Background: The dimeric immunoglobulin (Ig) chimeras used for drug targeting and delivery are
preferred biologics over their monomeric forms. Designing these Ig chimeras involves critical selection of a suitable
Ig base that ensures dimer formation. In the present study, we systematically analyzed several factors that
influence the formation of dimeric chimera. We designed and predicted 608 cytokine-Ig chimeras where we tested
the contributions of (1) different domains of Ig constant heavy chain, (2) length of partner proteins, (3) amino
acid (AA) composition and (4) position of cysteine in the formation of homodimer.
Method: The sequences of various Ig and cytokines were procured from Uniprot database, fused and submitted to
COTH (CO-THreader) server for the prediction of dimer formation. Contributions of different domains of Ig
constant heavy chain, length of chimeric proteins, AA composition and position of cysteine to the homodimer
formation of 608 cytokine-Ig chimeras were tested. Various in silico approaches were adopted for validating the
in silico findings. Experimentally we also validated our approach by expressing the chimeric design of shorter
cytokine with Ig domain in CHO cells and analyzing the protein by SDS-PAGE.
Results: Our results advocate that while the CH1 region and the Hinge region of Ig heavy chain are critical, the
length of partner proteins also crucially influences homodimer formation of the Ig-based chimera. We also report
that the CH1 domain of Ig is not required for dimer formation of Ig based chimera in the presence of larger partner
proteins. For shorter partner proteins fused to CH2-CH3, careful selection of partner sequence is critical,
particularly the hydrophobic AA composition, cysteine content & their positions, disulphide bond formation
property, and the linker sequences. We validated our in silico observation by various bioinformatics tools and
checked the ability of chimeras to bind with the receptors of native protein by docking studies. As a proof of
concept, we have expressed the chimeric proteins in CHO cells and found that our design favors the synthesis of
Conclusion: Our structural prediction study suggests that extra amino acids in the range of 15-20 added to the
CH2 domain of Ig is a critical requirement to make homodimer. This information from our study will have implication
in designing efficacious homodimeric chimera.