Computational and Experimental Evaluation of Linker Peptides and Thioredoxin Fusion Tag in CD20-rituximab Specific Interactions

Abstract

Background: Overexpression of CD20 protein on the surface of B cells in lymphoma can be targeted by several anti-CD20 molecules. The development of accessible interactive epitopes is more favorable than the full-length transmembrane CD20 in the affinity assessment of anti-CD20 monoclonal antibodies (mAbs).

Methods: The sequence of these epitopes was extracted, and the effects of different linker peptides and the location of histidine (His)-tag were computationally analyzed. The impact of thioredoxin (Trx)-tag on the folding of the selected construct and its interaction with rituximab was further investigated. The two final expression cassettes were expressed in Escherichia coli after optimization of culture conditions for incubation temperature, post-induction time, optical density at the induction time, and concentration of the inducer. ELISA evaluated the binding affinity of rituximab towards the recombinant proteins.

Results: By homology modeling studies, C-terminal His-tagged structures represented more desirable folded structures. Validation of the models revealed that CD20 extracellular domain linked by the G₄S polypeptide had better stereochemical quality and structural compatibility. It was selected due to its more effective interaction with rituximab showing the highest dissociation constant of 5.8E-09M, which improved after the fusion of Trx-tag (7.1E-10M). The most influential parameters in the expression of the two selected proteins were post-induction temperature and optical density at the induction time. Homemade ELISA assays revealed a slightly higher affinity of rituximab towards the Trx-CD20 protein than the CD20/G₄S molecule.

Conclusions: Experimental in vitro studies confirmed the computationally calculated affinity of rituximab towards the two designed CD20 constructs. Also, the cell-based binding assessment of anti-CD20 mAbs could be substituted by the engineered extracellular domain of human CD20 protein.

Keywords: Affinity; CD20; Molecular Dynamics; Response Surface Methodology; Thioredoxin.

Figure 1.. The designed expression cassettes using PAS or G₄S linkers connecting CD20-ECL1 and CD20-ECL2 regions. A - H, Expression cassettes possessing 20, 40, and 60 PAS amino acid polypeptide linkers with N- or C-terminal histidine (His)-tag, respectively. I and J, Expression cassettes with (G₄S)₃ linkers possessing N- or C-terminal His-tag, respectively.

Figure 2.. I-TASSER predicted tertiary structures of all designed constructs. A – H, CD20 structures with 20, 40, and 60 PAS amino acid polypeptide linkers with N-or C-terminal histidine (His)-tag, respectively. I and J, (G₄S)₃ linkers with N- or C-terminal His-tag, respectively.

Figure 3.. Molecular docking of designed CD20 constructs. A, CD20/20aa PAS; B, CD20/40aa PAS; C, CD20/60aa PAS; D, CD20/80aa PAS; E, CD20/G₄S. Designed constructs are colored green, and their ECL1 and ECL2 regions are colored red. Heavy and light chains of rituximab are colored magenta and cyan, respectively. The stick amino acids are residues located in the interface of CD20 and rituximab. Bolded amino acids belong to the antibody, while amino acids in regular font belong to the ECL1 and ECL2 regions of CD20.

Figure 4.. Thioredoxin (Trx)-CD20 structure. A, Schematic view; B, I-TASSER predicted tertiary structure of N-terminally fused Trx-CD20 fusion protein possessing C-terminal histidine (His)-tag. Thioredoxin, ECL1, G₄S linker, ECL2, and His-tag are colored orange, blue, yellow, green, and red, respectively.

Figure 5.. Molecular dynamic simulation of rituximab: CD20 complexes. A, Root mean square deviation (RMSD) plot; B, Radius of gyration (Rg) plot; C, Hydrogen bond plot; D, Solvent-accessible surface area (SASA) plot; E, Root mean square fluctuations (RMSF) plot. CD20/G₄S and thioredoxin (Trx)-CD20 proteins are in blue and orange, respectively.

Figure 6.. Indirect sandwich ELISA assessment. A, Zytux; B, AryoTrust against thioredoxin (Trx)-CD20 protein; C, Zytux; D, AryoTrust against CD20/G₄S protein; E, Rituximab affinity measurements against the two designed recombinant CD20 molecules using Beatty’s method; F, CD20-positive Raji cell-based ELISA assay. Data are given as mean ± SEM values of triplicate wells.