Ex vivo mass spectrometry-based biodistribution analysis of an antibody-Resiquimod conjugate bearing a protease-cleavable and acid-labile linker

Immune-stimulating antibody conjugates (ISACs) equipped with imidazoquinoline (IMD) payloads can stimulate endogenous immune cells to kill cancer cells, ultimately inducing long-lasting anticancer effects. A novel ISAC was designed, featuring the IMD Resiquimod (R848), a tumor-targeting antibody specific for Carbonic Anhydrase IX (CAIX) and the protease-cleavable Val-Cit-PABC linker. In vitro stability analysis showed not only R848 release in the presence of the protease Cathepsin B but also under acidic conditions. The ex vivo mass spectrometry-based biodistribution data confirmed the low stability of the linker-drug connection while highlighting the selective accumulation of the IgG in tumors and its long circulatory half-life.


SDS-PAGE
Protein samples were diluted to 0.2-0.3mg/ml in PBS and mixed with either Reducing or Non-Reducing 5x Loading buffer.Samples were denatured for 5 min at 95°C and loaded on NuPAGE 4-12% Bis-Tris Gel (NovexTM by Life Technologies).1x MES NuPAGE (NovexTM by Life Technologies) was used as running buffer.The electrophoresis was performed at 180 V, 110 mA for 1 h.The gel was then rinsed with deionized water and stained with Coomassie blue for 15 min on an orbital shaker.The staining solution was discarded.The gel was then rinsed with deionized water and immerged in destaining solution (10% AcOH / 30% MeOH / mQ water) for 3 h on an orbital shaker.
The destaining solution was discarded and recycled and the gel was rinsed with deionized water.Recipes for loading buffers and staining solution are described in Tables S1-S3.Table S3.Coomassie blue staining recipe

Gel Filtration
A 100 μL of diluted ISAC 3 sample (final concentration 0.1-0.5 mg/mL) was loaded on FPLC (Äkta, GE Healthcare) and the protein was separated by a Superdex200 Increase 10/300 GL column (GE Healthcare) previously equilibrated with 1 CV PBS, using PBS as the mobile phase at a flow rate of 0.6 mL/min (column pressure limit set at 5 MPa).The protein was detected by a UV-detector at a wavelength of 280 nm.

Mass Spectrometry
A sample of ISAC 3 was diluted to about 0.1 mg/mL and LC-MS was performed on a Waters Xevo G2XS QTof instrument (ESI-ToF-MS) coupled to a Waters Acquity UPLC H-Class System using a 2.1 × 50 mm Acquity BEH300 C4 1.7 μm column (Waters).H2O + 0.1% FA (solvent A) and MeCN + 0.1% FA (solvent B) were used as the mobile phase at a flow rate of 0.4 mL/min.The gradient was programmed as follows: after 1.5 min isocratic with 95% solvent A, stepwise change from 95% solvent A to 95% solvent B in 4.5 min (10% increase every 0.5 min), back to 95% solvent A in 0.5 min, linearly to 95% solvent B and back to 95% solvent A in 2.25 min (last step repeated twice).

HPLC Analysis
The samples were injected in into an analytical HPLC-PDA system (see General Remarks and Procedures).H2O + 0.1% TFA (solvent A) and MeCN + 0.1% TFA (solvent B) were used as the mobile phase at a flow rate of 1 mL/min.The gradient was programmed from 5% to 30% B over 26 min.
Areas under the curve (AUC) of the detected peaks were measured using software associated to the HPLC systems.The rate of R848 release from the starting carbamate was obtained by calculating the relative ratios of AUC values corresponding to the prodrug 2 and the free payload.Data were plotted versus time using GraphPad Prism software.
4.3.2.Pure Cys-ValCit-PABC-R848 2 LC profile (254 nm) P1 Mc-ValCit-PABC-PNP was purchased from MedChemExpress, Resiquimod (R848) was purchased from Fluorochem and Cathepsin B (from human placenta) was purchased from Merck.All other reagents were purchased from Merck and used as supplied.Solvents were used as supplied by Merck in HPLC or analytical grade.

5x Non-Reducing Loading Buffer (100 mL)
Samples at timepoints 0 and 4 h for the Cathepsin B release assay and stability in Acetate Buffer (2.2 M, pH 5.4) were submitted for High Resolution Mass Spectrometry (see General Remarks and Procedures).