<p>Diseases
are multifactorial, with redundancies and synergies between various pathways.
However, most of the antibody-based therapeutics in clinical trials and on the
market interact with only one target thus limiting their efficacy. The
targeting of multiple epitopes could improve the therapeutic index of treatment
and counteract mechanisms of resistance. To this effect, a new class of
therapeutics emerged: bispecific antibodies.</p><p>Bispecific formation
using chemical methods is rare and low yielding and/or requires a large excess
of one of the two proteins to avoid homodimerisation. In order for chemically prepared
bispecifics to deliver their full potential, high-yielding, modular and
reliable cross-linking technologies are required. Herein, we describe a novel
approach not only for the rapid and high-yielding chemical generation of
bispecific antibodies from native antibody fragments, but also for the
site-specific dual functionalisation of the resulting bioconjugates. Based on
orthogonal clickable functional groups, this strategy enables the assembly of
functionalised bispecifics with controlled loading in a modular and convergent
manner.</p>
Analysis of Assembly of Synthetic Antibody Fragments: Expression of Functional scFv with Predefined Specificity