A novel nanobody-based immunocytokine of a mutant interleukin-2 as a potential cancer therapeutic

Abstract

The immunotherapeutic application of interleukin-2 (IL-2) in cancer treatment is limited by its off-target effects on different cell populations and insufficient activation of anti-tumor effector cells at the site of the tumor upon tolerated doses. Targeting IL-2 to the tumor microenvironment by generating antibody-cytokine fusion proteins (immunocytokine) would be a promising approach to increase efficacy without associated toxicity. In this study, a novel nanobody-based immunocytokine is developed by the fusion of a mutant (m) IL-2 with a decreased affinity toward CD25 to an anti-vascular endothelial growth factor receptor-2 (VEGFR2) specific nanobody, denoted as VGRmIL2-IC. The antigen binding, cell proliferation, IFN-γ-secretion, and cytotoxicity of this new immunocytokine are evaluated and compared to mIL-2 alone. Furthermore, the pharmacokinetic properties are analyzed. Flow cytometry analysis shows that the VGRmIL2-IC molecule can selectively target VEGFR2-positive cells. The results reveal that the immunocytokine is comparable to mIL-2 alone in the stimulation of Primary Peripheral Blood Mononuclear Cells (PBMCs) and cytotoxicity in in vitro conditions. In vivo studies demonstrate improved pharmacokinetic properties of VGRmIL2-IC in comparison to the wild or mutant IL-2 proteins. The results presented here suggest VGRmIL2-IC could be considered a candidate for the treatment of VEGFR2-positive tumors.

Keywords: Immunocytokine; Immunotherapy; Mutant IL-2; Nanobody; VEGFR2.

Fig. 1

SDS-PAGE and western blotting analysis of protein expression a SDS-PAGE: M: protein molecular weight marker; #1, 2: Lysates of E. coli cells after and before induction of VGRmIL2-IC expression #3: Purified VGRmIL2-IC protein; #4: Lysate of E. coli cells after induction of wtIL-2 expression; #5, 6: Lysate of E. coli cells after induction of mIL-2 expression; 7: Purified mIL-2 b Western blotting analysis: #1; Purified wtIL-2; #2: Purified mIL-2; #3: Purified VGRmIL2-IC

Fig. 2

Flow cytometry analysis a VEGFR2 expression analysis on the surface of 293KDR and HEK-293 cells using commercially available anti-VEGFR2 PE-conjugated antibody: Blue and green graphs: Unstained and stained HEK-293 cells, respectively; Orange and red graphs: Unstained and stained 293KDR cells, respectively. b Binding potency analysis of VGRmIL2-IC and 3VGR19 Nb towards 293KDR cells using a homemade rabbit anti-camel antibody followed by FITC-conjugated anti-rabbit IgG antibody, gray graph: Untreated control cells; Red graph: Secondary antibody alone control; Light blue graph: LivIL2-IC as a non-specific Nb-IL2 control; Dark blue and green graphs: 3VGR19 Nb and VGRmIL2-IC treated cells, respectively

Fig. 3

a PBMC proliferation assay analysis by Alamar Blue reduction. Starved ConA-activated PBMCs were stimulated with different equimolar concentrations (1–105 pM) of VGRmIL2-IC or mIL-2 for 48 h. b IFN-γ secretion analysis by ELISA assay. PBMCs were stimulated with 30 pmol of VGRmIL2-IC or mIL-2. After 48 h of incubation, the supernatants were collected, and the secreted IFN-γ levels were measured. The results are the mean ± SD values of triplicate experiments. Data analysis was performed using a one-way ANOVA test (ns: not significant; ****P < 0.0001)

Fig. 4

Cellular cytotoxicity assay at different effector (PBMCs) and target cells (293KDR) ratios. Untreated PBMCs without exogenous cytokines served as control. Experiments were repeated three times and the results are presented as mean ± SD. Multiple comparisons were performed using 2-way ANOVA

Fig. 5

Pharmacokinetic profiles of VGRmIL2-IC, wtIL-2, and mIL-2 in mice. Monitoring of wtIL-2 and mIL-2 was performed for up to 48 h. Cytokines were not detectable in the plasma after 24 h