Immunomodulation to enhance the efficacy of an HPV therapeutic vaccine

Abstract

Background: While prophylactic human papillomavirus (HPV) vaccines will certainly reduce the incidence of HPV-associated cancers, these malignancies remain a major health issue. PDS0101 is a liposomal-based HPV therapeutic vaccine consisting of the immune activating cationic lipid R-DOTAP and HLA-unrestricted HPV16 peptides that has shown in vivo CD8+ T cell induction and safety in a phase I study. In this report, we have employed the PDS0101 vaccine with two immune modulators previously characterized in preclinical studies and which are currently in phase II clinical trials. Bintrafusp alfa (M7824) is a first-in-class bifunctional fusion protein composed of the extracellular domains of the transforming growth factor-β receptor type II (TGFβRII) fused to a human IgG₁ monoclonal antibody blocking programmed cell death protein-1 ligand (PDL1), designed both as a checkpoint inhibitor and to bring the TGFβRII 'trap' to the tumor microenvironment (TME). NHS-interleukin-12 (NHS-IL12) is a tumor targeting immunocytokine designed to bring IL-12 to the TME and thus enhance the inflammatory Th1 response.

Methods: We employed TC-1 carcinoma (expressing HPV16 E6 and E7 and devoid of PDL1 expression) in a syngeneic mouse model in monotherapy and combination therapy studies to analyze antitumor effects and changes in immune cell types in the spleen and the TME.

Results: As a monotherapy, the PDS0101 vaccine generated HPV-specific T cells and antitumor activity in mice bearing HPV-expressing mEER oropharyngeal and TC-1 lung carcinomas. When used as a monotherapy in the TC-1 model, NHS-IL12 elicited antitumor effects as well as an increase in CD8+ T cells in the TME. When used as a monotherapy, bintrafusp alfa did not elicit antitumor effects or any increase in T cells in the TME. When all three agents were used in combination, maximum antitumor effects were observed, which correlated with increases in T cells and T-cell clonality in the TME.

Conclusion: These studies provide the rationale for the potential clinical use of combinations of agents that can (1) induce tumor-associated T-cell responses, (2) potentiate immune responses in the TME and (3) reduce immunosuppressive entities in the TME.

Keywords: genital Neoplasms, female; head and neck neoplasms; immunotherapy; therapies, investigational; vaccination.

Figure 1

PDS0101 monotherapy reduced tumor volume in the TC-1 syngeneic tumor model, and the combination of PDS0101, bintrafusp alfa and NHS-IL12 resulted in further tumor control. TC-1 tumor bearing female C57BL/6J mice (n=8–16 per group) were treated with PBS control (100 µL s.c), R-DOTAP control (100 µL s.c), PDS0101 (s.c., 3 weekly doses starting on day 7), bintrafusp alfa (250 µg, i.p., days 7, 9, and 11) and/or NHS-IL12 (50 µg, s.c., day 7). (A) Individual growth curves for PBS control, R-DOTAP, and PDS0101 treated mice. (B) Individual growth curves for PDS0101, bintrafusp alfa, and PDS0101 plus bintrafusp alfa treated mice. (C) Individual growth curves for PDS0101, NHS-IL12, and PDS0101 plus NHS-IL12 treated mice. (D) Individual growth curves for bintrafusp alfa, NHS-IL12, and bintrafusp alfa plus NHS-IL12 treated mice. (E) Individual growth curves for PDS0101, bintrafusp alfa, NHS-IL12, and PDS0101 plus bintrafusp alfa plus NHS-IL12 treated mice. (F) Tumor weights at the end of study. (G) Table of ‘tumor control’, the number of mice with tumors below 300 mm³ at the end of study. A meta-analysis of two independent experiments is shown. **P<0.01, ****P<0.0001. IL12, interleukin-12; i.p. intraperitoneally; s.c., subcutaneously.

Figure 2

CD4 and CD8 T cell infiltration into tumors. TC-1 tumor bearing female C57BL/6J mice (n=4/group) were treated with PBS control (100 µL s.c.), R-DOTAP control (100 µg s.c.), PDS0101 (s.c., 3 weekly doses starting on day 7), bintrafusp alfa (250 µg, i.p., days 7, 9, and 11), and/or NHS-IL12 (50 µg, s.c., day 7). (A) Representative flow cytometry plots of CD4+ and CD8+ T cells in the spleen and tumor of PBS control treated mice, and PDS0101, NHS-IL12 plus bintrafusp alfa combination treated mice. (B) CD8+ T cell infiltration in tumor. (C) CD4+ T cell infiltration in tumor. (D) Activated and proliferating CD8+ T cells in tumor. (E) Activated and proliferating CD8+ T cells in spleen. (F) Activated effector CD8+ T cells in tumor. (G) Naïve CD8+ effector T cells in spleen. *P<0.05, **P<0.01. IL12, interleukin-12; i.p. intraperitoneally; s.c., subcutaneously.

Figure 3

Correlation between tumor weight and immune cell infiltration per milligram of tumor in the TC-1 syngeneic model. TC-1 tumor bearing female C57BL/6J mice were treated with PBS control (100 µL s.c.), PDS0101 (s.c., 3 weekly doses starting on day 7), bintrafusp alfa (250 µg, i.p., days 7, 9 and 11), and/or NHS-IL12 (50 µg, s.c., day 7). (A) A smaller tumor volume correlated with increased CD4+ and CD8+ T cells, regulatory T cells (Treg), and M1 macrophage infiltration into the tumor. Data are shown for all treatment groups combined. (B) Immunohistochemistry for CD4+ and CD8+ T cells in TC-1 tumors. Tumors were harvested, blocked, sectioned and stained with the Opal Immunology kit. Combination treatment with PDS0101, NHS-IL12, and bintrafusp alfa increased CD8+ and CD4+ T cell infiltration into the tumor compared with monotherapy treatments. IL12, interleukin-12; i.p. intraperitoneally; s.c., subcutaneously.

Figure 4

PDS0101 increased antigen-specific T cells against HPV16 E7 and reduced tumor volume in the syngeneic mEER tumor model. (A, B) TC-1 tumor bearing female C57BL/6J mice were treated with PBS control, R-DOTAP control, PDS0101 (s.c., 3 weekly doses starting on day 7), bintrafusp alfa (250 µg, i.p., days 7, 9 and 11), and/or NHS-IL12 (50 µg, s.c., day 7). (A) ELIspot assay for IFNγ in splenocytes stimulated with overlapping HPV16 E7 15-mers. Kruskall-Wallis analysis was performed. (B) Comparison between RF9 peptide and overlapping HPV16 E7 15-mer peptides as investigative antigens for IFNγ ELIspot. Mann-Whitney unpaired t-test. (C, D) mEER tumor bearing female C57BL/6J mice (n=11–12 per group) were treated with PDS0101 (s.c., 3 weekly doses starting on day 4) or PBS control (100 µL, s.c). (C) Tumor weights at the end of study. (D) IFNγ ELIspot data from splenocytes stimulated with overlapping HPV16 E7 15-mer peptides in PBS control versus PDS0101 treated mice. *P<0.05, **P<0.01. HPV, human papillomavirus; IFNγ, interferon-γ; i.p. intraperitoneally; s.c., subcutaneously.

Figure 5

TCR clonality significantly increased in all groups treated with PDS0101. TC-1 tumor bearing female C57BL/6J mice were treated with PBS control (100 µL s.c.), PDS0101 (s.c., 3 weekly doses starting on day 7), bintrafusp alfa (250 µg, i.p., days 7, 9, and 11), and/or NHS-IL12 (50 µg, s.c., day 7). Tumor infiltrating lymphocytes (TILs) were purified from whole tumor. DNA isolated from TILs was analyzed by Adaptive Biotechnology for TCR repertoire. (A) Number of T-cell clones that make up 25% of the TCR repertoire (n=3 mice per group). Red represents the most abundant T-cell clone in the individual mouse tumor; blue is the second most abundant; teal is the third. (B) Table of the average number of clones from three mice per group that make up 25% of the TCR repertoire. IL12, interleukin-12; i.p. intraperitoneally; s.c., subcutaneously; TCR, T-cell receptor.