From hit to vial: Precision discovery and development of an imidazopyrimidine TLR7/8 agonist adjuvant formulation

Abstract

Vaccination can help prevent infection and can also be used to treat cancer, allergy, and potentially even drug overdose. Adjuvants enhance vaccine responses, but currently, the path to their advancement and development is incremental. We used a phenotypic small-molecule screen using THP-1 cells to identify nuclear factor-κB (NF-κB)-activating molecules followed by counterscreening lead target libraries with a quantitative tumor necrosis factor immunoassay using primary human peripheral blood mononuclear cells. Screening on primary cells identified an imidazopyrimidine, dubbed PVP-037. Moreover, while PVP-037 did not overtly activate THP-1 cells, it demonstrated broad innate immune activation, including NF-κB and cytokine induction from primary human leukocytes in vitro as well as enhancement of influenza and SARS-CoV-2 antigen-specific humoral responses in mice. Several de novo synthesis structural enhancements iteratively improved PVP-037's in vitro efficacy, potency, species-specific activity, and in vivo adjuvanticity. Overall, we identified imidazopyrimidine Toll-like receptor-7/8 adjuvants that act in synergy with oil-in-water emulsion to enhance immune responses.

Fig. 1.. High-throughput in vitro screening of primary human PBMCs identified PVP-037, an imidazopyrimidine small-molecule that induces cytokine production.

(A) Schematic diagram depicting HTS of small-molecules with adjuvant effect using peripheral blood mononuclear cells (PBMCs) from adult human donors. A human bio-bank for PBMCs was created by drawing blood from donors over ~6 to 8 months. Downselecting from ~200,000 molecules via a HTS cell line screen, PBMC screening for immunomodulatory molecules was performed using a ~10,000 small-molecule library, via a no-wash 384-well quantitative TNF immunoassay from three adult healthy human donors. Preliminary hits were based on induced TNF production in at least two of the three donors screened. Using statistical analyses, ~250 potential hits were identified, among which ~25 established hits were confirmed with ELISA-based titration assays. (B) Titration of identified hits was performed using adult human PBMCs and TNF production over 18 hours of incubation. PVP-037 emerged as the lead molecule with activity similar to R848 at 33 and 11 μM, respectively. Chemical structure of PVP-037 is shown within the graph. n = 5 donors. (C) PVP-037 induced TNF in PBMCs as compared to R848. PVP-037 lacks NF-κB–inducing activity in cell lines (e.g., monocytic THP-1 cell line). n = 5 donors for PBMCs and two independent experiments for THP-1 screen. (D) Human adult PBMCs were stimulated with PVP-037 or R848 in an eight-point concentration titration manner for 18 hours and supernatant processed for TNF production or (E) 10 plex cytokines analysis. n = 8 donors.

Fig. 2.. Iterative PVP-037–based SAR identified highly potent analogs in vitro that were effective adjuvants in vivo.

(A) Commercially available analogs of PVP-037 were purchased. The activity of these analogs at 11 μM were compared against PVP-037 in human PMBCs, of which 37.37 was identified to have enhanced activity. Further analogs of Cpd 37.37 were then prepared to explore the SAR of the IMP series. The tables depict the amount of TNF produced by representative analogs in a human PBMC stimulation assay with an 18-hour incubation period. Cpd 2-144-3 referred to as PVP-037.1 was identified as the most potent molecule. (B) TNF production after stimulation of human adult PBMCs with PVP-037 and two analogs for 18 hours at 33 μM, presented as % of intradonor R848 response. Results are presented as box-and-whisker plots (n = 5). **P < 0.01 of PVP molecules versus DMSO by repeated measure one-way ANOVA of log-transformed data. Results represent median ± SEM. (C) Human adult PBMCs and murine splenocytes were stimulated with PVP-037 and its analogs for 18 hours at 33 μM. Each dot represents the median production of human TNF (n = 5) and murine IL-6 (n = 6) for each compound. Chemical structures of PVP-037 and PVP-037.1 are shown within the graph. (D) C57BL/6J adult mice (6 to 8 weeks old) were injected intramuscular (IM) prime (day 0)/boost (day 28) with saline, rHA alone, or rHA admixed with the indicated molecules. Titers for rHA-specific total IgG were measured by ELISA 42 days after immunization. Results are presented as box-and-whisker plots of 9 to 10 mice per group (saline, n = 4, alum, n = 2). *P < 0.05, **P < 0.01 of rHA + PVP molecules versus rHA by repeated measure one-way ANOVA of log₁₀-transformed data. Results represent median ± SEM.

Fig. 3.. Amine derivative PVP-037.2 has enhanced potency in vitro and adjuvanticity in vivo.

(A) An amine tag was added to PVP-037.1. (B) PVP-037.2 was identified as a potent analog. After stimulation of human adult PBMCs for 18 hours. n = 8 donors. (C) To evaluate PK (drug serum levels), C57BL/6J adult mice weighing 20 to 30 g received a single formulation administration intravenously of 20 μl per mouse, at 3 mg/kg. Blood was collected (tail vein) from the same mouse before (0 min) and 0.1, 0.25, 0.5, 2, 4, 6, and 8 hours after administration. Left indicates plasma concentration over time. Right indicates % plasma concentration relative to baseline. (D) C57BL/6J mice were injected IM prime (day 0) with saline, rHA, and rHA admixed with the indicated adjuvants. Graphs represents antibody titers at day 28. (E) C57BL/6J adult mice were injected IM prime (day 0) with saline, WT spike protein, and spike admixed with PVP-037.2 (100 nmoles per mouse). Graphs represent antibody titers at day 28 (effect of single-dose immunization). hACE2-RBD (WT) inhibition rate was determined at day 42. In vitro results are presented as line graph (n = 5). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 of small molecules versus DMSO by repeated measure one-way ANOVA of log-transformed data. In vivo results are 5 to 25 mice per group (except, sVNT n = 5). *P < 0.05, **P < 0.01 of rHA + PVP molecules versus rHA by repeated measure one-way ANOVA of log₁₀-transformed data.

Fig. 4.. PVP-037.2 demonstrates TLR7-dependent enhancement of T_H1-polarizing adjuvanticity.

(A) PBMCs from adult human donors were incubated with inhibitory (i)ODNs (TLR7/8/9 antagonists consisting of short single-stranded oligodeoxynucleotides) and either ODN 2088 (TLR7/8/9 antagonist) or ODN 20958 (TLR7 antagonist) for 1 hour. After inhibition with iODNs, cells were stimulated with CL264 (TLR7A) or PVP.037.2, and supernatants were harvested after 20 to 24 hours. TNF production was assayed in the supernatants via ELISA (n = 6 donors). (B) Bone marrow was isolated from WT or Tlr7^−/− mice to generate BMDMs, which were stimulated with 33 μM CL264 (TLR7A) or R848 (TLR7/8A), LPS (100 ng/ml) (TLR4A), 100 μM PVP-037.1, or PVP-037.2. The supernatants were collected after 20 to 24 hours, and TNF production was measured via ELISA (n = 5 samples). (C) WT or Tlr7^−/− mice (6 to 8 weeks old) were injected IM prime with saline, rHA alone, and rHA admixed with the 100 nmol of PVP-037.2. Ab titers for rHA-specific total IgG or IgG2c were measured by ELISA 28 days after immunization (n = 5 samples).

Fig. 5.. PVP-037.2 acts in synergy with a squalene-based oil in water formulation to enhance cytokine induction in vitro and vaccine immunogenicity in vivo.

(A) Graphical representation depicting incorporation of PVP-037.2 in a squalene-based O/W emulsion. (B) Representative pictures of formulation vials, (C) formulation properties, and (D) concentration, particle-size stability, of compositions manufactured for PVP-037.2/SE formulation and the vehicle (SE). (E) RAW-Blue cells [murine RAW264.7 macrophage cell line expressing secreted embryonic alkaline phosphatase (SEAP) reporter construct inducible by NF-κB and AP-1] were stimulated with indicated concentrations of PVP-037.2, PVP-037.2/SE, SE, R848, or DMSO. After 20 to 24 hours, supernatants were harvested, and SEAP production was measured using Quanti-Blue assay for assessing NF-κB activation. (F) BMDCs and (G) BMDMs were stimulated with indicated concentrations of PVP-037.2, PVP-037.2/SE, or SE for 20 to 24 hours and TNF production measured via ELISA (n = 5 samples). (H and I) In addition, cytotoxicity was measured in BMDCs and BMDMs using a lactate dehydrogenase (LDH) colorimetric assay kit, respectively. (J) WT mice (6 to 8 weeks old) were injected IM prime (day 0)/boost (day 28) with saline, rHA alone, and rHA admixed with the 10 nmol of PVP-037.2, PVP-037.2/SE, or SE. Ab titers for rHA-specific total IgG or IgG2c were measured by ELISA 42 days after immunization (n = 5 samples).