title: Preparation of biologically active single-chain variable antibody fragments that target the HIV-1 gp120 V3 loop.
abstract: KD-247 is a humanized monoclonal antibody that targets the third hypervariable (V3) loop of gp120. It can efficiently neutralize a broad panel of clade B, but not non-clade B, HIV-1 isolates. To overcome this limitation, we are seeking to prepare genetically-engineered single-chain variable fragments (scFvs) of KD-247 that will have broader neutralizing activity against both clade B and non-clade B HIV-1 isolates. Initial attempts of optimizing the expression of KD-247 scFv have resulted in the formation of insoluble protein. Therefore, we have established purification protocols to recover, purify, and refold the KD-247 scFv from inclusion bodies. The protocol involved step-wise refolding of denatured scFv by dilution, dialysis, and on-column nickel-affinity purification. Monomeric scFv was further purified by size-exclusion chromatography. Using far UV circular dichroism (CD) spectroscopy we confirmed the expected beta-sheet profile of the refolded KD-247 scFv. Importantly, the refolded KD-247 scFv showed neutralizing activity against replication-competent HIV-1 BaL and JR-FL Env pseudotyped HIV-1, at potency comparable to that of the native full-size KD-247 antibody. Ongoing studies focus on the application of this system in generating KD-247 scFv variants with the ability to neutralize clade B and non-clade B HIV-1 isolates.
authors.affiliation: Christopher S. Bond Life Sciences Center, University of Missouri School of Medicine, Columbia, MO 65211, USA.
authors.forename: Y T
authors.initials: YT
authors.lastname: Ong
authors.forename: K A
authors.initials: KA
authors.lastname: Kirby
authors.forename: A
authors.initials: A
authors.lastname: Hachiya
authors.forename: L A
authors.initials: LA
authors.lastname: Chiang
authors.forename: B
authors.initials: B
authors.lastname: Marchand
authors.forename: K
authors.initials: K
authors.lastname: Yoshimura
authors.forename: T
authors.initials: T
authors.lastname: Murakami
authors.forename: K
authors.initials: K
authors.lastname: Singh
authors.forename: S
authors.initials: S
authors.lastname: Matsushita
authors.forename: S G
authors.initials: SG
authors.lastname: Sarafianos
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000378:metabolism; D057134:Antibodies, Neutralizing / Q000276:immunology / Q000378:metabolism; D015483:HIV Antibodies / Q000276:immunology* / Q000378:metabolism; D015699:HIV Envelope Protein gp120 / Q000276:immunology*; D006801:Humans; D017510:Protein Folding; D057127:Single-Chain Antibodies / Q000276:immunology* / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D057134:Antibodies, Neutralizing; D015483:HIV Antibodies; D015699:HIV Envelope Protein gp120; D057127:Single-Chain Antibodies