Mouse antibody capture kit

SPR biosensor analysis was conducted on a Biacore T100 platform with CM5 Series S sensor chip (GE Healthcare, Life Science, Milan, Italy). Amine Coupling kit and Mouse Antibody Capture kit (GE Healthcare, Life Science, Uppsala, Sweden) were respectively exploited for standard amine coupling and the two capturing mediated immobilization strategies. The used oligonucleotides were purchased from Sigma-Aldrich (Milan, Italy) (Table S1). To allow the correct folding of secondary structures, each oligonucleotide was diluted to 20 μM in HEPES-KCl buffer (HEPES 10 mM pH 7.4, KCl 200 mM, EDTA 3 mM), denatured 5 min at 95 °C and then slowly cooled to RT. The mouse monoclonal 1H6 antibody (MW ~155 kDa) was kindly provided by P. M. Lansdorp [1 (link)].

The binding kinetics of the antibodies against human HER2 and human CD3 were assessed at pH 7.4 at 37 °C using the Biacore T200 (Cytiva, Marlborough, MA, USA). Anti-mouse IgG (Fc) antibody (Mouse antibody capture kit) was immobilized onto all flow cells of a CM4 chip using an amine coupling kit (Cytiva). Anti-HER2 and anti-CD3 antibodies and analytes were prepared in PB-P+ (Na-phosphate buffer 0.05 mol/L, NaCl 0.15 mol/L, 0.05 w/v% P-20) at pH 7.4. Antibodies were adjusted to concentrations corresponding to 200 RU for assessing binding with recombinant human HER2 (Sino Biological, 10004-H08H) and 630 RU for recombinant human CD3ε-CD3δ heterodimeric protein (CD3εδ) (In-house prepared, Table S1). Human HER2 was prepared by two-fold serial dilutions (6.3 nmol/L to 100 nmol/L). Human CD3 was prepared by two-fold serial dilutions (1.2 nmol/L to 4800 nmol/L) for mUCHT1 and mSP34, (18.8 nmol/L to 19,200 nmol/L) for mHIT3a and mOKT3, and (75 nmol/L to 76,800 nmol/L) for m12F6. The sensor surface was regenerated with Glycine 1.5 with 10 mmol/L glycine–HCl, pH 1.5 (Cytiva). Kinetic parameters were determined by processing and fitting the data to the 1:1 binding model for m4D5, m2C4, mUCHT1, and mSP34 and the steady state model for mHIT3a, mOKT3, and m12F6 using Biacore T200 Evaluation software, version 3.0 (Cytiva, Marlborough, MA, USA).