An Engineered IL15 Cytokine Mutein Fused to an Anti-PD1 Improves Intratumoral T-cell Function and Antitumor Immunity.

The use of cytokines for immunotherapy shows clinical efficacy but is frequently accompanied by severe adverse events caused by excessive and systemic immune activation. Here, we set out to address these challenges by engineering a fusion protein of a single, potency-reduced, IL15 mutein and a PD1-specific antibody (anti-PD1-IL15m). This immunocytokine was designed to deliver PD1-mediated, avidity-driven IL2/15 receptor stimulation to PD1+ tumor-infiltrating lymphocytes (TIL) while minimally affecting circulating peripheral natural killer (NK) cells and T cells. Treatment of tumor-bearing mice with a mouse cross-reactive fusion, anti-mPD1-IL15m, demonstrated potent antitumor efficacy without exacerbating body weight loss in B16 and MC38 syngeneic tumor models. Moreover, anti-mPD1-IL15m was more efficacious than an IL15 superagonist, an anti-mPD-1, or the combination thereof in the B16 melanoma model. Mechanistically, anti-PD1-IL15m preferentially targeted CD8+ TILs and single-cell RNA-sequencing analyses revealed that anti-mPD1-IL15m treatment induced the expansion of an exhausted CD8+ TIL cluster with high proliferative capacity and effector-like signatures. Antitumor efficacy of anti-mPD1-IL15m was dependent on CD8+ T cells, as depletion of CD8+ cells resulted in the loss of antitumor activity, whereas depletion of NK cells had little impact on efficacy. The impact of anti-hPD1-IL15m on primary human TILs from patients with cancer was also evaluated. Anti-hPD1-IL15m robustly enhanced the proliferation, activation, and cytotoxicity of CD8+ and CD4+ TILs from human primary cancers in vitro, whereas tumor-derived regulatory T cells were largely unaffected. Taken together, our findings showed that anti-PD1-IL15m exhibits a high translational promise with improved efficacy and safety of IL15 for cancer immunotherapy via targeting PD1+ TILs.See related Spotlight by Felices and Miller, p. 1110.

Small-molecule inhibitors of bacterial AddAB and RecBCD helicase-nuclease DNA repair enzymes.

The AddAB and RecBCD helicase-nucleases are related enzymes prevalent among bacteria but not eukaryotes and are instrumental in the repair of DNA double-strand breaks and in genetic recombination. Although these enzymes have been extensively studied both genetically and biochemically, inhibitors specific for this class of enzymes have not been reported. We developed a high-throughput screen based on the ability of phage T4 gene 2 mutants to grow in Escherichia coli only if the host RecBCD enzyme, or a related helicase-nuclease, is inhibited or genetically inactivated. We optimized this screen for use in 1536-well plates and screened 326,100 small molecules in the NIH molecular libraries sample collection for inhibitors of the Helicobacter pylori AddAB enzyme expressed in an E. coli recBCD deletion strain. Secondary screening used assays with cells expressing AddAB or RecBCD and a viability assay that measured the effect of compounds on cell growth without phage infection. From this screening campaign, 12 compounds exhibiting efficacy and selectivity were tested for inhibition of purified AddAB and RecBCD helicase and nuclease activities and in cell-based assays for recombination; seven were active in the 0.1-50 µM range in one or another assay. Compounds structurally related to two of these were similarly tested, and three were active in the 0.1-50 µM range. These compounds should be useful in further enzymatic, genetic, and physiological studies of these enzymes, both purified and in cells. They may also lead to useful antibacterial agents, since this class of enzymes is needed for successful bacterial infection of mammals.