The invention concerns homogenous
antibody-conjugates with high
payload loading (high DAR) obtained by site-specific conjugation to a single
antibody N-
glycan. The conjugates according to the invention are homogeneous, i.e. have a DAR at or close to the theoretical DAR with a narrow distribution, and do not require any genetic modification of the
antibody. The invention further concerns a modular, non-genetic preparation method for such conjugates, involving three simple steps and starting from any antibody. These steps are (a) enzymatic remodeling of the
glycan to give an antibody functionalized with two or four click probes per antibody, (b) strain-promoted
cycloaddition with a multivalent,
bifunctional reagent comprising one cyclic
alkyne and at least two click probes that are not reactive towards the cyclic
alkyne, and (c) inverse
electron-demand Diels-
Alder cycloaddition of the click probes with branched
linker-
drug constructs comprising one cyclic
alkyne or strained
alkene, connected to one or more payloads preferably connected through a
cleavable linker. The resulting conjugates, with DAR6 or higher, are rapidly generated with high homogeneity and with surprising stability. In addition, HIC profiles of the resulting ADCs indicate small relative
retention time and therefore show
high potential in the targeting of tumour cells and / or the treatment of
cancer.