ProteoCool Pills #30:How improve Specific heavy-light chain pairing in the production of bispecific mabs

 

A Bispecific antibodies (BsAb) is a single molecule that comprise two single binding entities that are physically connected, which enables simultaneous binding to two different epitopes  on two different antigens or even in some cases two different epitopes on the same antigens (named biparatopic antibodies). 

The ability of BsAbs to bind to different antigens was largelly exploited in immunotherapy to act as a linker beetween  a cytotoxic cell and a target (a tumour cell) to be killed. 

For example T-cell-redirecting bispecific antibodies  (named T-cell engagers) are specifically designed to bind to tumor-associated antigens, thereby engaging with CD3 on the T cell receptor. This linkage between tumor cells and T cells actively triggers T cell activation and initiates targeted killing of the identified tumor cells. These antibodies have emerged as one of the most promising avenues within tumor immunotherapy.

The main issue associated with asymmetric BsAbs is mispairing of polypeptide chains leading to product-related impurities. In fact BsAbs assemble require co-expression of 4 different chains (2 different heavy chains and 2 different light chains),  each heavy chain can form either homo- or heterodimers, which in turn can associate with the two light chains. From a stochastic view point, this leads to 16 chain arrangements with 10 unique combinations, including only one, that represent only the 10% of the total mixture, that is the desidered BsAb where heavy chain exhibit the correct heterodimer and pairing with distinct light chains and this low probability impairs the manufacturability of bsAbs 

Protein engineering was largelly applied in the last 20 years to solve this issue.

First of all, complementary mutations that favors heavy chain (HC) heterodimerization while disfavouring formation of HC homodimers were reported. The first reported, and most widely used, platform is the knob-into-hole (KiH) which introduces a large bulky tryptophan in one HC and smaller sterically complementary residues in the other HC. The KiH strategy is widely implemented because it is highly effective in suppressing the most of HC homodimers and because its patent has expired 

While the correct heavy chain heterodimerization can be enabled using the knobs-into-holes (KiH) approach, the correct association of generic light chains has remained a problem, in fact even in presence of KiH there are still 4 possible random combinations that limit the fraction of desidered form only to about 25%

To solve this issue the KiH could be coupled with the CrossMAb technology that was described and patented by Roche in 2011 (it is stil protected by IP). which is able to enforce correct light chain association in bispecific heterodimeric IgG antibodies. 

This technology could be applied to any existing antibody pair using domain crossover, without the need for the identification of common light chains, post-translational processing/in vitro chemical assembly or the introduction of a set of mutations enforcing correct light chain association.

In the past I had the opportunity to produce for R&D purpose 2 different bispecific containing both KiH and Crossmab and in both cases the antibodies were produced fron CHO with high yields (transient trasfection) and the an very good rate of HC-LC correct coupling (>90%)    was detected in both cases by mass-spectrometry.