Abstract
Bispecific antibodies (bsAbs) are often composed of several polypeptide chains that have to be expressed adequately to enable optimal assembly and yield of the bsAb. κλ bodies are a bispecific format with a native IgG structure, composed of two different light chains that pair with a common heavy chain. Introduction of non-optimal codons into the sequence of a particular polypeptide is an effective strategy for down modulating its expression. Here we applied this strategy but restricted the modification of the codon content to the constant domain of one light chain. This approach facilitates parallel optimization of several bsAbs by using the same modified constant domains. Partial sequence de-optimization reduced expression of the targeted polypeptide. Stable cell pools could be isolated displaying increased bispecific antibody titers as well as changes in the abundance of undesired by-products that require elimination during downstream processing. Thus, modulating the relative expression of polypeptides can have a significant impact on bsAb titer and product related impurities; which are important factors for large scale manufacturing for clinical supply.
Highlights
In the last two decades, bispecific antibodies have emerged as a new class of therapeutic molecules
Second and third promoter drove, respectively, the expression of the K2 kappa light chain which binds to human cluster of differentiation 47, the common heavy chain, and the different lambda light chains (O30, O35 and O41) which target human mesothelin
After purification of all IgG forms (IgGκκ, IgGκλ, IgGλλ) from the culture supernatant via affinity chromatography, the polypeptides were separated on an Agilent
Summary
In the last two decades, bispecific antibodies (bsAbs) have emerged as a new class of therapeutic molecules. Protein engineering efforts have led to a stunning variety of different possible approaches for the generation of bsAbs, with a current estimate of more than 100 different formats [5,6] These can be classified in different ways such as size, valency of binding sites, overall molecular structure and similarity to a standard IgG format. Blincyto, the approved anti-CD3, anti-CD19 (CD3xCD19) bispecific T cell engager (BiTE) is a tandem single chain variable fragment scFv format composed of a single polypeptide Another example is Hemlibra, which is the recently approved bsAb with Factor VIII mimetic activity that has a standard IgG structure and requires the co-expression of three polypeptides: two heavy chains and a single common light chain capable of Antibodies 2018, 7, 29; doi:10.3390/antib7030029 www.mdpi.com/journal/antibodies
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