Axially Substituted Silicon Phthalocyanine Payloads for Antibody–Drug Conjugates

Abstract

IR700, a silicon phthalocyanine (SiPc) photosensitizer, is an antibody–drug conjugate payload used clinically. It is, however, the sole SiPc payload to date, possibly due to the difficulty of its synthesis, resulting from its asymmetric phthalocyanine skeleton. Here, we report a new axially substituted SiPc payload that is more easily synthesized. Trastuzumab conjugated with the SiPc showed light- and antigen-dependent cytotoxicity in HER2-overexpressing cancer cell lines.

Key words

phthalocyanines - antibody–drug conjugates - photodynamic therapy - cancer - medicinal chemistry

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• 14 Conjugation of NHS-SiPcs with Antibodies; General Procedure Trastuzumab (8.0 mg; Roche) was purified by using a PD Minitrap G-25 column with pH 8.0 phosphate buffer to afford a 7.7 mg/mL trastuzumab solution. To 100 μL of this solution was added a solution of NHS-SiPc (50 nmol) in DMSO (20 μL), and the mixture was stirred at rt in darkness for 12 h. The mixture was then purified twice on a PD Minitrap G-25 column with phosphate-buffered saline (PBS) to afford an Ab-SiPc stock solution.
• 15 In Vitro Phototoxicity Studies The phototoxicities of Ab-NuSiPc, Ab-CyaSiPc, and Ab-IR700 were quantified by using Cell Counting Kit-8 (Dojindo Molecular Technologies, Inc.) according to the method described in the manufacturer’s manual. Briefly, ten thousand cells per well with 50 μL of medium were seeded into 96-well plates and cultured overnight. Subsequently, the cells were separately incubated for 24 h at 37 °C with the addition of 50 μL of various concentrations of Ab-NuSiPc, Ab-CyaSiPc, or Ab-IR700, prepared by three-times sequential dilution from 20 μg mL^–1. The drug-containing medium was then removed and the cells were washed with PBS once, and fresh medium was added. The cells were irradiated from the bottom of the 96-wells plate with a 690 nm LED lamp (Yamato Scientific Co., Ltd.) at 100 J cm^–2 at rt. After 24 h, Cell Counting Kit-8 reagent was added at 10 μL per well and the cells were incubated for 1.5 h. The absorbance of the samples was then measured on an iMark™ Microplate Absorbance Reader (BIO-RAD) with a 450 nm-wavelength filter.