Pharmacological activation of the STING (stimulator of interferon genes)–controlled innate immune pathway is a promising therapeutic strategy for cancer. Here we report the identification of MSA-2, an orally available non-nucleotide human STING agonist. In syngeneic mouse tumor models, subcutaneous and oral MSA-2 regimens were well tolerated and stimulated interferon-β secretion in tumors, induced tumor regression with durable antitumor immunity, and synergized with anti–PD-1 therapy. Experimental and theoretical analyses showed that MSA-2 exists as interconverting monomers and dimers in solution, but only dimers bind and activate STING. This model was validated by using synthetic covalent MSA-2 dimers, which were potent agonists. Cellular potency of MSA-2 increased upon extracellular acidification, which mimics the tumor microenvironment. These properties appear to underpin the favorable activity and tolerability profiles of effective systemic administration of MSA-2.

Neurobiology. In the article ''ORK1, a potassium-selective leak channel with two pore domains cloned from Drosophila melanogaster by expression in Saccharomyces cerevisiae '' by Steve A. N. Goldstein, Laura A. Price, David N. Rosenthal, and Mark H. Pausch, which appeared in number 23, November 12, 1996, of Proc. Natl. Acad. Sci. USA (93, 13256-13261), the authors request that the following sequence correction be noted. We have found errors in the ORK1 nucleotide sequence reported in this work. The correct sequence extends the ORF and predicts a protein of 1001 residues; the correct nucleotide and predicted protein sequences are deposited under the GenBank accession no. U55321. The errors do not otherwise alter the conclusions of the paper. We are grateful to Noam Zilberberg (Yale Univ. School of Medicine, New Haven, CT) for his efforts to establish the correct sequence.Neurobiology. In the article "A Rap guanine nucleotide exchange factor enriched highly in the basal ganglia" by Hiroaki Kawasaki, Gregory M. Springett, Shinichiro Toki, Juan J. Canales, Patricia Harlan, Justin P. Blumenstiel, Emy J. Chen, I. Amy Bany, Naoki Mochizuki, Amy Ashbacher, Michiyuki Matsuda, David E. Housman, and Ann M. Graybiel, which appeared in number 22, October 27, 1998, of Proc. Natl. Acad. Sci. USA (95, 13278-13283), due to a printer's error, the gene CalDAG-GEFII was referred to incorrectly in three places: in the heading of the second paragraph of Materials and Methods, in the first line of the Abbreviations footnote, and in line 11 of the second paragraph on page 13282. 318Corrections Proc. Natl. Acad. Sci. USA 96 (1999) Communicated by Vincent T. Marchesi, Yale University, New Haven, CT, August 21, 1996 (received for review July 5, 1996 ABSTRACT A K ؉ channel gene has been cloned from Drosophila melanogaster by complementation in Saccharomyces cerevisiae cells defective for K ؉ uptake. Naturally expressed in the neuromuscular tissues of adult flies, this gene confers K ؉

A detailed analysis of structural and functional aspects of G-protein-coupled receptors, as well as discovery of novel pharmacophores that exert their effects on members of this class of receptors, will be facilitated by development of a yeast-based bioassay. To that end, yeast strains that functionally express the rat somatostatin receptor subtype 2 (SSTR2) were constructed. High-affinity binding sites for somatostatin ([125I-Tyr-11]S-14) comparable to those in native tissues were detected in yeast membrane extracts at levels equivalent to the alpha-mating pheromone receptor (Ste2p). Somatostatin-dependent growth of strains modified by deletion of genes encoding components of the pheromone response pathway was detected through induction of a pheromone-responsive HIS3 reporter gene, enabling cells to grow on medium lacking histidine. Dose-dependent growth responses to S-14 and related SSTR2 subtype-selective agonists that were proportional to the affinity of the ligands for SSTR2 were observed. The growth response required SSTR2, G alpha proteins, and an intact signal transduction pathway. The sensitivity of the bioassay was affected by intracellular levels of the G alpha protein. A mutation in the SST2 gene, which confers supersensitivity to pheromone, was found to significantly enhance the growth response to S-14. In sst2 delta cells, SSTR2 functionally interacted with both a chimeric yeast/mammalian G alpha protein and the yeast G alpha protein, Gpa1p; to promote growth. These yeast strains should serve as a useful in vivo reconstitution system for examination of molecular interactions of the G-protein-coupled receptors and G proteins.

The advent of costimulation blockade provides the prospect for targeted therapy with improved graft survival in transplant patients. Perhaps the most effective costimulation blockade in experimental models is the use of reagents to block the CD40/CD154 pathway. Unfortunately, successful clinical translation of anti-CD154 therapy has not been achieved. In an attempt to develop an agent that is as effective as previous CD154 blocking antibodies but lacks the risk of thromboembolism, we evaluated the efficacy and safety of a novel anti-human CD154 domain antibody (dAb, BMS-986004). The anti-CD154 dAb effectively blocked CD40-CD154 interactions but lacked Fc binding activity and resultant platelet activation. In a non-human primate kidney transplant model, anti-CD154 dAb was safe and efficacious, significantly prolonging allograft survival without evidence of thromboembolism (MST 103 days). The combination of anti-CD154 dAb and conventional immunosuppression synergized to effectively control allograft rejection (MST 397 days). Furthermore, anti-CD154 dAb treatment increased the frequency of CD4+CD25+Foxp3+ regulatory T cells. This study demonstrates that the use of a novel anti-CD154 dAb that lacks Fc binding activity is safe without evidence of thromboembolism and is equally as potent as previous anti-CD154 agents at prolonging renal allograft survival in a non-human primate preclinical model.

CD40–CD40L interactions play a critical role in regulating immune responses. Blockade of CD40L by Abs, such as the anti-CD40L Ab 5c8, demonstrated positive clinical effects in patients with autoimmune diseases; however, incidents of thromboembolism (TE) precluded further development of these molecules. In this study, we examined the role of the Fc domain interaction with FcγRs in modulating platelet activation and potential for TE. Our results show that the interaction of the 5c8 wild-type IgG1 Fc domain with FcγRs is responsible for platelet activation, as measured by induction of PAC-1 and CD62P. A version of 5c8 with a mutated IgG1 tail was identified that showed minimal FcγR binding and platelet activation while maintaining full binding to CD40L. To address whether Fc effector function is required for immunosuppression, a potent Ab fragment, termed a “domain Ab” (dAb), against murine CD40L was identified and fused to a murine IgG1 Fc domain containing a D265A mutation that lacks Fc effector function. In vitro, this dAb–Fc demonstrated comparable potency to the benchmark mAb MR-1 in inhibiting B cell and dendritic cell activation. Furthermore, the anti-CD40L dAb–Fc exhibited a notable efficacy comparable to MR-1 in various preclinical models, such as keyhole limpet hemocyanin–induced Ab responses, alloantigen-induced T cell proliferation, “heart-to-ear” transplantation, and NZB × NZW F1 spontaneous lupus. Thus, our data show that immunosuppression and TE can be uncoupled and that a CD40L dAb with an inert Fc tail is expected to be efficacious for treating autoimmune diseases, with reduced risk for TE.