The precise time when the viral reservoir is seeded during acute HIV-1 infection remains unclear. We previously demonstrated that the viral reservoir was seeded by day 3 following SIVmac251 infection in rhesus monkeys. Here we report the impact of initiating ART on day 0 (6 h), 1, 2, or 3 following intrarectal SIVmac251 infection in 20 rhesus monkeys (N = 5/group). After 6 months of daily suppressive ART, antiretroviral drugs were discontinued, and viral rebound was monitored. 0% (0 of 5), 20% (1 of 5), 60% (3 of 5), and 100% (5 of 5) of animals that initiated ART on days 0 (6 h), 1, 2, or 3, respectively, showed viral rebound following ART discontinuation and correlated with integrated viral DNA in lymph node CD4+ T cells. These data demonstrate that the viral reservoir is seeded within the first few days of infection and that early ART initiation limits the viral reservoir.

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BackgroundIn contrast to immune checkpoint inhibitors, the use of antibodies as agonists of immune costimulatory receptors as cancer therapeutics has largely failed. We sought to address this problem using a new class of modular synthetic drugs, termed tumor-targeted immune cell agonists (TICAs), based on constrained bicyclic peptides (Bicycles).MethodsPhage libraries displaying Bicycles were panned for binders against tumor necrosis factor (TNF) superfamily receptors CD137 and OX40, and tumor antigens EphA2, Nectin-4 and programmed death ligand 1. The CD137 and OX40 Bicycles were chemically conjugated to tumor antigen Bicycles with different linkers and stoichiometric ratios of binders to obtain a library of low molecular weight TICAs (MW <8 kDa). The TICAs were evaluated in a suite of in vitro and in vivo assays to characterize their pharmacology and mechanism of action.ResultsLinking Bicycles against costimulatory receptors (e.g., CD137) to Bicycles against tumor antigens (e.g., EphA2) created potent agonists that activated the receptors selectively in the presence of tumor cells expressing these antigens. An EphA2/CD137 TICA (BCY12491) efficiently costimulated human peripheral blood mononuclear cells in vitro in the presence of EphA2 expressing tumor cell lines as measured by the increased secretion of interferon γ and interleukin-2. Treatment of C57/Bl6 mice transgenic for the human CD137 extracellular domain (huCD137) bearing EphA2-expressing MC38 tumors with BCY12491 resulted in the infiltration of CD8+ T cells, elimination of tumors and generation of immunological memory. BCY12491 was cleared quickly from the circulation (plasma t1/2 in mice of 1–2 hr), yet intermittent dosing proved effective.ConclusionTumor target-dependent CD137 agonism using a novel chemical approach (TICAs) afforded elimination of tumors with only intermittent dosing suggesting potential for a wide therapeutic index in humans. This work unlocks a new path to effective cancer immunotherapy via agonism of TNF superfamily receptors.

Zika virus infection in humans has been associated with serious reproductive and neurological complications. At present, no protective antiviral drug treatment is available. Here, we describe the testing and evaluation of the antiviral drug, galidesivir, against Zika virus infection in rhesus macaques. We conducted four preclinical studies in rhesus macaques to assess the safety, antiviral efficacy, and dosing strategies for galidesivir (BCX4430) against Zika virus infection. We treated 70 rhesus macaques infected by various routes with the Puerto Rico or Thai Zika virus isolates. We evaluated galidesivir administered as early as 90 min and as late as 72 hours after subcutaneous Zika virus infection and as late as 5 days after intravaginal infection. We evaluated the efficacy of a range of galidesivir doses with endpoints including Zika virus RNA in plasma, saliva, urine, and cerebrospinal fluid. Galidesivir dosing in rhesus macaques was safe and offered postexposure protection against Zika virus infection. Galidesivir exhibited favorable pharmacokinetics with no observed teratogenic effects in rats or rabbits at any dose tested. The antiviral efficacy of galidesivir observed in the blood and central nervous system of infected animals warrants continued evaluation of this compound for the treatment of flaviviral infections.

CD137 (4-1BB) is a co-stimulatory receptor on immune cells and Nectin-4 is a cell adhesion molecule that is overexpressed in multiple tumor types. Using a series of poly(ethylene glycol) (PEG)-based linkers, synthetic bicyclic peptides targeting CD137 were conjugated to Bicycles targeting Nectin-4. The resulting bispecific molecules were potent CD137 agonists that require the presence of both Nectin-4-expressing tumor cells and CD137-expressing immune cells for activity. A multipronged approach was taken to optimize these Bicycle tumor-targeted immune cell agonists by exploring the impact of chemical configuration, binding affinity, and pharmacokinetics on CD137 agonism and antitumor activity. This effort resulted in the discovery of BT7480, which elicited robust CD137 agonism and maximum antitumor activity in syngeneic mouse models. A tumor-targeted approach to CD137 agonism using low-molecular-weight, short-acting molecules with high tumor penetration is a yet unexplored path in the clinic, where emerging data suggest that persistent target engagement, characteristic of biologics, may lead to suboptimal immune response.

The 2015 Brazilian Zika virus outbreak sparked a rapid response to control the spread of the virus. What was first understood to be a mild self-resolving infection is now linked to significant neurological defects in both neonates and adults. The WHO declared the 2016 Zika epidemic a public health emergency and issued an unprecedented recommendation to women in affected regions to delay pregnancy until the risks surrounding Zika virus could be understood, or the epidemic contained. Since that time, considerable effort has been dedicated to understanding Zika transmission and pathogenesis to aid the development of drugs and vaccines. Several models have emerged to study numerous facets of Zika biology; this review details the various model systems.

Costimulatory molecules expressed on activated T and NK cells such as 4-1BB (CD137/TNFRSF9) can be leveraged for cancer immunotherapy. Despite compelling preclinical data, 4-1BB agonistic antibodies have been hampered by failure to delineate hepatoxicity from efficacy in the clinic [1,2]. Next generation strategies are focused on bispecific approaches aimed at promoting target-mediated clustering of 4-1BB to limit systemic and liver effects [3,4]. Bicycles® are fully synthetic, constrained bicyclic peptides that have antibody-like affinity and selectivity to their targets. Unlike traditional biologic approaches, the small size (~2 kDa) and tunable pharmacokinetic (PK) parameters of Bicycles enable superior tumor penetration and allow exploration into the relationship between pulsatile dosing and 4-1BB activation while de-risking hepatoxicity concerns due to a differentiated renal elimination mechanism combined with a tumor-localized immune response. We hypothesized that clustering and activation of 4-1BB could be achieved by conjugating a 4-1BB binding Bicycle to a tumor antigen targeting Bicycle. BT7480 is a tumor-targeted immune cell agonist (TICATM) targeting Nectin-4 and agonizing 4-1BB. Nectin-4 (PVRL4) is highly expressed on numerous tumors with unmet medical need, including bladder, pancreatic, breast, ovarian, esophageal, and lung. BT7480 exhibits highly potent 4-1BB agonism in an engineered 4-1BB reporter system that correlates with Nectin-4 surface expression on the co-cultured tumor cells. In addition, BT7480 induces robust production of interleukin-2 (IL-2) and interferon gamma (IFNγ) in primary PBMC/tumor cell co-culture assays. This activity is strictly dependent on the tumor cells expressing Nectin-4 and on the ability of the TICA to bind to both Nectin-4 and 4-1BB. Nectin-4/4-1BB TICAs are also target-specific immune cell stimulators of patient-derived lung tumors with an intact immune microenvironment. Intermittent dosing of BT7480 led to robust anti-tumor efficacy with 22 out of 24 complete responders (CRs) in a MC38 (Nectin-4-expressing) syngeneic mouse model. Importantly, a memory response was established as the CR mice were resistant to re-challenge with MC38 tumors. Additionally, BT7480 led to increased intratumoral T cell infiltration without elevation of liver enzymes in a CT26 (Nectin-4-expressing) syngeneic mouse model. In non-human primates (NHPs), BT7480 exhibits dose linear exposure and is well tolerated up to 10mpk. Further dose-range finding and safety analysis in NHPs is currently ongoing. BT7480 represents a new generation of chemically synthetic tumor antigen targeted 4-1BB agonists. References 1. Segal NH, Logan TF, Hodi FS, et al. Results from an integrated safety analysis of urelumab, an agonist anti-CD137 monoclonal antibody. Clin Cancer Res. 2017;23(8):1929-1936. 2. Chester et al. Immunotherapy targeting 4-1BB: mechanistic rationale, clinical results, and future strategies. Blood 2018;131(1): 49-57. 3. Hinner et al. Tumor-localized costimulatory T-cell engagement by the 4-1BB/HER2 bispecific antibody-anticalin fusion PRS-343. Clin Cancer Res. 2019; 25(19): 5878-5889. 4. Claus C, Ferrara, C, Xu W, et al. Tumor-targeted 4-1BB agonists for combination with T cell bispecific antibodies as off-the-shelf therapy. Sci Transl Med. 2019; 11(496): eaav5989. Citation Format: Kristen Hurov, Punit Upadhyaya, Johanna Lahdenranta, Jessica Kublin, Jun Ma, Elizabeth Repash, Marianna Kleyman, Julia Kristensson, Liuhong Chen, Eric Haines, Sailaja Battula, Kevin McDonnell, Nicholas Keen. BT7480, a novel fully synthetic tumor-targeted immune cell agonist (TICATM) induces tumor localized 4-1BB agonism [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 5552.

BackgroundAfter disappointing first clinical experiences with agonistic anti-CD137 (4-1BB) antibodies, a new generation of both systemic and targeted CD137 agonists is entering clinical development.1–3 These strategies rely on biologic agents with suboptimal properties for CD137 agonism due to their relatively large sizes and long circulating half-lives. These properties may limit their tissue penetration and cause sustained agonism resulting in overstimulation and activation-induced cell death of lymphocytes due to continuous exposure.Fully synthetic constrained bicyclic peptides (Bicycles™) with antibody-like affinities and target selectivity are uniquely suited to circumvent the above barriers to optimal targeted CD137 agonistic therapeutics. BT7480 is a tumor-targeted immune cell agonist (TICA) designed to deliver a highly potent CD137 agonist to Nectin-4 overexpressing tumor tissue with a flexible dosing schedule maximizing anti-tumor activity while circumventing the need for continuous systemic exposure.MethodsBT7480 functional activity in vitro was analyzed by measuring IL-2 and IFN gamma production from primary human PBMC/tumor cell co-cultures. BT7480 in vivo activity was determined in huCD137-syngeneic tumor models using tumor immune cell and transcriptional profiling by FACS, IHC, and Nanostring as well as tumor growth kinetics as read-outs.ResultsBT7480 binds potently and simultaneously to Nectin-4 and CD137 as assessed biochemically and caused Nectin-4-dependent CD137 agonism in primary human PBMC co-cultured with tumor cells. Treatment of Nectin-4 expressing tumors in immunocompetent mice with BT7480 leads to profound reprogramming of the tumor immune microenvironment including increased T cell infiltration and upregulation of a cytotoxic cell gene signature. BT7480 treatment induces complete tumor regressions and subsequent resistance to tumor re-challenge. TICA-dependent anti-tumor activity and established immunologic memory are dependent on cytotoxic T cells. Importantly, BT7480 in vivo activity is not dependent on continuous plasma exposure since once weekly dosing of BT7480 provides a maximum anti-tumor activity despite minimal BT7480 plasma exposure after day 2.BT7480 demonstrates linear pharmacokinetics in non-human primates and appears well tolerated at exposures in excess of the predicted efficacious exposure in humans.ConclusionsBT7480 is a highly potent Nectin-4 expression dependent CD137 agonist with optimal target binding, pharmacologic, and pharmacokinetic properties that enable intermittent dosing for curative effect through modulation of tumor immune microenvironment in syngeneic mouse tumor models. BT7480 is currently being evaluated in IND-enabling safety studies.Ethics ApprovalThe care and use of animals were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of WuXi AppTec and conducted in accordance with the regulations of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC).ReferencesHinner, et al. Tumor-localized costimulatory t-cell engagement by the 4-1BB/HER2 Bispecific antibody-anticalin fusion PRS-343. Clin. Cancer Res 2019 Oct 1;25(19):5878–5889.Claus, et al. Tumor-targeted 4-1BB agonists for combination with T cell bispecific antibodies as off-the-shelf therapy. Sci. Transl. Med 2019 Jun 12;11(496):eaav5989.Eskiocak, et al. Differentiated agonistic antibody targeting CD137 eradicates large tumors without hepatotoxicity. JCI Insight 2020 Mar 12;5(5):e133647.