Table 1.

Overview of the properties of the ADCs, synthesized from the HER2-targeting trastuzumab conjugated to LP1–8.

					Cell killing SKBR-3		Cell killing HCC-78
LP	Linker–payload	Yield after purification based on protein concentration	DAR (MS) After purification from HMWS	HMWS after conjugation (%)	EC50 (ng/mL)	Max. amount of dead cells	EC50 (ng/mL)	Max. amount of dead cells
LP1	P5(OEt)–VC–PAB–exatecan	59%	0.28	9.9%	251	37%	>3,000	N/A
LP2	P5(PEG2)–VC–PAB–exatecan	82%	5.51	2.3%	n.d.	n.d.	n.d.	n.d.
LP3	P5(PEG12)–VC–PAB–exatecan	80%	6.36	3.9%	15.5	86%	144.1	91%
LP4	P5(PEG12)–VA–PAB–exatecan	84%	4.39	7.5%	18.0	78%	121.2	68%
LP5	P5(PEG24)–VC–PAB–exatecan	99%	8.00	0.9%	12.5	96%	97.6	88%
LP6	MC–VC–PAB–exatecana	75%	0.12	19.2%	265	14%	>3,000	N/A
LP7	P5(PEG12)–VA–exatecan	77%	7.89	1.0%	114.7	44%	>3,000	N/A
LP8	P5(PEG12)–exatecan	97%	7.19	4.0%	182.4	35%	>3,000	N/A

Note: The conjugation has been performed with excess linker–payload (30 eq. with respect to the antibody) and TCEP (10 eq. with respect to the antibody). After purification, the ADCs have been analyzed for yield based on protein concentration before and after the conjugation, DAR via MS, ADC aggregation via analytic SEC and antiproliferative effect on HER2-positive cell-lines (SKBR-3 and HCC-78). The antiproliferative potency of the ADCs on the HER2-positive cells is reported as an EC₅₀ value of cell viability in ng/mL and the total amount of affected cells in percentage. None of the ADCs showed any effect on the HER2-negative cell line (MDA-MB-468, Supplementary Fig. S1).

^aConjugation conditions for LP6 were slightly adjusted to more typical maleimide reaction conditions: Reaction was conducted in PBS at pH 7.4 at room temperature for 2 hours; n.d., not determined; N/A, not applicable.