Ex vivo Hsp70-Activated NK Cells in Combination With PD-1 Inhibition Significantly Increase Overall Survival in Preclinical Models of Glioblastoma and Lung Cancer

Abstract

Heat shock protein 70 (Hsp70) which is expressed on the plasma membrane of highly aggressive tumors including non-small cell lung carcinoma and glioblastoma multiforme serves as a target for Hsp70-targeting NK cells. Herein, we aimed to investigate the antitumor effects of a combined therapy consisting of ex vivo Hsp70-peptide TKD/IL-2-activated NK cells in combination with mouse/human anti-PD-1 antibody in a syngeneic glioblastoma and a xenograft lung cancer mouse model. Mice with membrane Hsp70 positive syngeneic GL261 glioblastoma or human xenograft A549 lung tumors were sham-treated with PBS or injected with ex vivo TKD/IL-2-activated mouse/human NK cells and mouse/human PD-1 antibody either as a single regimen or in combination. Tumor volume was assessed by MR scanning and tumor-infiltrating CD8⁺ T, NK, and PD-1⁺ cells were quantified by immunohistochemistry (IHC). We could show that the adoptive transfer of ex vivo TKD/IL-2-activated mouse NK cells or the inhibition of PD-1 resulted in tumor growth delay and an improved overall survival (OS) in a syngeneic glioblastoma mouse model. A combination of both therapies was well-tolerated and significantly more effective with respect to both outcome parameters than either of the single regimens. A combined treatment in a xenograft lung cancer model showed identical effects in immunodeficient mice bearing human lung cancer after adoptive transfer of TKD/IL-2-activated human effector cells and a human PD-1 antibody. Tumor control was associated with a massive infiltration with CD8⁺ T and NK cells in both tumor models and a decreased in PD-1 expression on immune effector cells. In summary, a combined approach consisting of activated NK cells and anti-PD-1 therapy is safe and results in a long-term tumor control which is accompanied by a massive tumor immune cell infiltration in 2 preclinical tumor models.

Keywords: NK cell therapy; anti-PD-1 antibody; glioblastoma; immunophenotyping; lung carcinoma; membrane Hsp70.

Figure 1

The effect of TKD/IL-2 or Hsp70/IL-2 on the expression of CD56, CD94, and CD69 receptors in peripheral blood lymphocytes (PBLs) of healthy individuals. The expression of the receptors was examined within the fraction of stimulated and unstimulated PBLs derived from 10 healthy Caucasian individuals. (A) Representative figure of the healthy individual following stimulation with TKD/IL-2 or Hsp70/IL-2. (B) The amount and (C) the median fluorescence intensity (MFI) of the receptors expressed on NK cells. Data presented as means ± standard error (M ± SE).

Figure 2

Cytolytic activity of the ex vivo stimulated NK cells with anti-PD-1 antibody toward GL261, A549, and LLC tumor cells. Data presented as means ± standard error (M ± SE) for three independent experiments.

Figure 3

Therapeutic potency of a combined treatment with ex vivo TKD/IL-2- stimulated, mouse NK cells with anti-PD-1 antibody in the model of intracranial GL261 glioma. (A) Volumetric studies of the GL261 glioma. Tumor volume (mm³) as determined over time by T₁-weighted and T₂-weighted MRI scans in control (PBS, red lines; IgG control antibody, black lines) and treated (NK cells, green lines; PD-1 antibody, orange lines; NK cells + PD-1 antibody, blue lines) glioblastoma (GL261)-bearing C57/Bl6 mice (n = 3 per group). Tumor progression was calculated by measuring the cross-sectional areas on each slice and multiplying their sum as related to the thickness of the sections. (B) Kaplan-Meier analysis of the cumulative survival (days after treatment) in control (PBS, red lines; IgG control antibody, blue lines) and treated (NK cells, black lines; PD-1 antibody, green lines; NK cells + PD-1 antibody, orange lines) glioblastoma (GL261)-bearing C57/Bl6 mice (n = 8 per group). Solid lines: mean values; dotted lines: SD within 95% confidence interval.

Figure 4

Boxplots of the CD8+, NK1.1+ and PD-1+ cells infiltrating the GL261 tumor in control (PBS and IgG treated animals) and experimental groups. Tumor infiltrating CD8+ cytotoxic T cells, CD3-/CD56+ NK cells and PD1+ effector (T/NK) cells were counted in three representative IHC sections of the tumors of the different treatment groups (PBS ctrl, IgG ctrl, NK, NK + PD-1, PD-1) and infiltrating immune cells per mm² were calculated.

Figure 5

Therapeutic effect of ex vivo stimulated, human NK cells with anti-PD-1 antibody in the orthotopic xenograft model of A549 lung carcinoma. Kaplan-Meier analysis of the cumulative survival (days after treatment) in control (PBS, red lines; IgG control antibody, blue lines) and treated (NK cells, black lines; NK cells + PD-1 antibody, orange lines) lung cancer (A549)-bearing mice (n = 8 per group). Solid lines: mean values; dotted lines: SD within 95% confidence interval.

Figure 6

Boxplots of the CD8+, CD56+NK, and PD-1+ cells infiltrating the A549 lung carcinoma in control (PBS and IgG treated animals) and experimental groups. Tumor infiltrating CD8+ cytotoxic T cells, CD3-/CD56+ NK cells, and PD1+ effector (T/NK) cells were counted in three representative IHC sections of the tumors of the different treatment groups (PBS ctrl, IgG ctrl, NK, and NK + PD-1) and infiltrating immune cells per mm² were calculated.