In the present study, we analyzed the influence of different concentrations of anti-PD-1 antibodies on the vital function of T lymphocytes in an in vitro cell culture model. Nivolumab – an anti-PD-1 fully human monoclonal antibody has been widely used in clinical practice with high effectiveness; nonetheless, a relatively high percentage of adverse reactions have been reported. According to clinical data, PD-L1 expression on cancer cells does not constitute a critical and ideal prediction factor for PD-1/PD-L1 checkpoint blockade treatment in cancer patients. While blocking the PD-1/PD-L1 pathway, nivolumab should reverse the process of exhausting effector T lymphocytes and intensify T cell proliferation and lymphocyte elimination from the tumor microenvironment. Consequently, this action is intended to direct the dominant polarization of mature CD4-positive lymphocytes into CD8-positive cytotoxic T lymphocytes. The key step to achieving the therapeutic effect is restoring lymphocyte viability and cytotoxic capacity.
The first part of our study presented a method concerning obtaining mature dendritic cells that are capable of antigen presentation and activation of T cell polarization without the use of bacterial lipopolysaccharide (LPS) or staphylococcal enterotoxin B (SEB). According to published original research, LPS or SEB induces IFN-γ dependent on upregulation of PD-L1 expression on tumor cells and immune cells infiltrating the tumor microenvironment7. As interferon plays a dual role in tumor immunology, this technique does not appear to be advisable for obtaining mature dendritic cells, especially in cancer patients8. In the present study, we used CD14-positive cells isolated from the peripheral blood of patients with advanced lung adenocarcinoma. The method used to generate mature dendritic cells is the standard technique employed in laboratory practice (6-day incubation with the addition of GM-CSF and IL-4). As a novelty, we used tumor oligopeptides typical of lung adenocarcinoma and TNF-α stimulation on the 6th day of incubation. Generated DCs express markers involved in antigen presentation (CD80/86, CD83, CD1a, CD209 molecules) and helper T cell proliferation activation (CD11c). They also expressed PD-L1/2 molecules, but the expression of B7-H4 was not reported. The B7-H4 molecule has strong inhibitory properties on the activation and polarization of mature T cells into effector T cells. The lack of B7-H4 expression on DCs made it possible to analyze the effectiveness of PD-1/PD-L1/2 trial blockade without a strong coinhibitory signal. The main difference in the generation of mature DCs in our work is an additional application of TNF-α and tumor oligopeptides MUC 1.1, MUC 1.2, MAGE A.3 7−9. All oligopeptides were previously used for cancer vaccine production. MUC1 is a membrane protein typically expressed in lung cells and overexpressed with or without polarization in lung adenocarcinoma cells and is involved in cell-to-cell and cell-to-matrix interactions. MUC1 could also be involved in the exhaustion of T cell effector function in the tumor microenvironment 10−12. MAGE-A3 protein is involved in the epithelial-mesenchymal transformation process and is linked to faster disease progression 13−16. Third, a noncommonly used molecule for mature DC generation was TNF-α, which is involved in the upregulation of the NF-κB pathway and participates in tumor progression and chemo- and immunotherapy resistance in lung adenocarcinoma 17−19. Based on previously published data and the results of our study, we suggest that the use of MUC1 and MAGE-A3 in combination with TNF-α is a more reliable approach to generate autologous dendritic cells for basic research on cancer immunology. The use of the mentioned tumor oligopeptides allowed the possibility of generating dendritic cells fully capable of forming a complete immune synapse and initiating immature T cell polarization into the Th1 subpopulation. It also allowed a more "natural" immune phenotype than when LPS or SEB were used. We proved that the generation of fully functional dendritic cells from lung cancer patients possibly indicates a lack of functional exhaustion of the immune system in the peripheral blood.
In the present study, we observed a significantly increased percentage of activated T cells after nivolumab treatment for all tested concentrations compared to the control probe. However, the results showed considerably increased data for 10 µg/ml nivolumab. CD25 and CD69 markers are typical for early activated T lymphocytes. The CD95 marker is involved in tumor apoptosis activation in CD8-positive lymphocytes. We observed increased T cells in the early activation stage after nivolumab treatment at all tested doses compared to the control. We also analyzed the expression of the CD28 molecule, as it is a key particle in immune synapse formation. The percentage of cells expressing CD28 after nivolumab treatment was not significantly higher than that of the control probe. However, it was nearly four times higher for CD4-positive cells than for CD8-positive cells. We also observed a significantly higher percentage of cells expressing receptors for interleukin-2 (CD25) and interleukin-4 (CD124) in CD4-positive cells after nivolumab treatment compared with the control. IL-2 is critical for T cells to polarize into cytotoxic T cells and activate natural killer cells 20. Similarly, IL-4 is involved in activating NK cells, which play a leading role in tumor apoptosis activation 21. The expression of PD-1 on CD4- and CD8-positive cells was lower after nivolumab treatment, but it was not significant when compared with the nontreated control. Of note, the nivolumab antibody could have a different extracellular PD-1 target point than the anti-PD-1 antibody used for flow cytometric analysis.
Only a few original papers describe in vitro studies with nivolumab's influence on cells from lung adenocarcinoma-bearing patients. There is also a lack of data regarding the proper nivolumab dose for in vitro research. There was a previously reported nivolumab dose for in vitro analysis, but the methodology and aim of that study differed from those of the present study. Vetrei et al. reported a 15 µg/ml dose of nivolumab for an in vitro study 22. Wang Ch. et al. showed that nivolumab activates T cell proliferation even at a concentration of 1.5 ng/ml, but antigen presentation is required 9. Selby et al. pointed to nivolumab's ability to stimulate CD8+ cytotoxic function and CD4+ effector function, but it does not affect the proliferation of naïve T cells 7. Pharmacodynamic and pharmacokinetic analysis from the I phase trial, according to Centanni M. et al., for the ex vivo dose range > 0.1 µg/ml. They also confirmed no dose-related side effects in grade 3-5 tumors analyzed in 342 patients treated with 0.1 – 10 mg/kg nivolumab 23. The most important relation observed in basic research is not always confirmed clinically. Drug dose is an important factor for therapy effectiveness and safety depending on its pharmacological properties. The major challenge for ICI therapy is reducing several side effects, including life-threatening conditions or death, during therapy.
Accordingly, to data from phase III clinical trials, the median time for the appearance of side effects after nivolumab treatment, such as hyperthyroid, pneumonitis, colitis, hyperthyroid, and hepatitis renal dysfunction, took place between the fourth and tenth weeks after therapy started 24. Nevertheless, the more common adverse effects of ICI therapy in third or fourth grade are fatigue, dermatological changes, and gastrointestinal disorders. The intensity of the mentioned adverse effects often obliges discontinuing immunotherapy, which implicates a poor prognosis for patients. Analysis of antidrug antibody presence in patients with solid tumors from 6 clinical trials during therapy showed no clinical significance of nivolumab immunogenicity 25. Wang X. et al. described that the nivolumab time-averaged concentration after the first dose was not significantly related to objective response and overall survival in advanced-stage melanoma cases. They also confirmed a total of 3 mg/kg nivolumab once every two weeks (Q2 W) for solid tumor treatment, including NSCLC 26. Long G. et al. also confirmed no differences in treatment benefits and safety between lower doses (480 mg/kg every week) vs. 3 mg/kg every two weeks. 27. Desnoyer A. et al. revealed an association between changes in drug clearance and the effectiveness of nivolumab treatment, but there were no clinically significant factors involved. The authors also confirmed no-dose relation for nivolumab treatment28. A few retrospective multicenter studies showed no significant differences in progression-free survival (PFS) times beyond standard nivolumab treatment with 3 mg/kg nivolumab once every three-eight weeks and low-dose 240 mg/kg nivolumab every two weeks; 480 mg/kg nivolumab every four weeks; and 0.1 mg/kg or 100 mg/kg nivolumab every three weeks during treatment in patients with advanced NSCLC. Third phase clinical trials CheckMate-066, -025, -057, and -017 showed similar PFS times and frequencies of side effects of 480 mg/kg Q4 W nivolumab dose compared to the standard scheme in patients after disease progression. Additionally, preliminary data from CheckMate-384 in pretreated patients with advanced-stage IIIB/IV NSCLC showed no significant differences between subgroups treated with 480 mg/kg Q4 W vs. 240 mg/kg Q2 W. The recorded adverse effect percentage was similar, with data from patients treated with 3 mg/kg Q3 W 29. The more frequent use of lower doses might be more convenient for patients during the pandemic, according to cited research.
The PD-L1 expression level is the only basic predictive marker in clinical practice for immune checkpoint inhibitor therapies. Accordingly, in European registration, no PD-L1 expression is required for nivolumab treatment, as data from various clinical trials have shown clinical benefit even in patients with PD-L1 expression levels <1% 30. The effectiveness of nivolumab in first-line monotherapy was lower than that for chemotherapy. Hence, the recommendations advise the use of nivolumab in second-line cancer therapy. Another important point is combined immunotherapy. Significantly longer PFS was observed for patients treated with nivolumab in combination with ipilimumab as a first-line treatment. The percentage of side effects in patients treated with this combination was slightly lower than that after chemotherapy 31. The Food and Drug Agency approved using nivolumab in combination with ipilimumab for first-line treatment in two dose-time combination strategies. The first involves 3 mg/kg nivolumab every two weeks and ipilimumab at a dose of 1 mg/kg every six weeks administered intravenously. The second is 360 mg/kg nivolumab every three weeks and 1 mg/kg ipilimumab every six weeks and two weeks of platinum-doublet chemotherapy with the same type of administration. Accordingly, to the European Medicine Agency, the nivolumab dose for advanced NSCLC treatment should be 240 mg/kg distributed intravenously every two weeks in monotherapy and 360 mg/kg every three weeks in combination with 1 mg/kg ipilimumab every six weeks and platinum-based chemotherapy every three weeks. Despite the higher effectiveness of immunotherapy in lung cancer treatment, several specific side effects indicate the need for optimizing the drug dose 32. The failure of dependence in drug-response correlation for nivolumab treatment suggests a necessity to improve the methods of nivolumab use in clinical practice, e.g., in combination with other ICIs 7,25,31,33,34. Seldom-distributed higher doses of nivolumab might be more convenient for cancer-bearing patients, especially during a pandemic of SARS Covid-19. It is crucial to limit patients' hospitalization time without impacting their treatment program.
In conclusion, the possibility of generating autologous mature dendritic cells capable of activating Th cells from PBMCs of patients in stage IIIB/IV lung adenocarcinoma allows us to conclude that the patient's immunology system was not exhausted. Furthermore, data from clinical trials did not show a clear relationship between the occurrence and intensity of adverse effects and the administered dose of nivolumab. Therefore, we could conclude that the possibility of adverse effect development after immunotherapy mainly depends on the patients' immune system condition.
The key to improving immunotherapy efficiency by lowering side effects is to achieve the optimal T cell activation level. Currently, it is more important due to the possibility of subcutaneous anti-PD-1 drug administration. Johnson M et al. revealed the feasibility of monthly subcutaneous administration of the anti-PD-1 antibody. An intravenous dose escalation (0.5, 1, 3, or 10 mg/kg) of anti-PD-1 antibody was administered every 3 weeks, or a subcutaneous dose of 300 mg was administered every 4 weeks. The authors did not observe dose-limiting toxic effects, while grade 3 or higher treatment-related adverse events occurred in 4 (16%) patients treated intravenously and in 1 (6.7%) patient treated subcutaneously. Thus, monthly subcutaneous administration of anti-PD-1 offers a convenient and effective alternative to currently available intravenously administered checkpoint inhibitors 35.
The future perspective regarding the effectiveness of immunotherapy should be directed at minimizing the side effects while maintaining the stimulation of T lymphocyte activity. However, this requires in vitro tests, which should always be the basis for the development of new immunotherapy combinations and their administration.