Table 3.

Summary of engineering tips learned from previous cases studying macromolecule BBB crossing

Factor to consider	Description/details	Ref
Category: choice of receptors for the carrier
Highly expressed at BBB	Highly expressed in the BECs Identify novel RMTs by proteomics	[138,152–154]
Antibody cross-reactivity	Validate expression in species-of-interest Preclinical studies require cross-reactivity to nonhuman primates’ RMT	[103,121,155] [122,139–143,156]
Category: antibody format engineering
Fusion to naturally existing transcytosis ligands should be avoided	Being blocked by natural ligands present at high concentration Perturbing normal biological functions	[144,155,157]
Antibody and fusion format	Valency of the antibody targeting RMT is important Bivalency may not be preferred for the carrier, but may be preferred for the cargo Protein fusion order or configuration matters, depending on the optimal functions Fusion protein produced using genetic engineering is preferred over conjugation due to more homogeneity Overall structure stability Optimal activities of the cargo should be retained	[102,108,109,117–120,122]
Antibody affinity to RMT targets	High-affinity binding may induce receptor degradation and compromise further transcytosis thus limiting brain uptake High-affinity binding promotes peripheral receptor-mediated clearance, causes significant shorter circulation and lower BBB crossing efficiency High-affinity binding prevents the release of transported antibodies, instead, being trapped in the vasculature	[102,106,115–118,120,121,135,138]
Antibody effector function	Abolish Fc effector functions if causing toxicities	[119]
Antibody binding RMT receptor epitope	Avoid competing with natural ligands	[120,121]
Antibody endocytosis	Verify endocytosis by in vitro cell models	[107–109,120,122,142,158]
Category: in vivo characterization
Blood circulation time	Longer circulation time is preferred to increase exposure to brain Introducing Fc mutations that increase FcRn binding at pH = 6.0	[117,121,135,159]
Verify brain distribution	Quantifying antibody concentration from brain lysate is not sufficient Broad brain distribution should be confirmed using imaging	[102,107,117,121]
Quantification of brain uptake	Quantification using ELISA or radioactivity Brain capillary depletion or perfusion with PBS should be performed before quantification in order to exclude possible interferences from blood	[102,107,117,121]
Confirm biological effects or therapeutic efficacy	Using appropriate animal models to confirm improved therapeutic efficacy is required as the final verification of true brain entry	[120–123,126–129,131–133,135,138,141–143]