Targeted Molecular Construct for Bioorthogonal Theranostics of PD-L1-Expressing Cancer Cells

Molecular targeting of tumor-overexpressed oncoproteins can improve the selectivity and tolerability of anticancer therapies. The immunoinhibitory membrane protein programmed death ligand 1 (PD-L1) is highly expressed on certain tumor types, which masks malignant cells from T cell recognition and creates an optimal environment for the cancer to thrive and spread. We report here a ligand-tetrazine conjugate (LTzC) armed with a PD-L1 small molecule inhibitor to selectively target PD-L1-expressing cancer cells and inhibit PD-L1 function and conjugated to a tetrazine module and a lipoyl group to incorporate bioorthogonal reactivities and an oxidative stress enhancer into the construct. By pairing LTzC with an imaging probe, we have established a “track-&-tag” system for selective labeling of PD-L1 both on and in living cells using click chemistry. We have further shown the specificity and versatility of LTzC by click-to-release activation of prodrugs and selective killing of PD-L1-expressing breast cancer cells, offering a new multimodal approach to “track-&-treat” malignant cells that are capable of evading the immune system.

exposed (Supp. Fig. 1) and not engaged in binding to PD-L1 protein, and therefore identified as the suitable attachment site for the synthetic construction of LTzCs.
Purification was performed (0-10% MeOH in DCM) using column chromatography to afford non-targeting LTzC as a pink oil (7 mg, 42% yield were dissolved in anhydrous DCM (2 mL) under nitrogen atmosphere. The reaction was cooled to 0°C using an ice bath before triphosgene solution (17 mg, 0.055 mmol) in toluene was added dropwise. The reaction mixture was allowed warm to room temperature and stirred for 4 h. The reaction mixture was reduced to dryness by bubbling with argon before the residue was redissolved in anhydrous DCM and cooled to 0 °C (2 S7 mL). Compound 7 (19 mg, 0.11 mmol) in 1 mL of anhydrous DCM was added dropwise. The reaction was allowed to return to room temperature and stirred overnight under nitrogen atmosphere. The reaction was quenched with water and extracted with DCM. The resulting organic layer was dried over MgSO4, filtered and removed in vacuo to afford the crude product as a yellow oil. Purification was performed (0-20% EtOAc in Hexane) to afford PF 1 as a yellow solid (11 mg, 25% yield

Spectrophotometric analysis and calculation of cycloaddition reaction kinetics
The second-order rate constants of the cycloaddition between LTzC and benzonorbornadiene derivatives (compound 8 and 9) were determined using spectrophotometric analysis. A solution of LTzC (   Experiments were performed in triplicates. PrestoBlue TM cell viability reagent (10 % v/v) was added to each well and the plate incubated for 90 min. Fluorescence emission was detected using a PerkinElmer Victor multilabel reader (ex = 540nm; em = 590nm). All conditions were normalized to the untreated cells (100%) and analysis was performed using GraphPad Prism. 7. LTzC-mediated pro-drug activation in breast cancer cells MDA-MB-231 and MCF-7 cells were seeded in a 96-well plate (1000 cells per well), incubated or 24 h, followed by replacement with fresh media with or without IFN-γ (40 ng/mL). After 24 h, the media was replaced with fresh media containing respective LTzCs (3 μM) and incubated for 4 h. The media was removed from the wells, washed with PBS (3 times), and replaced with fresh media containing Pro-Dox (0.3 μM) or Pro-INK128 (0.1 μM) for 5 d. Control wells were incubated with DMSO (0.1 % v/v). Experiments were performed in triplicates.
PrestoBlue TM cell viability reagent (10 % v/v) was added to each well and the plate incubated for 90 min.
Fluorescence emission was detected using a EnVision Multimode Plate Reader (ex = 540 nm; em = 590 nm).
All conditions were normalized to the untreated cells (100%) and analysis was performed using GraphPad Prism.