Tyrosinase-Mediated Synthesis of Nanobody-Cell Conjugates

Abstract

A convenient enzymatic strategy is reported for the modification of cell surfaces. Using a tyrosinase enzyme isolated from Agaricus bisporus, unique tyrosine residues introduced at the C-termini of nanobodies can be site-selectively oxidized to reactive o-quinones. These reactive intermediates undergo rapid modification with nucleophilic thiol, amine, and imidazole residues present on cell surfaces, producing novel nanobody-cell conjugates that display targeted antigen binding. We extend this approach toward the synthesis of nanobody-NK cell conjugates for targeted immunotherapy applications. The resulting NK cell conjugates exhibit targeted cell binding and elicit targeted cell death.

Figure 1

General strategy for modifying cell surfaces with nanobodies. (a) Tyrosinase catalyzes the oxidation of small-molecule phenols to highly reactive o-quinones, which can modify nucleophiles present on proteins. Engineered tyrosine tags at protein termini can also be oxidized by tyrosinase, producing a site-specific o-quinone on the protein that reacts with protein-based nucleophiles. (b) Tyrosine-tagged nanobodies can be site-specifically oxidized by tyrosinase for attachment of these proteins to cells. The resulting linkage produces a well-defined point of attachment for installing nanobodies on cell surfaces while imbuing the target cell with novel antigen-binding functionality. (c) Nanobodies are low-molecular-weight (∼10–15 kDa) antigen-binders derived from the variable region of the camelid antibody (PDB ID 3K1K).

Figure 2

Modification of NK cell surfaces with nanobodies. (a) Tyrosinase enzyme produces a site-specific o-quinone at the C-terminal Ser–Gly₄–Tyr tag installed on nanobodies, as evidenced by a 14 Da mass shift detected via ESI-TOF MS. (b) To verify that NK cell surfaces can be decorated with nanobodies using tyorsinase, a Tyr-tagged nanobody against GFP (nbGFP_Tyr) was designed. Using tyrosinase, nbGFP_Tyr can be attached to the cell surface, and 2° labeling with GFP can be used to analyze the reaction using flow cytometry. (c) Labeling experiments with nbGFP_Tyr validated attachment of the nanobody to the cell surface, as only cells treated with both nbGFP_Tyr and tyrosinase showed an increase in GFP fluorescence (red trace) over controls (blue and orange traces). (d) Using a Cys point mutant, a single FITC dye can be attached to each nbGFP_Tyr (nbFITC). After attachment of 10 μM nbFITC to cell surfaces, comparison against FITC-calibration beads determined that a median value of ∼120,000 copies of the nanobody were linked to the cells. Data are represented as box plots, with the top of the box representing the 75th percentile of the data, the middle line representing the median of the data, and the bottom of the box representing the 25th percentile of the data.

Figure 3

Decoration of NK cells for nanobody-directed cell–cell interactions. (a) Using tyrosinase, a Tyr-tagged nanobody against HER2 (nbHER2_Tyr) was attached to NK cells. Secondary labeling with a soluble FITC–HER2 showed that only cells exposed to nbHER2_Tyr and tyrosinase exhibited a shift in FITC signal detected via flow cytometry (red trace) over controls. (b) To assess if tyrosinase-synthesized NK–nbHER2 conjugates can make targeted contacts with HER2+ cells, NK–nbHER2 cells were mixed with a HER2+ cell line (SK-BR-3) at a ratio of 2:1 (NK:target). Cells were allowed to bind and settle and then imaged using fluorscence microscopy. A nearest neighbor analysis was performed (CellProfiler), indicating that a statistically significant proportion of target cells (green) were bound to two or more NK–nbHER2 cells (red) only when the NK cells were pretreated with nbHER2_Tyr and tyrosinase (orange bar). (c) Fluorescence microscopy images confirm rosette formation is only seen when NK cells are pretreated with both nbHER2_Tyr and tyrosinase.

Figure 4

Targeted cell killing elicited by tyrosinase-synthesized nanobody–NK cell conjugates. (a) Schematic representation of the fluorescence-based cell assay used to determine NK cytotoxicity. HER2+ cells (SK-BR-3) were preloaded with calcein AM dye, which is retained by the cell membrane after uptake. Lysis of the HER2+ cell releases dye into the supernatant, providing a measurement for cell lysis. Only NK cells pretreated with both nbHER2_Tyr and tyrosinase (orange bar) show statistically significant specific cell lysis over control treatments. (b) To assess how the ratio of NK:target cell impacts specific cytotoxicity, NK–nbHER2 cells were synthesized using 10 μM nbHER2_Tyr and 400 nM tyrosinase and mixed with calcein AM loaded HER2+ cells (SK-BR-3). Statistically significant cell death was observed at ratios even as low as 2:1 (effector:target). (c) To assess the required concentration of nbHER2_Tyr needed to elict NK-mediated cell death, a variety of concentrations of nbHER2_Tyr were used to label NK cells with tyrosinase. Increased lysis was observed when using 5 and 10 μM nbHER2, while a sharp reduction of NK lytic activity was observed at the higher concentration of 20 μM nbHER2_Tyr.