CD169 (Siglec-1) as a Robust Human Cell Biomarker of Toll-Like Receptor 9 Agonist Immunotherapy

Abstract

Immunotherapy is a promising therapeutic area in cancer and chronic viral infections. An important component of immunotherapy in these contexts is the activation of innate immunity. Here we investigate the potential for CD169 (Siglec 1) expression on monocytes to serve as a robust biomarker for activation of innate immunity and, particular, as a proxy for IFN-α production. Specifically, we investigated the effects of Toll-like receptor 9 agonism with MGN1703 (lefitolimod) across experimental conditions ex vivo, in humanized mice, and in clinical trial participants. Ex vivo we observed that the percentage of classical monocytes expressing CD169 increased dramatically from 10% pre-stimulation to 97% 24 hrs after MGN1703 stimulation (p<0.0001). In humanized NOG mice, we observed prominent upregulation of the proportions of monocytes expressing CD169 after two doses of MGN1703 where 73% of classical monocytes were CD169 positive in bone marrow following MGN1703 treatment vs 19% in vehicle treated mice (p=0.0159). Finally, in a clinical trial in HIV-infected individuals receiving immunotherapy treatment with MGN1703, we observed a uniform upregulation of CD169 on monocytes after dosing with 97% of classical monocytes positive for CD169 (p=0.002). Hence, in this comprehensive evaluation ex vivo, in an animal model, and in a clinical trial, we find increases in the percentage of CD169 positive monocytes to be a reliable and robust biomarker of immune activation following TLR9 agonist treatment.

Keywords: CD169; HIV-1; biomarker; immunotherapy; toll-like receptor 9 (TLR9).

Figure 1

Upregulation of CD169 on human monocytes following ex vivo TLR9 agonist stimulation. (A) Levels of IFN-α in supernatants from human PBMCs stimulated with TLR9 agonist (MGN1703 3μM) for 24 hrs. Bar-graph depicts average IFN-α levels obtained from stimulation of five individual donors (+/- SEM). UT=untreated. (B) Flow plot demonstrating monocyte subset gating strategy with representative data. Classical monocytes CD14+/CD16^int, Intermediate monocytes CD14+/CD16+ and the non-classical monocytes CD14^int/CD16+. (C) Flow plots depicting the impact of MGN1703 on CD169 expression on classical monocytes after 24 hrs of stimulation for a representative donor (D) Time-dependent upregulation of CD169 on monocytes. Human PBMCs were stimulated with TLR9 agonist (MGN1703 at 3 μM) for 4, 8, 16 or 24 hrs. Levels of CD169 on monocyte subsets were measured using flow cytometry. Depicted in the graph is the average (+/- SEM) from five individual donors. Levels of CD169 on each monocyte subset were analyzed by paired ANOVA and Dunnett’s multiple comparison tests were performed for each time-point compared to baseline.

Figure 2

In vivo immunomodulatory impact on human monocytes of TLR9 agonist in humanized NOG mice. (A) Schematic depiction of humanized NOG mice experiment. Humanized NOG mice were dosed with MGN1703 in one of 3 different dosage groups (200, 2,000 and 10,000 μg/mouse) or with physiological saline on day 0 and day 3. Blood collection was performed at baseline and at the time of necropsy on day 4, where bone marrow, spleen, lymph nodes and lungs were collected as well. Graphic created with Biorender. (B) Tissue cells were isolated on day 4 from bone marrow, spleen, and lungs. These cells were evaluated for CD169 expression using flow cytometry. Percentages of CD169 positive classical monocytes are depicted in the bar graphs. Individual mice values are shown together with each bar that depicts the mean (+/- SEM). Data were analyzed by ANOVA and MGN1703 dose groups were compared with vehicle group using Dunnets multiple comparison tests. (C) Cytokines and chemokines in day 4 plasma were analyzed relative to baseline using O-link technology. Individual mice normalized protein expression (NPX) values are depicted as changes from baseline in light grey with the dose-group average denoted by large colored symbols.

Figure 3

In vivo upregulation of CD169 on human monocytes in a clinical TLR9 agonist study. (A) Schematic layout of a clinical Phase I/IIa trial (clinicaltrials.gov NCT02443935) investigating the immune-enhancing effects of TLR9 agonist (MGN1703) in HIV-infected individuals. Graphic created with Biorender. (B–D) Percentages of classical monocyte (B), intermediate monocytes (C), and non-classical monocytes (D) expressing CD169 are shown depicted with symbol and lines for averages (+/- SEM). Blue shaded areas indicate “early” and “late” dosing windows (dose 1 + 2 in the first dosing week and dose 7 + 8 in the last dosing week). The following Wilcoxon sign-ranked test statistical comparisons were made for each monocyte population: baseline vs. day 5 (Peak 1); baseline vs. day 7 (Trough); baseline vs. day 26 (Peak 2); and baseline vs. day 79 (Follow-up).