Adjuvant effect of the novel TLR1/TLR2 agonist Diprovocim synergizes with anti–PD-L1 to eliminate melanoma in mice

Diprovocim Induces Cytokine Production in both Human and Mouse Cells.

From a chemical library containing ∼100,000 members, we identified a class of compounds with bilateral symmetry capable of activating TNF biosynthesis in phorbol 12-myristate 13-acetate (PMA)-differentiated human THP-1 myeloid cells. The initial members of the class emerged from an undisclosed compound sublibrary designed to promote cell surface receptor dimerization (18). Diprovocim (Fig. 1A) was developed from this class after extensive structure–activity relationship (SAR) studies. It induced dose-dependent TNF production by THP-1 cells (EC₅₀ 110 pM) and human peripheral blood mononuclear cells (PBMC) (EC₅₀ 875 pM) (Fig. 1 B and C) and by mouse peritoneal macrophages (EC₅₀ 1.3 nM) and bone marrow-derived dendritic cells (BMDC) (EC₅₀ 6.7 nM) (Fig. 1 D and E). In addition to TNF, Diprovocim induced IL-6 production by mouse BMDC (Fig. 1F). However, Diprovocim failed to stimulate type I IFN production by mouse peritoneal macrophages (SI Appendix, Fig. S1).

Diprovocim Targets TLR1/TLR2 and Activates Downstream MAPK and NF-κB Signaling Pathway.

To determine the molecular target of Diprovocim, we analyzed its effects on peritoneal macrophages from wild-type C57BL/6J mice and C57BL/6J mice deficient in various TLR signaling components. Induction of TNF by Diprovocim was completely absent in TLR1- or TLR2-deficient macrophages but not in TLR-6 deficient macrophages (Fig. 2A). Diprovocim activity was also dramatically reduced in macrophages from MyD88-, TIRAP-, and IRAK4-deficient cells (Fig. 2A). These data suggest that Diprovocim targets the mouse TLR1/TLR2 heterodimer. TLR1 or TLR2 antibody significantly reduced the effect of Diprovocim on THP-1 cells, indicating that human TLR1/TLR2 is also a target of Diprovocim (Fig. 2B). Diprovocim induced phosphorylation of IKKα, IKKβ, p38, JNK, and ERK, as well as degradation of IκBα in THP-1 cells and mouse peritoneal macrophages, indicating that Diprovocim activates conventional TLR1/TLR2 signaling, including MAPK and canonical NF-κB signaling (Fig. 2 C and D).

Diprovocim Exhibits Adjuvant Activity in Vivo.

Intramuscular immunization of wild-type mice with ovalbumin (OVA) plus either alum or Diprovocim induced similar levels of serum OVA-specific IgG, which were highly elevated compared with levels induced by immunization with OVA plus vehicle (Fig. 3 A–C). Whereas immunization with OVA + alum induced primarily the Th2-related Ig subclass IgG1, OVA + Diprovocim induced both IgG1 and the Th1-related IgG2b (Fig. 3 B and C).

DCs purified from draining lymph nodes and spleens 24 h after immunization of mice with OVA + Diprovocim activated OT-I CD8 T cells cocultured with them, as evidenced by CD69 up-regulation on the OT-I cells (Fig. 3D). In contrast, DCs from mice immunized with OVA + vehicle failed to induce CD69 expression on OT-I CD8 T cells (Fig. 3D). This finding suggests that Diprovocim activates antigen cross-presentation by DCs and cross-priming of CD8 T cells in vivo. To further investigate whether cross-priming stimulated by Diprovocim results in the development of killing ability by CD8+ T cells in vivo, a CTL killing assay was performed. Fluorescent labeled and OVA peptide (aa 257–263)-pulsed target cells were injected i.v. into mice immunized with OVA + Diprovocim, and the number of live target cells was measured by flow cytometry 2 d later. About 70% of target cells had been eliminated in mice immunized with OVA + Diprovocim, compared with ∼10% in mice immunized with OVA + vehicle (Fig. 3E). These data demonstrate that Diprovocim exhibits adjuvant activity in antigen-specific antibody production and CTL killing, which was abrogated in TLR1- and/or TLR2-deficient mice (Fig. 3 A–C, F, and G).

Complete Inhibition of B16 Tumor Growth by Combined Checkpoint Blockade and Anticancer Vaccine Adjuvanted with Diprovocim.

We tested the adjuvant activity of Diprovocim in preventive immunization of wild-type mice against B16 melanoma expressing OVA (B16-OVA) (Fig. 4A). Mice were injected i.m. distal to the tumor cell injection site with OVA with or without Diprovocim on the same day but before inoculation with B16-OVA cells. Tumor growth rates and survival times were similar for mice immunized with vehicle alone, Diprovocim alone, or OVA alone (Fig. 4 B and C). Relative to OVA alone, immunization with Diprovocim + OVA modestly but significantly slowed tumor growth rate but failed to prolong survival; a similar effect was observed with OVA immunization combined with anti–PD-L1 treatment (Fig. 4 B and C). Strikingly, although without effect by itself (SI Appendix, Fig. S2), when anti–PD-L1 treatment was added to Diprovocim + OVA immunization, there was complete inhibition of tumor growth and 100% survival through 8 wk of observation (Fig. 4 B and C). This dramatic antitumor effect was dependent on OVA immunization because Diprovocim alone combined with anti–PD-L1 treatment had no effect on tumor growth or mouse survival (Fig. 4 D and E); this finding is consistent with poor immunogenicity of B16 melanoma (19–21).

To determine whether surviving mice were endowed with specific and long-term memory directed against the cancer antigen, we rechallenged the 5-wk survivors from Fig. 4C with B16-OVA cells and B16 cells lacking OVA (B16). In the absence of any further therapy, we observed complete failure of B16-OVA tumor growth, whereas B16 tumors grew rapidly (Fig. 4F). Both B16 and B16-OVA tumor cells grew at similar rates in naïve C57BL/6J mice (Fig. 4F). Taken together, these data indicate that when used as an adjuvant in a cancer vaccine, Diprovocim promotes antigen-specific antitumor immunity, which is greatly enhanced when combined with T cell checkpoint blockade in mice. Immunization with Diprovocim as an adjuvant produces antigen-specific memory responses that protect the host from relapse of tumor growth.

We tested the antitumor effect of Diprovocim in therapeutic immunization of mice with already established B16-OVA tumors. C57BL/6J mice were immunized with OVA with or without Diprovocim on the day of or 3 d after tumor inoculation and received a booster immunization 7 d later (Fig. 4A). In some mice, alum was substituted for Diprovocim to permit direct comparison between these two adjuvants. For all conditions, anti–PD-L1 treatment was initiated on day 3 after tumor inoculation and repeated every 3 d thereafter for 12 d. Mice immunized with OVA alone on the day of tumor inoculation survived an average of 24 d, and 100% of mice (8/8) died by 38 d after tumor inoculation. As expected, Diprovocim + OVA administered on the day of tumor inoculation completely inhibited tumor growth, permitting 100% of mice (8/8) to survive through 54 d of observation (Fig. 4 G and H). When alum was used instead of Diprovocim in the same experiment, tumor growth was partially inhibited, and the average survival time was 37 d, with 25% of mice (2/8) surviving past 54 d (Fig. 4 G and H). When immunization was delayed until 3 d after tumor inoculation, Diprovocim + OVA still significantly inhibited tumor growth and prolonged average survival compared with OVA alone (41 d vs. 22 d) (Fig. 4 I and J). In contrast, alum + OVA inhibited tumor growth but only slightly increased average survival time (30 d); the survival curve for alum + OVA was not significantly different from the curve for OVA alone (P = 0.082) (Fig. 4 I and J). In pretumor and posttumor treatment, the effect of Diprovocim on tumor growth, survival rate, and survival time was superior to that of alum.

Diprovocim Enhances Antitumor CTL Responses.

To investigate the cellular mechanism by which combined anti–PD-L1 treatment and Diprovocim-adjuvanted immunization eliminates tumors, we analyzed the tumor-infiltrating leukocytes (TILs) of mice immunized with Diprovocim + OVA or alum + OVA 3 d after tumor inoculation (Fig. 5A). Tumors were collected 14 d after inoculation, and single-cell suspensions were antibody stained and analyzed by flow cytometry to detect total leukocytes, CD4 and CD8 T cells, NK cells, DCs, and macrophages. The leukocytes were also stained with antibody to the H-2K^b MHC-class I tetramer bound to the OVA peptide (aa 257–264), as well as antibody against CD8 to identify tumor-specific CD8 T cells. OVA immunizations containing Diprovocim significantly increased the frequency of leukocytes in tumors compared with vehicle + OVA (Fig. 5B). Further analysis of these TILs revealed that Diprovocim increased the frequencies of CD4 and CD8 T cells including activated CD4 and CD8 T cells (CD44^high) and OVA-specific CD8 T cells, as well as the frequency of NK cells (Fig. 5 C–H). Alum + OVA immunization showed a trend toward increasing TILs (Fig. 5B), which reached statistical significance for total and CD44^high CD8 T cells (Fig. 5 E and F); however, the magnitude of the increase was reduced compared with that induced by Diprovocim + OVA. OVA-specific CD8 T cells were not increased by alum + OVA immunization (Fig. 5G) on day 14 after tumor inoculation; neither were total and CD44^high CD4 T cells (Fig. 5 C and D), nor NK cells compared with vehicle + OVA (Fig. 5H). The frequencies of intratumor DCs and macrophages were similar for mice immunized with vehicle + OVA, Diprovocim + OVA, and alum + OVA (Fig. 5 I and J). Overall, these data indicate that the intratumor frequencies of CD4 and CD8 T cells, activated CD4 and CD8 T cells, OVA-specific CD8 T cells, and NK cells, but not DCs or macrophages, correlated with the antitumor effects of Diprovocim and alum in immunized mice.

To determine the immune cell population(s) necessary for the antitumor effect of Diprovocim + OVA plus anti–PD-L1, we depleted mice of CD8 T cells, CD4 T cells, NK cells, or all three cell populations using cell type-specific antibodies. The depletion antibodies were administered i.p. on the day of B16-OVA tumor inoculation (day 0) and every 3 d thereafter for 15 d (Fig. 5K). The effect of Diprovocim + OVA on both tumor growth and mouse survival was abrogated when mice were depleted of CD8 T cells or all three cell types together (CD4 T, CD8 T, and NK cells) (Fig. 5 L and M). In contrast, depletion of CD4 T cells or NK cells had little effect on the antitumor activity of Diprorocim + OVA (Fig. 5 L and M). We noted a slight, statistically significant difference between the effects of CD8 T cell depletion vs. CD4 + CD8 + NK cell depletion on tumor growth in mice treated with Diprovocim + OVA plus anti–PD-L1, in which tumor growth was greater in mice depleted of all three cell types. However, this difference did not translate to a difference in either survival rate or time. This finding suggests a minor role of either CD4 T cells, NK cells, or both, in mediating the antitumor effects of Diprovocim + OVA plus anti–PD-L1. These data demonstrate that CD8 T cells are necessary for tumor eradication by therapeutic Diprovocim + OVA immunization and checkpoint inhibition in mice.

Synthesis of Diprovocim.

The protocol for synthesis of Diprovocim is provided in SI Appendix.

Mice.

C57BL/6J, Tlr2^−/−, Myd88^−/−, and OT-I mice were purchased from The Jackson Laboratory. Ly96^−/− (MD-2^−/−) mice were from Riken BioResource Research Center. Tlr4^lps3/lps3, Tlr6^int/int, Tlr7^rsq1/rsq1, Tirap^tor/tor, Ticam1^Lps2/Lps2, and Ticam1^Lps2/Lps2/Irak4^{otiose/otiose} mice were generated on a pure C57BL/6J background by ENU mutagenesis and are described at mutagenetix.utsouthwestern.edu.

Tlr1^−/− mice were created by CRISPR/Cas 9 gene targeting. Female C57BL/6J mice were superovulated by injection of 6.5 U pregnant mare serum gonadotropin (PMSG; Millipore), followed by injection of 6.5 U human CG (hCG; Sigma–Aldrich) 48 h later. The superovulated mice were subsequently mated overnight with C57BL/6J male mice. The following day, fertilized eggs were collected from the oviducts, and in vitro-transcribed Cas9 mRNA (50 ng/μL) and Tlr1 small base-pairing guide RNA (50 ng/μL; 5′-CAAACCGATCGTAGTGCTGA-3′) were injected into the cytoplasm or pronucleus of the embryos. The injected embryos were cultured in M16 medium (Sigma–Aldrich) at 37 °C in 5% CO₂. For the production of mutant mice, two-cell stage embryos were transferred into the ampulla of the oviduct (10–20 embryos per oviduct) of pseudopregnant Hsd:ICR (CD-1) female mice (Harlan Laboratories).

All experimental procedures using mice were approved by the Institutional Animal Care and Use Committee of the University of Texas Southwestern Medical Center and were conducted in accordance with institutionally approved protocols and guidelines for animal care and use. All of the mice were maintained at the University of Texas Southwestern Medical Center in accordance with institutionally approved protocols.

Isolation of Peritoneal Macrophages, BMDC, Human PBMC, and Cell Culture.

Thioglycollate-elicited macrophages were recovered 4 d after i.p. injection of 2 mL BBL thioglycollate medium, brewer modified (4% wt/vol; BD Biosciences) by peritoneal lavage with 5 mL PBS. The peritoneal macrophages were cultured in DMEM cell culture medium [DMEM containing 10% vol/vol FBS (Gemini Bio Products), 1% vol/vol penicillin and streptomycin (Life Technologies)] at 37 °C and 95% air/5% CO₂. For murine BMDCs, bone marrow cells were cultured in Petri dishes in 10 mL DMEM cell culture medium containing 10 ng/mL of murine GM-CSF (R&D Systems). On day 3 of culture, this was replaced with fresh GM-CSF medium. Loosely adherent cells were transferred to a fresh Petri dish and cultured for an additional 4 d. Human PBMC were purchased from Stemcell Technologies. THP-1 (American Type Culture Collection) cells were differentiated by treatment with 100 nM PMA (Sigma) in Roswell Park Memorial Institute (RPMI) cell culture medium [RPMI containing 10% vol/vol FBS (Gemini Bio Products), 1% penicillin and streptomycin (Life Technologies)] for 24 h. After that, cells were washed with PBS and cultured in fresh RPMI cell culture medium for 24 h before use in experiments.

Measurement of Cytokine Production.

Cells were seeded onto 96-well plates at 1 × 10⁵ cells per well and stimulated with Diprovocim [dissolved in DMSO, and final DMSO concentrations (≤0.2%) were kept constant in all experiments] for 4 h. Mouse TNF, IL-6, or IFN-β or human TNF in the supernatants were measured by ELISA kits according to the manufacturer’s instructions (eBioscience and PBL Assay Science). Pretreatment with 20 μg/mL anti-TLR1, anti-TLR2, or isotype control antibody (eBioscience) was for 1 h. Unless otherwise indicated, mouse cells were from wild-type C57BL/6J mice.

Western Blotting.

Mouse peritoneal macrophages or human THP-1 cells (1 × 10⁶ per well) were stimulated in 12-well plates with Diprovocim at 500 nM for mouse cells or 5 nM for human cells for the indicated times and lysed directly in sample buffer (Sigma). Cell lysates were separated by SDS/PAGE and transferred to nitrocellulose membranes. Membranes were probed with the following antibodies: phospho-IKKα (Ser176)/IKKβ (Ser177), IκBα, phospho-p38 (Thr180/Tyr182), phospho-JNK (Thr183/Tyr185), phospho-ERK1/2 (Thr202/Tyr204) (Cell Signaling Technology), and β-Actin (Sigma).

Immunization and Measurement of Antibody Response.

EndoFit ovalbumin (OVA) with ≥98% purity minimum (SDS/PAGE) and endotoxin levels <1 EU/mg was purchased from Invivogen. Mice (four mice per group) were immunized i.m. with 100 μg OVA mixed with vehicle (DMSO:Tween 80:saline = 1:1:8), with 10 mg/kg Diprovocim, or with 2 mg/kg Alum (Alhydrogel adjuvant 2%; Invivogen). After 14 d, serum titers of OVA-specific IgG, IgG1, or IgG2b (SouthernBiotech) were measured by ELISA.

In Vivo CTL Killing Assay.

C57BL/6J male mice were injected i.m. with 100 μg OVA plus 10 mg/kg Diprovocim (n = 4 mice per group). One week later, naive C57BL/6J mice were killed, and splenocytes were collected. Half of the splenocytes were left unpulsed, and half were pulsed with OVA257–263 peptides for 2 h in complete medium (RPMI containing 10% vol/vol FBS, 1% penicillin and streptomycin) at 37 °C. The unpulsed and peptide-pulsed cells were labeled with 0.5 μM (low) or 5 μM (high) CellTrace Violet (Invitrogen), respectively, in serum-free medium for 20 min. Equal numbers (2 × 10⁶) of CellTrace Violet^high (OVA pulsed) and CellTrace Violet^low (unpulsed) cells were mixed together and injected i.v. into the immunized mice. After 48 h, blood from treated mice was collected and subjected to flow cytometry analysis. The numbers of remaining live CellTrace Violet^high and CellTrace Violet^low cells were determined and used to calculate the percentage of OVA peptide-pulsed target cells killed. Specific killing was defined as the ratio = CellTrace Violet^low cells/CellTrace Violet^high cells. The percentage of target cell lysis = (1 − unimmunized ratio/immunized ratio) × 100.

Tumor Inoculation, Immunization, and Tumor Measurement.

B16-OVA cells (B16F10 melanoma cells stably expressing chicken ovalbumin) were grown in DMEM containing 10% vol/vol FBS. A total of 2 × 10⁵ B16-OVA cells in 100 μL PBS were injected s.c. into the right flank of 8- to 12-wk-old male C57BL/6J mice to establish tumors (n = 8 mice per group). For pretreatment, 10 mg/kg Diprovocim or 2 mg/kg alum with or without OVA (100 μg) was injected i.m. into mice on the same day as tumor inoculation (day 0). Mice received a booster shot 7 d after the first immunization. On day 3, 6, and 9, some groups were injected i.p. with 200 μg checkpoint inhibitor (anti–mPD-L1; BioXcell) in 100 μL saline. For posttreatment, 10 mg/kg Diprovocim or 2 mg/kg Alum with OVA (100 μg) was injected i.m. into mice on day 3 after tumor inoculation. Mice received a booster shot 7 d after the first immunization. On day 3, 6, 9, 12, and 15 after tumor inoculation, mice were also injected i.p. with 200 μg anti–mPD-L1 in 100 μL saline. For depletion of CD4 T cells, CD8 T cells, and/or NK cells, 300 μg anti-mCD4 (BioXcell), 300 μg anti-mCD8 (BioXcell), 300 μg anti-mNK1.1 (BioXcell), or the three antibodies together in 200 μL saline were injected i.p. into mice on day 0, 3, 6, 9, 12, and 15 after tumor inoculation. On day 3 after tumor inoculation, 10 mg/kg Diprovocim with OVA (100 μg) or vehicle was injected i.m. into mice. Mice received a booster shot 7 d after the first immunization. On day 3, 6, 9, 12, and 15 after tumor inoculation, mice were also injected i.p. with 200 μg anti–mPD-L1 in 100 μL saline. Tumors were measured with a digital caliper (Fisher), and the tumor sizes were calculated using the following formula: volume = 0.5 × length × width². Mice were killed when the tumor length or width reached 2 cm.

Tumor-Infiltrating Leukocyte Separation and Staining.

A total of 2 × 10⁵ B16-OVA cells in 100 μL PBS were injected s.c. into the flank of each mouse to establish tumors (n = 6 mice per treatment). On day 3 after tumor inoculation, 10 mg/kg Diprovocim or 2 mg/kg alum with OVA (100 μg) was injected i.m. into mice. Mice received a booster shot 7 d after the first immunization. On day 3, 6, 9, and 12 after tumor inoculation, mice were also injected i.p. with 200 μg anti–mPD-L1. On day 14 after tumor inoculation, tumors were harvested, minced, and filtered through a 40-μm strainer to obtain single-cell suspensions. Red blood cells were lysed with RBC lysis buffer (Sigma). After pelleting, cells were stained with a mixture of antibodies for 45 min, including anti-mouse CD45.2-PE or CD45.2-APC (BioLegend), anti-mouse CD3-FITC (BD Biosciences), anti-mouse CD4-BV786 (BD Biosciences), anti-mouse CD8-BV510 (BioLegend), anti-mouse CD44-PE-CF594 (BioLegend), APC-conjugated H-2Kb/OVA (SIINFEKL) tetramer (Baylor College of Medicine), anti-mouse F4/80-PE (Tonbo Bioscience), anti-mouse CD11b-BV605 (BioLegend), anti–CD11c-BV711 (BD Biosciences), and anti–NK1.1-BV650 (BD Biosciences). Then, the cells were washed twice with PBS. Stained cells were analyzed with an LSR II flow cytometer (BD Biosciences), and the flow cytometry data were analyzed using FlowJo software.

Measurement of Cross-Priming of CD8 T Cells.

C57BL/6J male mice were injected i.m. with 100 μg OVA mixed with vehicle or with 10 mg/kg Diprovocim (n = 4 mice per treatment). Twenty-four hours later, DCs from draining lymph nodes and spleen were purified by Mouse Pan Dendritic Cell Isolation Kit (Miltenyi Biotech). CD8 T cells from OT-I transgenic mice were purified by Mouse CD8+ T Cell Isolation Kit (Miltenyi Biotech). Then, 3 × 10⁵ DCs were cocultured with 3 × 10⁵ OT-I CD8 T cells in RPMI medium containing 10% vol/vol FBS and 1% vol/vol penicillin and streptomycin for 24 h. Then, cells were collected and stained with anti-mouse CD3-FITC, anti-mouse CD8-BV510, and anti-mouse CD69-PE-CF594 (BioLegend) for 45 min. Then, the cells were washed twice with PBS. Stained cells were analyzed with an LSRII instrument, and the flow cytometry data were analyzed using FlowJo software.

Statistical Analyses.

Data represent means ± SEM in all graphs depicting error bars. The statistical significance of differences between experimental groups was determined using GraphPad Prism 7 and the indicated statistical tests. For comparisons of differences between two unpaired experimental groups, an unpaired Student’s t test was used, and two-tailed P values are reported. P values are indicated by *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001. P ≤ 0.05 was considered statistically significant.