SUMMARY Bacterial and host cyclic dinucleotides (cdNs) mediate cytosolic immune responses through the STING signaling pathway, though evidence suggests alternative pathways exist. We used cdN-conjugated beads to biochemically isolate host receptors for bacterial cdNs, and identified the oxidoreductase RECON. High-affinity cdN binding inhibited RECON enzyme activity by simultaneously blocking the substrate and co-substrate sites, as revealed by structural analyses. During bacterial infection of macrophages, RECON antagonized STING activation by acting as a molecular sink for cdNs. Bacterial infection of hepatocytes, which do not express STING, revealed that RECON negatively regulates NF-κB activation. Loss of RECON activity, via genetic ablation or inhibition by cdNs, resulted in increased NF-κB activation and reduced bacterial survival, suggesting that cdN inhibition of RECON promotes a proinflammatory, antibacterial state that is distinct from the anti-viral state associated with STING activation. Thus, RECON functions as a cytosolic pattern recognition receptor specific for bacterial cdNs, shaping inflammatory gene activation via its effects on STING and NF-κB.

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Background: Despite significant clinical benefit of checkpoint inhibitors (CPI) in some settings, unfortunately, the majority of patients still fail to respond and/or develop resistance. This is likely due to a complex set of factors including, but not limited to, the presence of immunosuppressive myeloid cells and/or T cell exhaustion due to chronic TCR activation in the absence of sufficient costimulation. ALPN-202, a variant CD80 vIgD™-Fc fusion protein that mediates PD-L1-dependent CD28 costimulation and blocks PD-L1 and CTLA-4, was designed to overcome several of these suppressive mechanisms. The objective of these studies was to measure effects of ALPN-202 on the suppression of T cell activation by M2c macrophages and to determine in vivo effects of ALPN-202 on T cell exhaustion in an adoptively transferred human TCR transgenic tumor model. Methods: M2c macrophages differentiated from primary monocytes with M-CSF and IL-10 were cocultured for 72hr with T cells, anti-CD3 and a titration of ALPN-202, anti-PD-1, anti-PD-L1, or anti-CTLA-4 antibodies. Cytokine concentrations in the culture media were measured at 24 and 72 hrs, and T cells and macrophages characterized at 72 hrs by flow cytometry. To measure its anti-tumor activity and effect on T cell exhaustion in vivo, ALPN-202 was evaluated in a humanized model using anti-HPV E6 TCR-transduced human T cells transferred into immunodeficient NSG mice bearing HPV+ SCC152 squamous cell tumors stably expressing PD-L1. Tumor volume was measured twice weekly and on day 38 tumors were harvested, digested, and intratumoral T cells characterized for expression of exhaustion markers. Results: In the in vitro coculture assay, ALPN-202 increased T cell proliferation and production of IL-2, IFNγ, TNFα, GM-CSF, IL-6, and IL-21 significantly more potently than CPI alone. Additionally, the M2c macrophages in the presence of ALPN-202 displayed a dose-dependent elevation of MHC II, CD80, and CD86, indicative of a more pro-inflammatory, M1-like phenotype. In the SCC152 tumor model, ALPN-202 induced a more robust anti-tumor response than CPI while the intratumoral T cells expressed lower levels of exhaustion markers. Conclusions: As a dual checkpoint inhibitor and conditional CD28-costimulator, ALPN-202 induced robust and selective T cell costimulation in vitro which overcame M2c macrophage-mediated suppression more potently than CPI alone. Intriguingly, not only were T cells vigorously activated, but M2c macrophages transitioned to a more M1-like proinflammatory phenotype. In a humanized tumor model, ALPN-202 treatment resulted in potent anti-tumor activity with reduced signs of T cell exhaustion in the TME. The data suggest that by combining CD28 costimulation with CPI, ALPN-202 may provide a more robust and persistent anti-tumor T cell response compared to CPI alone. Citation Format: Mark F. Maurer, Siddarth Chandrasekaran, Katherine Lewis, Sherri Mudri, Kayla Kleist, Fariha Ahmed-Qadri, Chelsea Gudgeon, Steven D. Levin, Stacey R. Dillon, Kristine M. Swiderek, Stanford L. Peng. ALPN-202 combines checkpoint inhibition with conditional T cell costimulation to overcome T cell suppression by M2c macrophages and improve the durability of engineered T cell anti-tumor responses [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-085.

Background:BAFF and APRIL are TNF superfamily members that bind both TACI and BCMA on B cells; BAFF also binds BAFF-R. Together, BAFF and APRIL support B cell development, differentiation, and survival. Their co-neutralization dramatically reduces B cell function, including antibody production, whereas inhibition of either BAFF or APRIL alone mediates relatively modest effects.Objectives:While CTLA-4-based therapeutics that block T cell costimulation provide safe and moderately effective T cell inhibition in many disease settings, and while B cell targeting therapies have demonstrated promising therapeutic potential, we postulate that improved, combined BAFF and APRIL inhibition, either alone or coupled with inhibition of T cell costimulation, will provide more effective and durable relief from severe B cell-related autoimmune diseases like SLE.Methods:We used our directed evolution platform to identify variant domains of the TNF family receptors TACI or BCMA that exhibit enhanced affinity for BAFF and APRIL as compared to their wild-type (WT) counterparts. These variant TACI or BCMA domains (vTD), alone or together with platform-derived CTLA-4 domains (vIgD), were fused to a modified human IgG1 Fc lacking effector function, yielding a panel of immunomodulatory molecules: TACI vTD-Fc, BCMA vTD-Fc, TACI vTD/CTLA-4 vIgD-Fc, & BCMA vTD/CTLA-4 vIgD-Fc. All were evaluated for functional activity: 1)in vitroin primary human B cell & MLR assays and in a Jurkat/NF-kB reporter cell line expressing TACI, and 2)in vivoin standard immunization models, and in the bm12-induced and NZB/NZW spontaneous mouse models of lupus.Results:The novel engineered TACI vTD-Fc or BCMA vTD-Fc fusion proteins significantly inhibited BAFF- and APRIL-mediated signalingin vitroin TACI+Jurkat cells. TACI (or BCMA) vTD/CTLA-4 vIgD-Fc proteins also attenuated T cell activation in primary human lymphocyte assays. When administered to mice, these molecules rapidly and potently reduced key B and T cell subsets, including plasma cells, follicular T helper cells, germinal center cells, & memory T cells. Treatment with TACI vTD-Fc or TACI vTD/CTLA-4 vIgD-Fc proteins also significantly reduced titers of antigen-specific antibodies in immunized mice more so than abatacept or WT TACI-Fc, and potently suppressed anti-dsDNA autoantibodies, blood urea nitrogen levels, proteinuria, and renal immune complex deposition in the bm12 & NZB/W lupus models.Conclusion:Directed evolution of TNFR and IgSF domains has successfully facilitated the development of Fc fusion proteins containing TACI or BCMA vTDs, with or without fusion to CTLA-4 vIgDs. These novel immunomodulators consistently demonstrate potent immunosuppressive activity and efficacyin vitroandin vivo, appearing superior to existing and/or approved immunomodulators like belimumab, abatacept, or atacicept. Such biologics may therefore be attractive candidates for the treatment of serious autoimmune diseases, particularly B cell-related diseases such as SLE, Sjogren’s syndrome, etc.Disclosure of Interests: :Stacey R. Dillon Shareholder of: Shareholder of Alpine Immune Sciences, Inc., Employee of: Employee of Alpine Immune Sciences, Inc., Lawrence S. Evans Shareholder of: Shareholder of Alpine Immune Sciences, Inc., Employee of: Employee of Alpine Immune Sciences, Inc., Mark W. Rixon Shareholder of: Shareholder of Alpine Immune Sciences, Inc., Employee of: Employee of Alpine Immune Sciences, Inc., Joe Kuijper Shareholder of: Shareholder of Alpine Immune Sciences, Inc., Employee of: Employee of Alpine Immune Sciences, Inc., Dan Demonte Shareholder of: Shareholder of Alpine Immune Sciences, Inc., Employee of: Employee of Alpine Immune Sciences, Inc., Katherine E. Lewis Shareholder of: Shareholder of Alpine Immune Sciences, Inc., Employee of: Employee of Alpine Immune Sciences, Inc., Steve Levin Shareholder of: Shareholder of Alpine Immune Sciences, Inc., Employee of: Employee of Alpine Immune Sciences, Inc., Kayla Kleist Shareholder of: Shareholder of Alpine Immune Sciences, Inc., Employee of: Employee of Alpine Immune Sciences, Inc., Sherri Mudri Shareholder of: Shareholder of Alpine Immune Sciences, Inc., Employee of: Employee of Alpine Immune Sciences, Inc., Susan Bort Shareholder of: Shareholder of Alpine Immune Sciences, Inc., Employee of: Employee of Alpine Immune Sciences, Inc., Janhavi Bhandari Shareholder of: Shareholder of Alpine Immune Sciences, Inc., Employee of: Employee of Alpine Immune Sciences, Inc., Fariha Ahmed-Qadri Shareholder of: Shareholder of Alpine Immune Sciences, Inc., Employee of: Employee of Alpine Immune Sciences, Inc., Jing Yang Shareholder of: Alpine Immune Sciences, Inc., Employee of: Alpine Immune Sciences, Inc., Michelle A. Seaberg Shareholder of: Shareholder of Alpine Immune Sciences, Inc., Employee of: Employee of Alpine Immune Sciences, Inc., Rachel Wang Shareholder of: Shareholder of Alpine Immune Sciences, Inc., Employee of: Employee of Alpine Immune Sciences, Inc., Russell Sanderson Shareholder of: Alpine Immune Sciences, Inc., Employee of: Alpine Immune Sciences, Inc., Martin F. Wolfson Shareholder of: Shareholder of Alpine Immune Sciences, Inc., Employee of: Employee of Alpine Immune Sciences, Inc., Jan Hillson Shareholder of: Alpine Immune Sciences, Inc., Employee of: Alpine Immune Sciences, Inc., Stanford L. Peng Shareholder of: Alpine Immune Sciences, Inc., Employee of: CMO and President of Alpine Immune Sciences, Inc., Kristine M. Swiderek Shareholder of: Shareholder of Alpine Immune Sciences, Inc., Employee of: Employee of Alpine Immune Sciences, Inc.

Host cells have evolved a sophisticated arsenal of germ-line encoded pattern recognition receptors that detect a vast array of microorganisms in distinct tissues and cellular compartments. Cytoplasmic sensors monitor for microbes that gain access to the host cell cytosol following breach of the plasma membrane or intracellular invasion. This sensing is achieved through detection of invariant molecular patterns associated with microorganisms or cell stress responses that indicate the presence of a pathogen. Cyclic dinucleotides (cdNs) of both bacterial and host origin have emerged as important molecules that are sensed during infection. Details about the receptors cdNs engage in host cells and the responses those interactions elicit are just coming to light. We have recently discovered the first bacterial cdN-specific pattern recognition receptor, the oxidoreductase RECON. Our work on this receptor has revealed its central role in directing innate immune responses when engaged by bacterial cdNs. These findings reveal a new pattern recognition receptor specific for bacterial cdNs that orchestrates cytosolic immune surveillance by shaping downstream inflammatory gene activation.