Development and characterization of single domain monoclonal antibody against programmed cell death ligand-1; as a cancer inhibitor candidate

Abstract

Objectives: One of the important interactions in controlling the human immune system is the reaction between checkpoint proteins such as programmed cell death-1 (PD-1) and its ligand, PD-L1. These are negative immunoregulatory molecules that promote immune evasion of tumor cells. PD-L1 expression is an immune-mediated mechanism used by various malignant cells in order to down-regulate the immune system. Checkpoint inhibitors (CPIs) are a new class of anti-cancer agents that stimulate immune cells to elicit an antitumor response by blocking the ligand and receptor interactions. Nanobody (Nb) as a new type of antibody fragment, has some potential as CPI.

Materials and methods: A female camel was immunized with recombinant PD-L1 protein, nanobody library was constructed and PD-L1 specific Nb was selected. The selected Nb was characterized in terms of affinity, specificity, and binding potency in ELISA, Western blotting, and flow cytometry.

Results: Developed nanobody, A22 binds to its cognate target with high specificity and affinity. Western blot and flow cytometry techniques showed that nanobody A22 was able to specifically detect and attach to human PD-L1 protein on the cell surface and in the cell lysate. MTT assay showed the inhibitory effect of PD-L1 by specific Nb on A431 and HEK293 cells, with no cytotoxic effect on cell growth.

Conclusion: The results highlighted the potential of anti-PD-L1 Nb as a novel therapeutic in cancer therapy without undesirable cytotoxicity.

Keywords: Cancer; Checkpoint inhibitors; Nanobody; Programmed cell death - ligand-1; Single domain antibody.

Figure 1

Antibody titration against recombinant PD-L1 after camel immunization procedure

Figure 2

VHH gene amplification. Amplification of VH and VHH genes by the 1st PCR (a); the second nested PCR for VHH gene (b)

Figure 3

Polyclonal phage-ELISA for evaluation of the biopanning procedure

Figure 4

PE-ELISA assay for selection of individual nanobodies

Figure 5

Protein expression; SDS-PAGE (a) and Western blotting (b) of Nb. A22. #1, 4: Lysate of recombinant clones after induction; #2, 3: Purified Nb. A22 (13kDa)

Figure 6

Specificity analysis of Nb. A22 by ELISA

Figure 7

Binding analysis of nanobody. A22 by flow cytometry; HEK293 (PD-L1 negative) (a) and A431 (PD-L1 positive) (b) cell lines. Solid and dotted histograms indicated cells treated with and without Nb. A22, respectively

Figure 8

Cytotoxicity assay of A431 (a) and HEK293 (b) cells treated with nanobody (Nb)