Enhanced anti-melanoma efficacy through a combination of the armed oncolytic adenovirus ZD55-IL-24 and immune checkpoint blockade in B16-bearing immunocompetent mouse model

Abstract

Although the recent treatment in melanoma through the use of anti-PD-1 immunotherapy is successful, the efficacy of this approach remains to be improved. Here, we explore the feasibility of combination strategy with the armed oncolytic adenovirus ZD55-IL-24 and PD-1 blockade. We find that combination therapy with localized ZD55-IL-24 and systemic PD-1 blockade leads to synergistic inhibition of both local and distant established tumors in B16-bearing immunocompetent mouse model. Our further mechanism investigation reveals that synergistic therapeutic effect is associated with marked promotion of tumor immune infiltration and recognition in both local and distant tumors as well as spleens. PD-1 blockade has no obvious effect on promotion of tumor immune infiltration and recognition. Localized therapy with ZD55-IL-24, however, can help PD-1 blockade to overcome the limitation of relatively low tumor immune infiltration and recognition. This study provides a rationale for investigation of such combination therapy in the clinic.

Keywords: Combination therapy; Melanoma; Oncolytic virus; PD-1 blockade; Tumor immune infiltration; Tumor immune recognition.

Fig. 1

ZD55-IL-24 synergizes with PD-1 blockade to reject local established melanomas in B16-bearing immunocompetent mouse model. C57BL/6 mice were inoculated with 1 × 10⁶ B16 melanoma cells s.c. in the right flank and treated 9 d later (the average tumor volume was about 80 mm³) with PBS, anti-PD-1 antibody (0.2 mg/dose), ZD55-IL-24 (7.5 × 10⁸ PFU/dose), or the combination via intraperitoneal or intratumoral injection as indicated. a Scheme of tumor engraftment and treatments. b In vivo tumor growth curves. c Photograph of tumors resected from the sacrificed mice at the end of the experiment. d Weight of tumors resected from the sacrificed mice at the end of the experiment. e Body weight changes of mice monitored during the therapy period. s.c. subcutaneous injection, i.t. intratumoral injection, i.p. intraperitoneal injection, D Death, n = 10 C57BL/6 mice per group. Data are presented as Mean ± SEM

Fig. 2

ZD55-IL-24 helps PD-1 blockade to overcome the limitation of relatively low tumor immune infiltration in local established tumors. C57BL/6 mice were treated as indicated in Fig. 1a, and then the tumor cells were isolated for flow cytometry analysis. Tumor immune infiltration of innate immune cells in local tumors. a Representative flow cytometry plots of tumor-infiltrating total myeloid cells (CD11b⁺) and neutrophils (CD11b⁺Ly-6G⁺Ly-6C^low). b Representative flow cytometry plots of tumor-infiltrating NK cells (NK1.1⁺CD3⁻) and NKT cells (NK1.1⁺CD3⁺). c Representative flow cytometry plots of tumor-infiltrating M1 macrophages (F4/80⁺CD206⁻) and M2 macrophages (F4/80⁺CD206⁺). d Percentages of various innate immune cells in local tumors. Tumor immune infiltration of adaptive immune cells in local tumors. e Representative flow cytometry plots of tumor-infiltrating total T cells (CD3⁺), CD8⁺CD3⁻ cells and CD8⁺ T cells (CD8⁺CD3⁺). f Representative flow cytometry plots of tumor-infiltrating CD4⁺CD3⁻ cells and CD4⁺ T cells (CD4⁺CD3⁺). g Representative flow cytometry plots of tumor-infiltrating plasma cells (B220⁺CD19⁻) and B cells (B220⁺CD19⁺). h Percentages of various adaptive immune cells in local tumors. Mean ± SEM is shown. Data represent cumulative results from seven independent experiments

Fig. 3

ZD55-IL-24 also synergizes with PD-1 blockade to reject distant uninjected melanomas in B16-bearing immunocompetent mouse model. C57BL/6 mice were inoculated with 1 × 10⁶ B16 cells s.c. in the right flank on day 0 and 2 × 10⁵ B16 cells s.c. in the left flank on day 4. The B16-bearing C57BL/6 mice were subsequently treated with PBS, isotype IgG (0.2 mg/dose), anti-PD-1 antibody (0.2 mg/dose), ZD55-IL-24 (7.5 × 10⁸ PFU/dose), or the combination via intraperitoneal or intratumoral injection as indicated, starting when the average tumor volume was about 80 mm³. a Scheme of tumor engraftment and treatments. b Growth of local ZD55-IL-24-injected (right flank) and c distant ZD55-IL-24-uninjected (left flank) tumors. d Overall survival. e Body weight changes of the mice monitored during the therapy period. f Local and distant tumor growth curves of Group PBS, g Group IgG, h Group ZD55-IL-24, i Group ZD55-IL-24/IgG, j Group anti-PD-1 and k Group ZD55-IL-24/anti-PD-1, respectively. s.c. subcutaneous injection, i.t. intratumoral injection, i.p. intraperitoneal injection, n = 10 C57BL/6 mice per group. Data are presented as Mean ± SEM

Fig. 4

ZD55-IL-24 helps PD-1 blockade to overcome the weakness of relatively low immune cell infiltration in not only local injected but also distant uninjected tumors. C57BL/6 mice were treated as indicated in Fig. 3a, and then the left flank tumors were resected from the sacrificed mice for flow cytometry analysis. Tumor immune infiltration of innate immune cells in distant tumors. a Representative flow cytometry plots of tumor-infiltrating total myeloid cells and neutrophils in distant tumors. b Representative flow cytometry plots of tumor-infiltrating NK cells and NKT cells in distant tumors. c Representative flow cytometry plots of tumor-infiltrating M1 macrophages and M2 macrophages in distant tumors. d Percentages of various innate immune cells in distant tumors. Tumor immune infiltration of adaptive immune cells in distant tumors. e Representative flow cytometry plots of tumor-infiltrating total T cells, CD8⁺CD3⁻ cells and CD8⁺ T cells in distant tumors. f Representative flow cytometry plots of tumor-infiltrating CD4⁺CD3⁻ cells and CD4⁺ T cells in distant tumors. g Representative flow cytometry plots of tumor-infiltrating plasma cells and B cells in distant tumors. h Percentages of various adaptive immune cells in distant tumors. Mean ± SEM is shown. Data represent cumulative results from seven independent experiments

Fig. 5

ZD55-IL-24 fails to help PD-1 blockade to further facilitate immune cell recruitment and activation in spleens. C57BL/6 mice were treated as indicated in Fig. 3a, and then the spleen cells were isolated for flow cytometry analysis. Recruitment and activation of innate immune cells in spleens. a Representative flow cytometry plots of tumor-infiltrating total myeloid cells and neutrophils in spleens. b Representative flow cytometry plots of tumor-infiltrating NK cells and NKT cells in spleens. c Representative flow cytometry plots of tumor-infiltrating M1 macrophages and M2 macrophages in spleens. d Percentages of various innate immune cells in spleens. Recruitment and activation of adaptive immune cells in spleens. e Representative flow cytometry plots of tumor-infiltrating total T cells, CD8⁺CD3⁻ cells and CD8⁺ T cells in spleens. f Representative flow cytometry plots of tumor-infiltrating CD4⁺CD3⁻ cells and CD4⁺ T cells in spleens. g Representative flow cytometry plots of tumor-infiltrating plasma cells and B cells in spleens. h Percentages of various adaptive immune cells in spleens. Mean ± SEM is shown. Data represent cumulative results from seven independent experiments

Fig. 6

ZD55-IL-24 helps PD-1 blockade to overcome the limitation of relatively low tumor immune recognition in B16-bearing immunocompetent mouse model. C57BL/6 mice were treated as indicated in Fig. 3a, and then the tumor and spleen cells were isolated for flow cytometry analysis. a Representative flow cytometry plots of tumor-infiltrating MHC II⁺CD11c⁻ APCs and DCs (MHC II⁺CD11c⁺) in local tumors. b Percentages of MHC II⁺CD11c⁻ APCs and DCs in local tumors. c Representative flow cytometry plots of tumor-infiltrating MHC II⁺CD11c⁻ APCs and DCs in distant tumors. d Percentages of MHC II⁺CD11c⁻ APCs and DCs in distant tumors. e Representative flow cytometry plots of MHC II⁺CD11c⁻ APCs and DCs in spleens. f Percentages of MHC II⁺CD11c⁻ APCs and DCs in spleens. Mean ± SEM is shown. Data represent cumulative results from seven independent experiments

Fig. 7

Mechanistic model of combination therapy with ZD55-IL-24 and PD-1 blockade