Table 1.

DARs of Fc donors and ADC products after two-step Fc donor conjugation and pair-FORCE

Acceptor	Format	pHAb (NHS)	MMAE (Q221)	MMAE (Q297)	MMAE (Q221 + Q297)
	Fc donor DAR	2.2	0.6	0.8	1.3
Trastuzumab (HER2)	N	1.2	0.7	0.8	1.6
Trastuzumab (HER2)	C	1.2	0.6	0.6	0.5*
Trastuzumab (HER2)	N + C	1.1	0.7	0.8	1.3
Pertuzumab (HER2)	N	1.1	0.7	0.8	1.6
Pertuzumab (HER2)	C	1.2	0.6	0.7	1.4
Pertuzumab (HER2)	N + C	1.2	0.7	0.8	1.4

Acceptor	Format	pHAb (NHS)	AF488 (Q297)	MMAE (Q297)
	Fc donor DAR	2.2	1.0	0.8
Cetuximab (EGFR)	N	1.0	1.0	0.7
Cetuximab (EGFR)	C	1.1	1.0	0.8
Cetuximab (EGFR)	N + C	1.0	1.0	0.9
Imgatuzumab (EGFR)	N	1.1	0.9	0.8
Imgatuzumab (EGFR)	C	1.1	0.9	0.7
Imgatuzumab (EGFR)	N + C	1.1	1.0	0.8

Listed above are DAR values determined by mass spectrometry (MS) of different Fc donors or pair-FORCE products after either random conjugation of pHAb to the Fc donor (NHS chemistry), or after the two-step MTG-mediated azide-DBCO conjugation (AF488, MMAE). See the Methods section for details of the MS analysis. The DAR of AF488-conjugated HER2 antibody derivatives was not measured, but is expected to be similar to EGFR-binding antibody derivatives, as the same Fc donor was used. The DAR in Fc donor modules can be further increased by adding additional structurally compatible and accessible transglutaminase recognition sites. Exemplary intact MS spectra for MMAE-conjugated Fc donors and HER2-targeting MMAE ADCs are provided in Supplementary Fig. 6. *Strong adducts were observed for this molecule, which influenced the DAR determination (likely induced during MS measurement).