Team:SMMU-China/Improvement

Background of the improvement parts

This part is a mutant of the AR185-T2A-EGFP designed by team SMMU in 2018. AR185-T2A-EGFP (BBa_K2865001) is a RyR2-specific VHH (heavy chain variable domain) antibody which is designed to treat heart failure by inhibiting RyR2 phosphorylation. Previous researches have revealed that the phosphorylation of RyR2, a calcium ion releasing channel located in the sarcoplasmic reticulum, will increase the probability of the calcium ion channel opening, resulting in calcium leakage in the cytoplasm and initiating the pathological mechanism of myocardial injury and heart failure development. By inhibiting the phosphorylation of the RyR2 subunit S2808 with a nanobody AR185-T2A-EGFP, the FKBP12.6 can bind to the RyR2 again and the probability of the calcium ion channel opening will decrease, blocking the initiating of the pathological mechanism of heart failure.

Design

The limited dephosphorylation efficiency of the AR185-T2A-EGFP lowers the potential potency of the heart failure treatment. We consequently came up with an idea to increase the dephosphorylation efficiency of the AR185-T2A-EGFP.

Characterization of the improvement parts

Proof of successful AR185-T2A-EGFP mutational nanoantibody expressing in H9C2 cell:

The CDR (complementarity determining region) is the key site for RyR2 recognition of the AR185-T2A-EGFP nanoantibody. By random mutation of the AR185-T2A-EGFP with an exiting random mutation kit, we finally obtained 54 specific mutants. We then transfected the AR185-T2A-EGFP mutational nanoantibody to H9C2 cells using lentiviral technology and the results showed that we finally acquired the AR185-T2A-EGFP mutational nanoantibody expressing H9C2 cell.

Proof of the improved dephosphorylation efficiency of the AR185-T2A-EGFP mutational nanoantibody:

To obtain antibodies that functionally inhibit RyR2 phosphorylation, each of the antibody fragments was tested for its effect in an ELSA based RyR2 phosphorylation assay. And the results showed that the dephosphorylation efficiency of mutants 11 is nearly doubled than the previous AR185-T2A-EGFP nanoantibody. This diagram indicated that we obtained a potential medicine for the treatment of intractable heart failure.