CLS-484 (AC484), a first-in-class, orally bioavailable, potent PTPN2 and PTPN1 active-site inhibitor. AC484 treatment in vitro amplifies the response to interferon and promotes the activation and function of several immune cell subsets. In mouse models of cancer resistant to PD-1 blockade, AC484 monotherapy generates potent anti-tumour immunity. We show that AC484 inflames the tumour microenvironment and promotes natural killer cell and CD8 + T cell function by enhancing JAK–STAT signalling and reducing T cell dysfunction. Inhibitors of PTPN2 and PTPN1 offer a promising new strategy for cancer immunotherapy and are currently being evaluated in patients with advanced solid tumours (ClinicalTrials.gov identifier NCT04777994 ). More broadly, our study shows that small-molecule inhibitors of key intracellular immune regulators can achieve efficacy comparable to or exceeding that of antibody-based immune checkpoint blockade in preclinical models. Finally, to our knowledge, AC484 represents the first active-site phosphatase inhibitor to enter clinical evaluation for cancer immunotherapy and may pave the way for additional therapeutics that target this important class of enzymes.

Crystallization is a unique process that occurs at the expense of entropy, including the conformational entropy of surface residues, which become ordered in crystal lattices during formation of crystal contacts. It could therefore be argued that epitopes free of amino acids with high conformational entropy are more thermodynamically favorable for crystal formation. For a protein recalcitrant to crystallization, mutation of such surface amino acids to residues with no conformational entropy might lead to enhancement of crystallization. This paper reports the results of experiments with an important cytosolic regulator of GTPases, human RhoGDI, in which lysine residues were systematically mutated to alanines. Single and multiple mutations were introduced into two different variants of RhoGDI, NDelta23 and NDelta66, in which the first 23 and 66 residues, respectively, were removed by recombinant methods. In total, 13 single and multiple mutants were prepared and assessed for crystallization and all were shown to crystallize using the Hampton Research Crystal Screens I and II, in contrast to wild-type NDelta23 and NDelta66 RhoGDI which did not crystallize. Four crystal structures were solved (the triple mutants NDelta23:K135,138,141A and NDelta66:K135,138,141A, and two single mutants NDelta66:K113A and NDelta66:K141A) and in three cases the crystal contacts of the new lattices were found precisely at the sites of mutations. These results support the notion that it is, in principle, possible to rationally design mutations which systematically enhance proteins' ability to crystallize.

The BET family of proteins consists of BRD2, BRD3, BRD4, and BRDt. Each protein contains two distinct bromodomains (BD1 and BD2). BET family bromodomain inhibitors under clinical development for oncology bind to each of the eight bromodomains with similar affinities. We hypothesized that it may be possible to achieve an improved therapeutic index by selectively targeting subsets of the BET bromodomains. Both BD1 and BD2 are highly conserved across family members (>70% identity), whereas BD1 and BD2 from the same protein exhibit a larger degree of divergence (∼40% identity), suggesting selectivity between BD1 and BD2 of all family members would be more straightforward to achieve. Exploiting the Asp144/His437 and Ile146/Val439 sequence differences (BRD4 BD1/BD2 numbering) allowed the identification of compound 27 demonstrating greater than 100-fold selectivity for BRD4 BD2 over BRD4 BD1. Further optimization to improve BD2 selectivity and oral bioavailability resulted in the clinical development compound 46 (ABBV-744).