To overcome the disadvantage of trifunctional antibody production as a mixture, Bio.services has introduced knobs-into-holes amino acid changes to perform rational design in antibody engineering. The principle is that heavy chains (H) of human immunoglobulin IgG interact at the level of their CH3 domains directly, whereas, at the level of their CH2 domains, they interact via the carbohydrate attached to the Asn N84.4 in the DE turn. Therefore, the interface between CH3 domains can play a key role for the efficient formation of heterodimers.

The figure above presents the obtained products from the knobs-into-holes engineering for trifunctional antibody production. Comparing to quadromas, this method avoids the impurity problem of produced tri functional antibody.

Heterodimerization of antibody heavy chains specific for different antigens can be enforced through knobs-into-holes mutations in CH3 domains; as a result, correctly assembled bispecific antibodies can be expressed either in E.coli or mammalian cells [e.g. human embryonic kidney (HEK) cells] co-transfected with constructs encoding all four chains.

Moreover, it is very possible that the modification on Fab region to introduce the knobs-into-holes area may affect the specificity and binding affinity of the antibodies. Usually, a library of antibody chain with engineered mutations will be constructed. And directed protein evolution methods such as phage display will be applied to select the stable and functional antibody chain to construct the tri functional antibody.