Differential combination immunotherapy requirements for inflamed (warm) tumors versus T cell excluded (cool) tumors: engage, expand, enable, and evolve

Abstract

Background: Different types of tumors have varying susceptibility to immunotherapy and hence require different treatment strategies; these cover a spectrum ranging from 'hot' tumors or those with high mutational burden and immune infiltrates that are more amenable to targeting to 'cold' tumors that are more difficult to treat due to the fewer targetable mutations and checkpoint markers. We hypothesized that an effective anti-tumor response requires multiple agents that would (1) engage the immune response and generate tumor-specific effector cells; (2) expand the number and breadth of the immune effector cells; (3) enable the anti-tumor activity of these immune cells in the tumor microenvironment; and (4) evolve the tumor response to widen immune effector repertoire.

Methods: A hexatherapy combination was designed and administered to MC38-CEA (warm) and 4T1 (cool) murine tumor models. The hexatherapy regimen was composed of adenovirus-based vaccine and IL-15 (interleukin-15) superagonist (N-803) to engage the immune response; anti-OX40 and anti-4-1BB to expand effector cells; anti-PD-L1 (anti-programmed death-ligand 1) to enable anti-tumor activity; and docetaxel to promote antigen spread. Primary and metastatic tumor growth inhibition were measured. The generation of anti-tumor immune effector cells was analyzed using flow cytometry, ELISpot (enzyme-linked immunospot), and RNA analysis.

Results: The MC38-CEA and 4T1 tumor models have differential sensitivities to the combination treatments. In the 'warm' MC38-CEA, combinations with two to five agents resulted in moderate therapeutic benefit while the hexatherapy regimen outperformed all these combinations. On the other hand, the hexatherapy regimen was required in order to decrease the primary and metastatic tumor burden in the 'cool' 4T1 model. In both models, the hexatherapy regimen promoted CD4+ and CD8+ T cell proliferation and activity. Furthermore, the hexatherapy regimen induced vaccine-specific T cells and stimulated antigen cascade. The hexatherapy regimen also limited the immunosuppressive T cell and myeloid derived suppressor cell populations, and also decreased the expression of exhaustion markers in T cells in the 4T1 model.

Conclusion: The hexatherapy regimen is a strategic combination of immuno-oncology agents that can engage, expand, enable, and evolve the immune response and can provide therapeutic benefits in both MC38-CEA (warm) and 4T1 (cool) tumor models.

Trial registration: ClinicalTrials.gov NCT03493945.

Keywords: cytokines; immunomodulation; immunotherapy; translational medical research; vaccination.

Conflict of interest statement

Competing interests: None declared.

© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY. Published by BMJ.

Figures

Figure 1

MC38-CEA colorectal carcinoma has an immune-inflamed phenotype compared with 4T1 breast carcinoma. (A, B) Female C57BL/6-CEA-Tg mice (8–12 weeks old; n=5) were implanted with 3×105 MC38-CEA cells on the flank and female Balb/c mice (8–12 weeks old) were implanted with 5×104 4T1 cells on the mammary fat pad. Fourteen to fifteen days after tumor implantation, the tumors were harvested and analyzed via (A) flow cytometry and (B) immunofluorescence staining for CD8+ T cell infiltration. (C, D) RNA was isolated from three MC38-CEA and three 4T1 tumor explants harvested 28 days post-tumor implantation and the immune-related transcriptome for each tumor was analyzed using the nCounter PanCancer Immune Profiling Panel. Heatmap showing select genes with data presented as fold change values compared with housekeeping genes suite of that particular tumor sample on scale of 0 (light blue) to (C) 800 (red) and (D) 3500 (red). All genes reported are significantly different. CEA, carcinoembryonic antigen; Tg, transgenic.

Figure 2

The hexatherapy treatment regimen results in enhanced therapeutic effects in the ‘warm’ MC38-CEA model. (A) Female C57BL/6-CEA-Tg mice (8–12 weeks old) were inoculated s.c. on the flank with 3×105 MC38-CEA cells on day 0. Ad-CEA (1×1010 VP) was administered s.c. on days 7, 14, and 21; 1 µg N-803 s.c. on days 14 and 21; 100 µg anti-OX40 and 20 µg anti-4-1BB i.p. on days 7, 14, and 21; 200 µg anti-PD-L1 i.p. on days 14 and 21; and 500 µg docetaxel on day 21. In this hexatherapy regimen, the IO agents were grouped into four modalities: Ad-CEA+N-803, OX40+4-1BB, PD-L1, and docetaxel. (B, C) The single modality treatments were cumulatively combined and used to treat MC38-CEA tumor-bearing mice (n=6–8/group) using the schedule described above. Tumor volumes were monitored; (B) tumor growth curve and (C) mean tumor volumes on day 25 were plotted. Another set of MC38-CEA tumor-bearing mice (n=6–10/group) were treated with the hexatherapy regimen or with corresponding empty adenoviral vector and antibody isotype. Tumor volumes on day 28 were plotted (B inset). (D, E) MC38-CEA tumor-bearing female C57BL/6-CEA-Tg mice (n=8–10/group) were treated with the hexatherapy regimen or the hexatherapy regimen minus one treatment modality. Tumor volumes were monitored; (D) tumor growth curve and (E) mean tumor volumes on day 26 were plotted. (F, G) The hexatherapy regimen was administered to CD8+ T cell-depleted MC38-CEA tumor-bearing female C57BL/6 mice (n=6–10). (F) Tumor volumes on day 28 and (G) survival were monitored. The same untreated and hexatherapy-treated groups are presented in figure 2B inset and figure 2F. (H) For each treatment combination tested in the MC38-CEA model, the percentage of mice with tumor volume <300 mm3 was calculated and plotted against the number of IO agents received. Meta-analysis of three to four independent experiments is shown. Statistical test: Analysis of variance with Tukey’s post hoc test. Error bars, SEM. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. CEA, carcinoembryonic antigen; IO, immuno-oncology; i.p., intraperitoneal; s.c., subcutaneous; Tg, transgenic; VP, viral particle.

Figure 3

Treatment with hexatherapy induces enhanced CD4+ and CD8+ T cell activity in the ‘warm’ MC38-CEA tumor model. In a separate experiment, MC38-CEA tumor-bearing mice (n=6–10/group) were treated as in figure 2A. Three spleens from each animal cohort were collected on day 28 (7 days after the last treatment). (A–D) Splenocytes from different treatment groups were stimulated in vitro with 1 µg/mL plate-bound CD3 antibody for 4 hours. Intracellular expression of Ki67 and IFNγ in (A, B) CD4+ T cells and (C, D) CD8+ T cells was analyzed by flow cytometry as frequency of CD4+ and CD8+ T cells, respectively. (E–I) 1×106 splenocytes were stimulated with H2-Kb-restricted peptide epitopes for (E, F) CEA, (G) gp70 (p15E), the neo-epitopes (H) JAK1 and (I) Ptgfr, and HIV-gag. Antigen-specific IFN-γ production was measured via ELISpot. HIV-gag values were subtracted from the values obtained with the other antigens to normalize the data. Statistical test: Analysis of variance with Tukey’s post hoc test. Error bars, SEM. *p<0.05; **, p<0.01; ***p<0.001; ****p<0.0001. CEA, carcinoembryonic antigen; IFN, interferon; PD-L1, programmed death-ligand 1.

Figure 4

The hexatherapy regimen results in enhanced therapeutic effects associated with expanded effector immune cell populations in the ‘cool’ 4T1 tumor model. (A) Female Balb/c mice (8–12 weeks old) were implanted with 5×104 4T1 cells on the mammary fat pad on day 0. Ad-Twist (1×1010 VP) was administered s.c. on days 7, 14, and 21; 1 μg N-803 s.c. on days 14 and 21; 100 µg anti-OX40 and 20 µg anti-4-1BB i.p. on days 7, 14, and 21; 200 µg anti-PD-L1 i.p. on days 14 and 21; and 500 µg docetaxel on day 21. In this hexatherapy regimen, the IO agents were grouped into four modalities: Ad-CEA+N-803, OX40+4-1BB, PD-L1, and docetaxel. (B–D) 4T1 tumor-bearing mice were treated with the hexatherapy regimen or hexatherapy regimen minus one treatment modality (n=18–20/group). (B) Primary tumor volumes were monitored and (C) mean tumor volumes on day 25 were plotted. On day 28 post-tumor implantation the lungs (n=13–15/group) were collected from the different animal treatment cohorts. (D) Single cell suspension samples of lungs were incubated in complete RPMI supplemented with 6 µM 6-thioguanine for 10–12 days, after which clonogenic metastatic cell colonies were enumerated. Another set of 4T1 tumor-bearing female Balb/c mice (n=8–10/group) were treated as described above. (E–H). On day 28, splenocytes from the different treatment groups were harvested and stimulated in vitro with 1 µg/mL plate-bound CD3 antibody overnight. Intracellular expression of Ki67 and IFN-γ in (E, F) CD4+ T cells and (G, H) CD8+ T cells was analyzed by flow cytometry as frequency of CD4+ and CD8+ T cells, respectively. (I, J) The splenocytes were also stimulated in vitro with H2-Kd-restricted peptide epitopes for (I) Twist, (J) AH1 and β-gal. Antigen-specific IFN-γ production was measured via ELISpot. β-gal values were subtracted from the values obtained with the other antigens to normalize the data. Statistical tests: Analysis of variance with Tukey’s post hoc test for group analyses. Student’s t-test for comparing two groups. Error bars, SEM. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. CEA, carcinoembryonic antigen; IFN, interferon; IO, immuno-oncology; PD-L1, programmed death-ligand 1; s.c., subcutaneous; VP, viral particle.

Figure 5

Treatment with the hexatherapy regimen results in tumor-infiltrating T cells that are more proliferative and less exhausted in the ‘cool’ 4T1 tumor model. Primary tumors (n=4–5/group) from figure 4B, C were collected on day 28 post-tumor implantation (7 days after the last treatment). (A–C) Flow cytometry was performed to determine (A) the frequency of FoxP3neg CD3+CD4+ T cells in the CD45+ tumor-infiltrating immune population, (B) the frequency of Ki67+ expression in the FoxP3neg CD4+ T cells, and the (C) frequency of CTLA-4 and PD-1 expression in the CD4+ T cells. (D) Likewise, flow cytometry was performed to determine the frequency of CD3+CD8+ T cells in the CD45+ compartment. (E) Immunofluorescence staining of CD8+ T cells (green) and PECAM1+ cells (red) was performed on untreated and hexatherapy regimen-treated tumors to further elucidate T cell infiltration. (F, G) Flow cytometry was done to assess (F) the frequency of Ki67+ expression, and the (G) frequency of CTLA-4 and PD-1 expression in the CD8+ T cells. Statistical test: Analysis of variance with Tukey’s post hoc test. Error bars, SEM. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. PD-1, programmed cell death protein-1; PECAM1+, platelet endothelial cell adhesion molecule-1 positive.

Figure 6

The hexatherapy treatment regimen reduces PMN-MDSC and improves effector T cell-to-Treg cell ratios in the 4T1 tumor microenvironment. (A–C) Primary tumors (n=4–5/group) from figure 4B, C were collected on day 28 post-tumor implantation (7 days after the last treatment) and were assessed through flow cytometry for frequency of (A) PMN-MDSC (CD11b+Ly6G+Ly6Clo), (B) M-MDSC (CD11b+Ly6G−Ly6Chigh), and (C) Tregs (FoxP3+CD4+ T cells) in the CD45+ population. (D, E) Flow cytometric analysis was used to determine the (D) CD4-to-Treg ratio and (E) CD8-to-Treg ratio in the tumor. Analysis of variance with Tukey’s post hoc test. Error bars, SEM. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. M-MDSC, mononuclear myeloid derived suppressor cell; PD-L1, programmed death-ligand 1; PMN-MDSC, polymorphonuclear MDSC; Treg, regulatory T cell.

Figure 7

Treatment with the hexatherapy regimen results in enhanced therapeutic effects associated with expanded effector immune cell populations in the 4T1 tumor model. (A) For each treatment combination tested in the 4T1 tumor model, the percentage of mice with tumor volume 3 was calculated and plotted against the number of IO agents received. Meta-analysis of three independent experiments is shown. (B) 4T1 tumor-bearing female Balb/c mice (8–12 weeks old) were treated with hexatherapy regimen or single IO agent as described in figure 4A. Two to three tumor samples from each animal cohort were harvested on day 28 post-tumor implantation and were used to analyze the immune-related transcriptome using the nCounter PanCancer Immune Profiling Panel. Heatmap showing select genes with data presented as fold change on scale of −2 (blue) to +2 (red) relative to the gene expression in the untreated tumors. Statistical test: Student’s t-test. *p<0.05. IO, immuno-oncology; PD-1, programmed cell death protein-1; PD-L1, programmed death-ligand 1.