Q&A: How bYlok® Technology Improves Bispecific Antibody Development

Q: What is bYlok®?

bYlok® technology is a design engineering approach for bsAb that facilitates correct chain pairing. It modifies a bsAb sequence to move a disulphide bond from the constant region of the Fab arm to the variable region. This drives the correct pairing of the LCs and HCs in a bsAb, and Lonza have demonstrated >95 % correct HC-LC pairing with bYlok® technology modified bsAbs expressed in-house.

Q: What kind of bsAb formats can bYlok® be used to construct?

The bYlok® platform is suitable for IgG-like bsAb formats where there are two different light and heavy chains. By repositioning the disulphide bond from a constant to a variable region, this format, that requires heterodimeric assembly of binding domains, can be reliably constructed.

Q: Does bYlok® work with both kappa and lambda LCs?

We have assessed the use of bYlok® technology with kappa LCs and expect that lambda light chains would perform equally as well.

Q: Which upstream processes can bYlok® technology modified bsAbs be produced in?

We have expressed bYlok® bsAbs using standard processes for typical mAbs in CHO cells. The bYlok® technology is compatible with any expression system, but for the highest titers, we recommend using Lonza's GS Xceed® Gene Expression System combined with GS piggyBac® transposon technology.

Q: What titers do bYlok® modified bsAbs achieve?

It depends on the molecule, but we have seen recent bsAbs titres with GS piggyBac® range from ~ 4 – 8 g/L. The bYlok® technology does not impact the expression levels of bsAbs.

Q: What benefits does bYlok® technology have in terms of reducing impurities?

At Lonza, we have generated data demonstrating that, when used with the GS Gene Expression System® and GS Xceed® CHOK1SV GS-KO® cell line, bYlok® technology drives >95% correct heterodimer pairing for multiple molecules. This means that there are fewer unwanted homodimeric species.

Q: Does the bYlok® technology modification affect the safety profile of bsAbs?

Our in-house data demonstrates that bYlok® does not have an impact on immunogenicity scores using in-silico predictions. We hypothesise that this is because the natural mAb structure and amino acid sequence are subject to such a minor modification when bYlok® technology is used, and immunogenic epitopes have not been created. Furthermore, since bYlok® drives correct HC-LC pairing, fewer unwanted homodimeric species are produced which can streamline downstream processing.

Q: What effects does the bYlok® modification have on stability?

The stability of IgG-like bYlok® bsAbs tested in-house is not negatively affected compared to the parental mAbs. In accelerated thermal stability assays, these molecules did not show any significant degradation.

Q: What impact does bYlok® have on overall bsAb development timelines and costs?

bYlok® can de-risk the clone selection process as clones show more consistent product assembly compared to alternative pairing technologies. It can also increase the correctly assembled product ratio and assist with the downstream process. Additionally, a number of bispecific design engineering platforms require specialized purification strategies which can mean substantial loss of yield of the correct species. With bYlok® bsAbs, standard purification methods can be used with no sacrifice to yields.

Q: How can scientists access this technology?

With our Lonza in Your Lab® offering, bYlok® is available via a license for scientists to evaluate in their own labs.

It can also be accessed as part of Lonza's 13-month* CMC program, where we can construct bsAbs drug candidates using bYlok® alongside our bsAb toolbox of expression, purification, and formulation technologies to provide the highest titer bsAb with an appropriate PQA profile. It is also accessible through our early development services.