Triggering of lymphocytes by CD28, 4-1BB, and PD-1 checkpoints to enhance the immune response capacities

Abstract

Tumor infiltrating lymphocytes (TILs) usually become exhausted and dysfunctional owing to chronic contact with tumor cells and overexpression of multiple inhibitor receptors. Activation of TILs by targeting the inhibitory and stimulatory checkpoints has emerged as one of the most promising immunotherapy prospectively. We investigated whether triggering of CD28, 4-1BB, and PD-1 checkpoints simultaneously or alone could enhance the immune response capacity of lymphocytes. In this regard, anti-PD-1, CD80-Fc, and 4-1BBL-Fc proteins were designed and produced in CHO-K1 cells as an expression host. Following confirmation of the Fc fusion proteins' ability to bind to native targets expressed on engineered CHO-K1 cells (CHO-K1/hPD-1, CHO-K1/hCD28, CHO-K1/hCTLA4, and CHO-K1/h4-1BB), the effects of each protein, on its own and in various combinations, were assessed in vitro on T cell proliferation, cytotoxicity, and cytokines secretion using the Mixed lymphocyte reaction (MLR) assay, 7-AAD/CFSE cell-mediated cytotoxicity assay, and a LEGENDplex™ Human Th Cytokine Panel, respectively. MLR results demonstrated that T cell proliferation in the presence of the combinations of anti-PD-1/CD80-Fc, CD80-Fc/4-1BBL-Fc, and anti-PD-1/CD80-Fc/4-1BBL-Fc proteins was significantly higher than in the untreated condition (1.83-, 1.91-, and 2.02-fold respectively). Furthermore, anti-PD-1 (17%), 4-1BBL-Fc (19.2%), anti-PD-1/CD80-Fc (18.6%), anti-PD-1/4-1BBL-Fc (21%), CD80-Fc/4-1BBL-Fc (18.5%), and anti-PD-1/CD80-Fc/4-1BBL-Fc (17.3%) significantly enhanced cytotoxicity activity compared to untreated condition (7.8%). However, concerning the cytokine production, CD80-Fc and 4-1BBL-Fc alone or in combination significantly increased the secretion of IFN-γ, TNF-α, and IL-2 compared with the untreated conditions. In conclusion, this research establishes that the various combinations of produced anti-PD-1, CD80-Fc, and 4-1BBL-Fc proteins can noticeably induce the immune response in vitro. Each of these combinations may be effective in killing or destroying cancer cells depending on the type and stage of cancer.

Fig 1. Western blot analysis of purified Fc fusion proteins by probing with goat anti-human IgG (Fc specific)-peroxidase antibody under reducing conditions.

M: Protein ladder, Lane 1: anti- PD-1, Lane 2: CD80-Fc, and Lane 3: 4-1BBL-Fc.

Fig 2. The flow cytometry binding analysis of Fc fusion proteins with engineered CHO-K1 cells in 0, 0.2, 1, 5, and 10 μg/mL concentrations.

Binding capacity was detected by FITC mouse anti-human IgG and using stable cell lines engineered A) human PD-1 (CHO-K1/hPD-1) B) human CD28 (CHO-K1/hCD28) C) human CTLA4 (CHO-K1/hCTLA4), and D) human 4-1BB (CHO-K1/h4-1BB).

Fig 3. T cell proliferation induction of anti-PD-1, CD80-Fc, and 4-1BBL-Fc proteins in mono and combination treatments.

For an allogeneic MLR, responder cells from 6 healthy donors were stained with CFSE and mixed with γ-irradiated PBMCs at a 1:1 ratio. PBMCs were harvested after 5 days and analyzed by flow cytometry. (A) Histogram plots depict examples of two populations used in this study for MLR analysis: M1 (divided) and M2 (highly divided) populations of untreated (Light gray) and PHA conditions (dark gray). M2 is a subset of the M1 population that contains cells with a high division rate. (B) Percentage of CFSE intensity was measured in different conditions and statistical analysis was performed using a one-way ANOVA test (*p≤ 0.05, **p≤0.01). The column height represents the average percentage of the CFSE- labeled cells and error bars are ± SEM.

Fig 4. Potential of mono and combination treatments of anti-PD-1, CD80-Fc, and 4-1BBL-Fc proteins on cytokine release.

An allogeneic MLR was performed and supernatants were harvested on day 4 for cytokine analysis in triplicates. This figure shows concentration of 6 cytokines: (A) IFN‐γ (B) TNF-α (C) IL-2 (D) IL-10 (E) IL-17A (F) IL-17F. Data is reported as mean ± SEM (*p≤ 0.05, **p≤0.01, ***P≤0.0001, ****P≤0.0001).

Fig 5. T cells cytotoxicity activity in the presence and absence of anti-PD-1, CD80-Fc, and 4-1BBL-Fc proteins in two types of treatment, namely, mono and combination.

CFSE-target cells and effector cells from 6 healthy donors were co-cultured (A) at 1:1 and 10:1 ratios (two-way ANOVA was applied for the data collected) (B) At 10:1 ratio, all of the conditions were compared with the untreated using one-way ANOVA. (*p≤ 0.05, **p≤0.01, ***P≤0.0001). The column height represents the average percentage of the 7-AAD-positive CFSE-target cells and error bars are ± SEM.