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Correlation between anti-PD-L1 tumor concentrations and tumor-specific and nonspecific biomarkers in a melanoma mouse model - PubMed

  • ️Fri Jan 01 2016

Correlation between anti-PD-L1 tumor concentrations and tumor-specific and nonspecific biomarkers in a melanoma mouse model

Ana M Contreras-Sandoval et al. Oncotarget. 2016.

Abstract

Blockade of PD-L1 with specific monoclonal antibodies (anti-PD-L1) represents a therapeutic strategy to increase the capability of the immune system to modulate the tumor immune-resistance. The relationship between anti-PD-L1 tumor exposition and anti-tumor effect represents a challenge that has been addressed in this work through the identification of certain biomarkers implicated in the antibody's mechanism of action, using a syngeneic melanoma mouse model. The development of an in-vitro/in-vivo platform has allowed us to investigate the PD-L1 behavior after its blockage with anti-PD-L1 at cellular level and in animals. In-vitro studies showed that the complex PD-L1/anti-PD-L1 was retained mainly at the cell surface. The antibody concentration and time exposure affected directly the recycling or ligand turnover. In-vivo studies showed that anti-PD-L1 was therapeutically active at all stage of the disease, with a rapid onset, a low but durable efficacy and non-relevant toxic effect. This efficacy measured as tumor shrinkage correlated with tumor-specific infiltrating lymphocytes (TILs), which increased as antibody tumor concentrations increased. Both, TILS and antibody concentrations followed similar kinetic patterns, justifying the observed anti-PD-L1 rapid onset. Interestingly, peripheral lymphocytes (PBLs) behave as infiltrating lymphocytes, suggesting that these PBLs might be considered as a possible biomarker for antibody activity.

Keywords: anti-PD-L1 mAb; biomarkers; immunomodulation; melanoma model; preclinical study.

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Conflict of interest statement

CONFLICTS OF INTEREST

No potential conflicts of interest were disclosed.

Figures

Figure 1
Figure 1. Effect of different anti-PD-L1 concentrations on PD-L1 availability and cells imaged by confocal microscopy to visualize complex localization

(A) In-vitro design: Cells seeded at density of 1 × 106 cells/well and exposure for 4 or 24 h at 5, 25 and 50 μg/mL of anti-PD-L were analysed by flow cytometer to quantify the PD-L1 availability over 48 h; at the same time, cells treated with 50 μg/mL for different times of exposure were fixed, stained and analyzed by con-focal microscopy. (B and C) represent the percentage of cells expressing PD-L1 over 48 h in control and after 4 h and 24 h exposure to three different antibody concentrations. Statistical differences were calculated between control and treatments and across times for each treatment (***p < 0.001; **p < 0.01; *p > 0.5). Data represent the mean ± SD of three independent experiments. (D and E) Immunohistochemical images show the location of PD-L1/ mAb in culture cells (green). Cells, incubated with 50 μg/mL anti-PD-L1 mAb at 37°C, were fluorescently labeled with mAb against anti-PD-L1 and with dapi for nuclei; upper panels correspond to cells treated for 4 h and visualized just after treatment, 4 h, and during 24 h post-treatment (panels D), and cells exposed continuously to the treatment for 24 h (panels E).

Figure 2
Figure 2. Pharmacodynamic of anti-PD-L1 administered to tumor bearing mice

(A) experimental design for one cycle of treatment. Data are represented for control and treated groups according to different stages of the disease, small, medium and large tumor size; (B) Time profile of the mean tumor growth. Arrows show the starting time of the treatment at day 3, 7 and 11, respectively; (C) Time profile of the mean body weight; (D) Kaplan-Meier curve representing the results from the first cycle of treatment; (E–G) individual tumor growth kinetics. Symbols represent observations, solid lines mean tendency of control (black), responders and non-responders (different colours) and coloured dashed lines animals with a delayed effect. Arrows show the four doses corresponding to first cycle; (H) Individual tumor size represented by circles for control and treated groups (small, medium and large) just at the day of first and fourth dose administration and 24 h before starting the second cycle of treatment. Statistical differences were calculated across groups (*P < 0.05; **P < 0.01; ***P < 0.001). Overall data for the two cycles of treatment are shown in panels: (I) time profile of the mean body weight, and (J) Kaplan-Meier curve for the complete study.

Figure 3
Figure 3. Anti-PD-L1 treatment induces specific biomarkers expression in-vivo

Observations were individually collected after first mAb dose administration (100 μg/mouse i.v.). (A) experimental design; (B) time profile of mAb concentrations in serum and tumor, respectively; (C) Specific tumor-infiltrating lymphocytes, OVA-CD8+, vs. mAb tumor concentrations throughout 72 h post-dosing; (D) non-specific CD8+ vs. specific, OVA-CD8+ and (E) Individual time profiles of OVA-CD8+ vs. peripheral lymphocytes over 72 h. Symbols represent experimental data, solid lines the mean tendency, arrows dosing time and in panels C, D and E, black circles correspond to control data.

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