Engineering of a Lectibody Targeting High-Mannose-Type Glycans of the HIV Envelope

A novel anti‐HIV‐1 bispecific bNAb‐lectin fusion protein engineered in a plant‐based transient expression system

“…The second ELISA utilized a mannosidase‐treated HIV gp120 (SF162, clade B). Given that Avaren likely binds to the terminal mannose residues of HMGs on the surface of gp120 (Hamorsky et al ., submitted; Matoba et al ., ), mannosidase treatment would eliminate Avaren's ability to bind gp120. On the contrary, VRC01 Fab binds to the CD4‐binding site and thus should bind gp120 regardless of mannosidase treatment.…”

“…This transgene contained the subtilisin‐like processing protease kex2p positioned between the heavy and light chains of the Fab whereby during expression the heavy and light chain would be translated as a single polypeptide followed by separation in the trans‐Golgi via cleavage by the kex2p protease. Once cleaved, the heavy and light chains would assemble into a functional Fab (Hamorsky et al ., ; submitted). The overexpressed VRC01 Fab ‐Avaren fusion was then extracted from the plants and purified using a three‐step chromatography procedure.…”

“…The yield may be improved by further optimization of expression and/or purification conditions, thereby making it more viable for scale up and manufacturing. The synergism displayed in the mixture of the bivalent parental molecules VRC01 IgG and Avaren‐Fc (Figure ), both of which we have previously produced in N. benthamiana (Matoba et al ., ; Hamorsky et al ., submitted), provided a rationale for, and strengthened our confidence in, utilizing these molecules in a fusion. To obtain a proof of concept, we opted for a simpler approach in this foundational study, creating a bispecific molecule monovalent for each antigen, VRC01 Fab ‐Avaren.…”

“…In this paper, we engineered a novel bispecific molecule based on the CD4 binding site‐specific bNAb VRC01 and the antiviral lectin Avaren, which showed superior potency compared to its parental proteins. The fusion protein, VRC01 Fab ‐Avaren, was expressed in N. benthamiana at approximately 40 mg/kg of leaf biomass, which was lower than those achieved for the parental molecules (~150 mg/kg for VRC01 (Matoba et al ., ; Hamorsky et al ., submitted), and over 100 mg/kg for Avaren). The yield may be improved by further optimization of expression and/or purification conditions, thereby making it more viable for scale up and manufacturing.…”

“…VRC01 Fab and Avaren were each purified by a two‐step purification scheme consisting of Talon Immobilized Metal Affinity Chromatography (IMAC) from Clontech followed by CHT chromatography from BioRad. Production of VRC01 and Avaren‐Fc were done as described previously (Hamorsky et al ., ; submitted).…”