A. Yamaguchi et al.
Bioorganic & Medicinal Chemistry 32 (2021) 116013
and mix-FA-conjugated bispecific FR/HER2 ADCs and bispecific integ-
rin/HER2 ADCs were prepared in the same manner. Purified ADCs were
formulated in PBS and stored at 4 ◦C.
blocking buffer (0.2% BSA in PBS) with agitation at room temperature
for 2 h. After the blocking buffer was discarded, serially diluted ADC
samples (in 100 µL PBS containing 0.1% BSA) were added and the plate
was incubated overnight at 4 ◦C with agitation. The buffer was discarded
4.4. HIC analysis
and the cells were washed three times with 100
μL of PBS containing
0.25% Tween 20. Cells were then incubated with 100
μ
L of donkey anti-
Each ADC (1 mg mLꢀ 1, 10 µL in PBS) was analyzed using an Agilent
1100 HPLC system equipped with a MAbPac HIC-Butyl column (4.6 ×
100 mm, 5 µm, Thermo Scientific). Elution conditions were as follows:
mobile phase A = 50 mM sodium phosphate containing ammonium
sulfate (1.5 M) and 5% isopropanol (pH 7.4); mobile phase B = 50 mM
sodium phosphate containing 20% isopropanol (pH 7.4); gradient over
human IgG–HRP conjugate (diluted 1:10,000 in PBS containing 0.1%
BSA, Jackson ImmunoResearch) at room temperature for 1 h. The plate
was washed three times with PBS containing 0.25% Tween 20, and 100
μ
L of TMB substrate (0.1 mg mLꢀ 1) in phosphate–citrate buffer/30%
H2O2 (1:0.0003 vol to volume, pH 5) was added. After color was
developed for 10–30 min, 25 μL of 3 N-HCl was added to each well and
30 min from A:B = 99:1 to 1:99; flow rate = 0.5 mL minꢀ 1
.
then the absorbance at 450 nm was recorded using a plate reader
(BioTek Synergy HTX). Concentrations were calculated based on a
standard curve. KD values were then calculated using Graph Pad Prism 8
software. All assays were performed in triplicate.
4.5. Long-term stability test
Each ADC (1 mg mLꢀ 1, 100 µL) in PBS was incubated at 37 ◦C. Ali-
quots (10 µL) were taken at each time point (7 and 14 days) and
immediately stored at ꢀ 80 ◦C until use. Samples were analyzed using an
Agilent 1100 HPLC system equipped with a MAbPac SEC analytical
column (4.0 × 300 mm, 5 µm, Thermo Scientific). Elution conditions
were as follows: flow rate = 0.2 mL minꢀ 1; solvent = PBS.
4.9. Cell viability assay
Cells (KB, U-87ΔEGFR, KPL-4 or HEK293) were seeded in a culture-
treated 96-well clear plate (5,000 cells per well in 50 μL culture me-
dium) and incubated at 37 ◦C under 5% CO2 for 24 h. Serially diluted
samples (50 µL) were added to each well and the plate was incubated at
37 ◦C for 72 h. After the old medium was replaced with 80 µL fresh
4.6. Human cathepsin cleavage assay
medium, 20
μ
L of a culture medium containing WST-8 (1.5 mg mLꢀ 1
,
Each ADC (1 mg mLꢀ 1) in 30 µL of MES buffer (10 mM MES-Na, 40
µM DTT, pH 5.0) was incubated at 37 ◦C for 10 min. To the solution was
added pre-warmed human cathepsin B (20 ng µLꢀ 1, EMD Millipore) in
30 µL MES buffer, followed by incubation at 37 ◦C. Aliquots (20 µL) were
collected at each time point (4, 8, and 24 h) and treated with EDTA-free
protease inhibitor cocktails (0.5 µL of 100X solution, Thermo Scientific).
All samples were analyzed using a Thermo Vanquish UHPLC coupled
with a Q Exactive™ Hybrid Quadrupole-Orbitrap™ Mass Spectrometer
equipped with a C18 reverse-phase column (MabPac™ RP column, 4
µm, 2.1 × 50 mm, Thermo Scientific). Elution conditions were as fol-
lows: mobile phase A = water (0.1% formic acid); mobile phase B =
acetonitrile (0.1% formic acid); gradient over 4 min from A:B = 75:25 to
1:99; flow rate = 0.4 mL minꢀ 1. Average L/DAR values were determined
based on mass intensities.
Cayman chemical) and 1-methoxy-5-methylphenazinium methylsulfate
(1-methoxy PMS, 100 μM, Cayman Chemical) was added to each well,
and the plate was incubated at 37 ◦C for 2 h. After gently agitating the
plate, the absorbance at 460 nm was recorded using a plate reader
(BioTek Synergy HTX). EC50 values were calculated using Graph Pad
Prism 9 software. All assays were performed in quadruplicate.
4.10. Clonogenicity assay
Sulforhodamine B (SRB) colorimetric assay28 was performed to
evaluate clonogenicity after treatment with our ADCs. CAL51 cells were
plated into 24-well plates (2,000 cells per well) and incubated over-
night. The cell culture medium was removed and the cells were washed
with a fresh folate-free culture medium. The cells were treated with each
ADC in a folate-free medium (60
μL) and incubated for 3 h. Subse-
4.7. Cell culture
quently, 190 L of a folate-containing complete medium was added and
μ
the cells were incubated for 5 days. Subsequently, cells were fixed with
5% trichloroacetic acid and then stained with 0.03% of sulforhodamine
B solution (Sigma) at room temperature for 30 min. The stained cells
were imaged using a GelCount system (Oxford Optronix) and then dis-
solved in Tris buffer (10 mM). Optical density was determined fluoro-
metrically using a VICTOR X3 plate reader (Ex: 488 nm, Em: 585 nm).
KB (ATCC) was cultured in folic acid-free RPMI1640 (Corning)
supplemented with 10% EquaFETAL® (Atlas Biologicals), GlutaMAX®
(2 mM, Gibco), sodium pyruvate (1 mM, Corning), and pen-
icillin–streptomycin (penicillin: 100 units mLꢀ 1; streptomycin: 100 µg
mLꢀ 1, Gibco). CAL51 cells (Leibniz Institute DSMZ) was cultured under
the same conditions except that the culture medium contained folic acid.
KPL-4 (provided by Dr. Junichi Kurebayashi at Kawasaki Medical
School), U-87ΔEGFR (provided by Dr. Balveen Kaur at the University of
Texas Health Science Center at Houston), and HEK293 (ATCC) were
cultured in DMEM (Corning) supplemented with 10% EquaFETAL®,
GlutaMAX® (2 mM), and penicillin–streptomycin (penicillin: 100 units
mLꢀ 1; streptomycin: 100 µg mLꢀ 1). All cells were cultured at 37 ◦C
under 5% CO2 and passaged before becoming fully confluent up to 10
passages. All cell lines were periodically tested for mycoplasma
contamination.
Declaration of Competing Interest
The authors declare the following financial interests/personal re-
lationships which may be considered as potential competing interests: Y.
A., N.Z., Z.A., and K.T. are named inventors on a patent application
relating to the work filed by the Board of Regents of the University of
Texas
System
(PCT/US2018/034363;
US-2020-0115326-A1;
EU18804968.8-1109/3630189). The remaining authors declare no
competing interests.
4.8. Cell-based ELISA
Acknowledgements
Cells (KB, U-87ΔEGFR, KPL-4 or HEK293) were seeded in a culture-
We gratefully acknowledge Prof. Junichi Kurebayashi (Kawasaki
Medical School) and Prof. Balveen Kaur (The University of Texas Health
Science Center at Houston) for kindly providing the cell lines KPL-4 and
U-87ΔEGFR. This work was supported by the Department of Defense
Breast Cancer Research Program (W81XWH-18-1-0004 and W81XWH-
19-1-0598 to K.T.), the Cancer Prevention and Research Institute of
treated 96-well clear plate (10,000 cells per well in 100
μL culture
medium) and incubated at 37 ◦C under 5% CO2 for 24 h. Para-
formaldehyde (8%, 100 L) was added to each well and incubated for 15
min at room temperature. The medium was aspirated and the cells were
washed three times with 100 L of PBS. Cells were treated with 100 L of
μ
μ
μ
8
Yamaguchi, Aiko
