Table 1. Intrinsic and apparent binding affinity to target antigens.

Antibody	Intrinsic binding kinetics determined by Octetc			Apparent cell binding affinity parental NCI-H358d	Apparent cell binding affinity NCI-H358.HER2.kod
	Kon (M−1 s−1)	Koff (s−1)	KD (nM)	EC50 (nM)	EC50 (nM)
Trastuzumab IgGa	2.3 × 105	2.6 × 10−4	1.1	1.6	ND
GA201 IgGb	2.5 × 105	1.6 × 10−4	0.6	0.5	0.5
VκF94A IgGb	2.0 × 105	1.4 × 10−3	7.2	NDe	ND
VκS93A + VHP97A IgGb	1.4 × 105	3.3 × 10−3	24	ND	ND
VκF94A + VHP97A IgGb	1.1 × 105	1.6 × 10−2	148	ND	ND
EGFR/HER2 DuetMabb	2.3 × 105	1.7 × 10−4	0.7	0.9	1.1
VκF94A/HER2 DuetMabb	2.2 × 105	1.8 × 10−3	8.1	1.7	2.3
VκS93A + VHP97A/HER2 DuetMabb	1.8 × 105	5.7 × 10−3	32	5.6	>50
VκF94A + VHP97A/HER2 DuetMabb	1.4 × 105	2.3 × 10−2	167	8.3	>200
VκS93A + VHP97A/HER2 IgG-scFvb	1.5 × 105	4.1 × 10−3	27	5.1	6.5
VκF94A + VHP97A/HER2 IgG-scFvb	1.2 × 105	1.9 × 10−2	159	7.7	9.8

The amino acid residues in variable regions (V_H or V_L) are numbered by Kabat numbering system.

^aBinding measured against HER2.

^bBinding measured against EGFR.

^cKinetic measurements to soluble monomeric forms of EGFR and HER2 were performed using an Octet384 instrument. The dissociation constants, K_D, were calculated as the ratio of k_off/k_on from a non-linear fit of the data.

^dApparent cell binding affinities to parental NCI-H358 and NCI-H358.HER2.ko cells were determined by flow cytometry. Half maximal effective concentration (EC₅₀) values were calculated as the antibody concentration that generates 50% of the maximal MFI signal.

^eNot determined.