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The blood brain barrier (BBB) is a challenge when developing monoclonal antibodies for neurological diseases as antibodies do not cross the BBB making brain distribution challenging. An antibody construct which binds mTfR1 to pass the BBB, scFc-scFv8D3, have previously been developed. The aim of the project was to design six scFc-scFv8D3 affinity-mutants, determine their mTfR1 affinity and evaluate their in vitro BBB transcytosis (transport). Six scFc-scFv8D3 affinity-mutants were designed by investigating possible interactions between scFc-scFv8D3 and mTfR1 and then exchanging important residues to remove some possible interactions. The mutants were T100V, S101A, H102A, Y103F, Y103A and S101A+Y103F. Plasmids expressing the affinity mutants were amplified in E. Coli and the mutants were expressed in Expi293 cells. The mutants were purified by protein G affinity chromatography. The mTfR1 affinity for the affinity mutants were tested with indirect mTfR1 ELISA and the BBB transcytosis in vitro was tested with an In-Cell BBB trans assay. The assay used mouse cerebral endothelial cells to mimic the BBB.

The H102A mutant showed higher, the T100V and S101A mutants showed similar, the Y103F and S101A+Y103F mutants showed lower and the Y103A showed drastically lower mTfR1 affinity compared to scFc-scFv8D3. Only the Y103A affinity mutant showed a significant lower BBB transcytosis while the other mutants showed no significant difference in BBB transcytosis.The conclusion is that reducing mTfR1 affinity drastically also lower the in vitro BBB transcytosis suggesting that the in-cell BBB-trans assay may be useful as a screening method for identifying affinity-mutants with to low affinity before in vivo studies. The S101A affinity-mutant showed increased BBB transcytosis in vitro, it is currently unknown why but may be due to escaping lysosomal degradation or a small change in affinity undetected in the ELISA. The other affinity mutants neither increased or decreased the BBB transcytosis, therefore no further relationship between mTfR1 affinity and in vitro BBB transcytosis could be deduced. A further relationship could be theorized looking at indicated (but not significant) differences in BBB transcytosis, in this theorized relationship an optimal mTfR1 affinity higher than the Y103F mutant but lower than the S101A mutant (assuming its affinity is lower compared to scFc-scFv8D3). The in vitro BBB transcytosis cannot be directly translated to transcytosis in vivo, this due to some factors not being modelled in vitro like plasma half-life. Therefore in vivo studies need to be performed to determine a relationship between mTfR1 affinity and in vivo BBB transcytosis.