Cell lines

Peripheral blood mononuclear cells (PBMC)

Preparation Method

PBMC were isolated from fresh whole blood. PBMC were suspended at 2 x 10⁶cells/well in 2 ml complete medium (RPMI-1640 supplemented with 2 mM L-glutamine, 10% FCS and 1% penicillin/streptomycin); 24-well non-treated culture plates were precoated with plate-bound 2 μg/ml anti-CD3 antibody and 2 μg/ml anti-CD28 antibody for 2.5 h at 37℃.

Reaction Conditions

PBMC were either plated as‘non-activated’ in non-coated wells or ‘activated’ in precoated wells. Plated cells were then treated with anti-PD-1 monoclonal antibody, pembrolizumab, at 2 μg/ml and were incubated for 24 h in a humidified incubator at 37℃ and 5% CO².

Applications

Pembrolizumab treatment could reduce PD-1 expression significantly in CD4⁺T cells in non-activated and activated PBMC. Pembrolizumab treatment resulted in a significant reduction in PD-1 expression in both CD4⁺CD25⁺T cells and CD4⁺CD25^-T cells.

Animal models

NSG mice; Humanized NSG mice

Preparation Method

Humanized NSG mice were generated by intravenous injection of 105 human CD34⁺(hCD34⁺) HPSCs into 3-week-old female NSG mice, 4 h post-140 cGy total body irradiation using the RS-2000 irradiator. Humanized NSG mice that had over 25% hCD45⁺cells in the peripheral blood were considered as engrafted and humanized. Then patient-derived tumors were finely minced and loaded into 1-cc syringes with 14-gauge needles. Depending on the tumor model, 20–40 μl of homogenized tumor tissue was inoculated subcutaneously at the right flank of mice while under anesthesia.

Dosage form

10 mg/kg i.p. for the first dose, followed by 5 mg/kg, i.p. dosage on day 5, 10, 15, 20, and 25.

Applications

Pembrolizumab treatment significantly decreased PD^-1⁺cell detection in hCD45⁺cells in tumor models. But the percentage of PD^-L1⁺cells in hCD45+ or hCD45^-cell populations was not affected by pembrolizumab treatment. Moreover, pembrolizumab treatment obviously delayed TNBC CDX growth compared with the vehicle control group and inhibited tumor growth in both TNBC and NSCLC PDX Onco-Humanized NSG models.

• Bioengineered 13.1 (2022): 917-929.PMID: 34968160

Pembrolizumab, an anti–programmed death-1 monoclonal antibody, has demonstrated clinically significant anti-tumor activity with acceptable safety in patients with advanced solid cancers and was approved by the U.S. FDA for the treatment of advanced melanoma, NSCLC, head and neck squamous cell cancer, and other malignant tumors.^[1]

In vitro study demonstrated that pembrolizumab had a greater effect on PD-1 expression in CD4+CD25- T cells which expressed most of the PD-1, and that are comprised of non-Treg and/or non-activated T cells. However, pembrolizumab did not affect the expression levels of Treg-related markers, including cytotoxic T lymphocyte antigen-4 (CTLA-4), CD15s, latency-associated peptide (LAP) and Ki-67 as well as the levels of FoxP3+/-Helios+/- Treg subsets in both cohorts.^[2]

In vivo study of pembrolizumab indicated that in Onco-Humanized NSG mice, pembrolizumab could inhibit tumor growth, not only in CDX but also in various PDX tumor models. Results showed that the efficacy of pembrolizumab is dependent on the engraftment of an adaptive human immune system in Onco-Humanized NSG mice, specifically hCD8+ T cells. Furthermore, pembrolizumab increased both CD4+ and CD8+ T-cell numbers in the blood of the 2 NSCLC Onco-Humanized NSG models but decreased both CD4+ and CD8+ T-cell numbers in the blood of the TNBC Onco-Humanized NSG model.^[3]

References:
[1]. Mo DC, et al. Safety and efficacy of pembrolizumab plus lenvatinib versus pembrolizumab and lenvatinib monotherapies in cancers: A systematic review. Int Immunopharmacol. 2021 Feb;91:107281.
[2]. Toor SM, et al. In-vitro effect of pembrolizumab on different T regulatory cell subsets. Clin Exp Immunol. 2018 Feb;191(2):189-197.
[3]. Wang M, et al. Humanized mice in studying efficacy and mechanisms of PD-1-targeted cancer immunotherapy. FASEB J. 2018 Mar;32(3):1537-1549.