Characterization and in vitro data of antibody drug conjugates (ADCs) derived from heterotrifunctional linker designed for the site-specific preparation of dual ADCs

Experimental procedures and 1H and 13C NMR of the heterotrifunctional linker used for preparation of dual drug conjugates and PBD payload are included. Procedure for carrying preparation of antibody linker conjugate via thiol maleimide conjugation and antibody drug conjugates (ADCs) using copper assisted click reaction and oxime ligation, their cell viability assay and western blotting procedures of the resultant conjugates are detailed. Also, reduced mass spectroscopy results and in vitro cytotoxicity of antibody drug conjugates used in this article are shown.


Value of the data
In the data presented here we describe the synthesis of a heterotrifunctional linker serving as a flexible platform for the preparation of dual-cytotoxic drug conjugates in a site-specific manner [1]. As a proof of concept, we detail the preparation and evaluation of dual drug ADCs carrying monomethyl auristain E (MMAE) and pyrrolobenzodiazepine dimer (PBD).

Data
Experimental procedures and NMR of the linker used for preparation of dual drug conjugates and PBD payload are included. Procedure for carrying preparation of antibody drug conjugates (ADCs), their cell viability assay and western blotting procedures are detailed. Also, reduced mass spectroscopy results of antibody drug conjugates used in this article are listed.

General information
All reagents were purchased through VWR or Sigma Aldrich and were used without further purification. 1 H and 13 C NMR spectra were obtained on a Bruker Ascend 400 spectrometer. Coupling constants are quoted in hertz (Hz). Mass Spectrometry was obtained using a Waters Acquity UPLC LCMS. O-vc-PAB-MMAE was purchased from Concortis.

Synthesis of SG3457
Synthesis of SG3457.

General information for the synthesis of SG3457
Reaction progress was monitored by thin-layer chromatography (TLC) using Merck Kieselgel 60 F254 silica gel, with fluorescent indicator on aluminium plates. Visualisation of TLC was achieved with UV light. Flash column chromatography was performed using Merck Kieselgel 60 F254 silica gel. Extraction and chromatography solvents were bought and used without further purification from Fisher Scientific, U.K. All chemicals were purchased from Aldrich, Lancaster or BDH. Azido-dPEG s 8acid was purchased from Quanta biodesign. 1 H and 13 C NMR spectra were obtained on a Bruker Avance 400 spectrometer. Coupling constants are quoted in hertz (Hz). Chemical shifts are recorded in parts per million (ppm) downfield from tetramethylsilane. Spin multiplicities are described as s (singlet), bs (broad singlet), d (doublet), t (triplet), q (quartet), p (pentuplet) and m (multiplet).
The LC/MS conditions were as follows:

Method 1 (3 min run)
The HPLC (Shimadzu Nexera s /Prominence s LCMS-2020) was run using a mobile phase of water containing 0.1% formic acid (A) and acetonitrile containing 0.1% formic acid (B). Gradient: 5% B held over 25 s, then increased from 5% B to 100% B over a 1 min 35 s' period. The composition was held for 50 s at 100% B, then returned to 5% B in 5 s and held there for 5 s. The total duration of the gradient run was 3.0 min.

Method 2 (15 min run)
The HPLC (Shimadzu Nexera s /Prominence s LCMS-2020) was run using a mobile phase of water containing 0.1% formic acid (A) and acetonitrile containing 0.1% formic acid (B). Gradient: 5% B held over 1.0 min, then increased from 5% B to 100% B over 9 min. The composition was held for 2 min at 100% B, then returned to 5% B in 10 s and held for 2 min 50 s. The total duration of the gradient run was 15.0 min.

General procedure for linker-antibody conjugation
Heterofunctional linker 1 was conjugated to desired antibody in multiple steps. First, antibodies were mildly reduced to generate free thiols by adding 50 mM TCEP solution to 5 mL of 3.6 mg/mL antibody solution in 10 mM PBS, pH 7.4, 1 mM EDTA. The resulting solution was gently mixed at 37°C for 1 h. Reduced antibody was transferred to a slide-a-lyzer dialysis cassette (10 K MWCO) and dialyzed against PBS, 1 mM EDTA, pH 7.4, 4°C for 24 h with several buffer changes. Reduced antibody was oxidized to reform internal disulfides by addition of dehydroascorbic acid (50 mM stock in DMSO, 20 eq.) followed by gentle mixing for 4 h at room temperature. To the oxidized antibody solution was then added a solution of heterofunctional linker 1 (10 mM, DMSO, 4 eq.) The resulting reaction mixture was briefly vortexed and further incubated for the desired amount of time followed by addition of N-acetyl cysteine (10 μL of a 100 mM solution in water, 50 eq) and further incubation for 15 min to quench unreacted maleimide. All conjugation reactions were performed at room temperature (22°C) under ambient atmosphere. To the resulting deep blue solution was added sodium ascorbate (0.72 mL, 500 mM) and the mixture vortexed until the color disappeared. The final concentration of this cocktail was as follows: CuSO 4 ¼ 6 mM, BTTAA ¼ 30 mM, sodium ascorbate ¼ 90 mM. In a separate tube containing a solution (10 mM PBS, pH 7.4) of antibody conjugated with linker 1 (8.5 mg/mL) was added the payload equipped with the azido group (10 mM, DMSO, 8 eq.). The final concentration of DMSO in the resultant solution was adjusted to 10% by adding free DMSO. To this solution was added the catalyst cocktail to attain a final concentration of CuSO4 as 1 mM. The resulting solution was gently mixed at room temperature for 5 h followed by purification using CHT (Ceramic hydroxyapatite) column.

General procedure for oxime ligation
In a tube containing a solution (10 mM PBS, pH 7.4) of antibody conjugated with linker 1 (8.3 mg/mL) was added the payload equipped with the aminoxy group (10 mM, DMSO, 8 eq.). The final concentration of DMSO in the resultant solution was adjusted to 10% by adding free DMSO. To this solution was added the m-phenylenediamine catalyst (1 M, pH ¼ 7.2) to attain a final concentration of catalyst as 100 mM. The resulting solution was gently mixed at room temperature for 12 h followed by purification using CHT (Ceramic hydroxyapatite) column.

ADC characterization
Reduced liquid chromatography mass spectrometry analysis (rLCMS), which was used to determine conjugation at the light or heavy chain and drug to antibody ratio (DAR), was performed on an Agilent 1290 series uHPLC coupled to an Agilent 6230 TOF. 2 μg of reduced antibodies or ADCs were loaded onto a Zorbax RRHD 300-Diphenyl (2.1 Â 50 mm, 1.8 μm, Agilent) and eluted at a flow rate of 0.5 mL/min using a step gradient of 80% B after 2.1 min (mobile phase A: 0.1% Formic acid in water and mobile phase B: 0.1% Formic acid in acetonitrile). A positive time-of-flight MS scan was acquired, and data collection and processing were carried out using MassHunter software (Agilent). Conjugation efficiencies were calculated based on intensity of mass spectrometry signals of unconjugated vs conjugated ( Figs. 1 and 2).

Determination of in vitro cell viability
Human cancer cell lines SK-BR-3 and MDA-MB-453 were seeded into white polystyrene tissueculture treated 96-well plates (Costar) at a density of 3000 cells/well in RPMI þ 10% FBS (Invitrogen). On the following day, antibodies and ADCs were spiked into triplicate wells using an 8-point dose curve of 1:4 serial dilutions starting from 0.5 μg/mL. Cell viability was determined 6 days later using the Cell Titer-Glo Luminescent Cell Viability Assay kit (Promega) following the manufacturer's protocol. Luminescence was measured using an EnVision 2104 Multilabel Reader (Perkin Elmer). Cell viability was calculated as a percentage of control untreated cells.

Transparency document. Supporting information
Transparency data associated with this article can be found in the online version at https://doi.org/ 10.1016/j.dib.2018.11.005.