Site-Specific Modification of Single-Chain Antibody Fragments for Bioconjugation and Vascular Immunotargeting

Abstract

The conjugation of antibodies to drugs and drug carriers improves delivery to target tissues. Widespread implementation and effective translation of this pharmacologic strategy awaits the development of affinity ligands capable of a defined degree of modification and highly efficient bioconjugation without loss of affinity. To date, such ligands are lacking for the targeting of therapeutics to vascular endothelial cells. To enable site-specific, click-chemistry conjugation to therapeutic cargo, we used the bacterial transpeptidase, sortase A, to attach short azidolysine containing peptides to three endothelial-specific single chain antibody fragments (scFv). While direct fusion of a recognition motif (sortag) to the scFv C-terminus generally resulted in low levels of sortase-mediated modification, improved reaction efficiency was observed for one protein, in which two amino acids had been introduced during cloning. This prompted insertion of a short, semi-rigid linker between scFv and sortag. The linker significantly enhanced modification of all three proteins, to the extent that unmodified scFv could no longer be detected. As proof of principle, purified, azide-modified scFv was conjugated to the antioxidant enzyme, catalase, resulting in robust endothelial targeting of functional cargo in vitro and in vivo.

Bivalent engagement of endothelial surface antigens is critical to prolonged surface targeting and protein delivery in vivo

“…14,22 Sortagged scFv-LPETGG proteins-αPECAM (clone 390), αICAM (clone YN1), and an untargeted control (clone R6.5, binds human ICAM-1 with no cross-reactivity to mouse)-were produced as previously described in stably transfected Drosophila S2 cells or in the periplasmic space of E coli using the pBAD expression system (ThermoFisher Scientific). 20 Sortagged αICAM mAb-ss (YN1 mAb-LPETGG) was produced and purified from a CRISPR-Cas9-modified hybridoma, as previously described. 23 Finally, sortagged sTM-LPETGG was produced by inserting its cDNA between Bgl II and Sal I sites in the previously described vector, pMT/linker-LPETGG.…”

“…23 Finally, sortagged sTM-LPETGG was produced by inserting its cDNA between Bgl II and Sal I sites in the previously described vector, pMT/linker-LPETGG. 20 The vector was co-transfected with pCoBLAST in S2 cells and selected with blasticidin to generate a stable cell line. S2 cells were maintained in Schneider's medium (Thermo Fisher Scientific) and transitioned to Insect-Xpress (Lonza, Walkersville, MD) for protein production, as previously described.…”

“…S2 cells were maintained in Schneider's medium (Thermo Fisher Scientific) and transitioned to Insect-Xpress (Lonza, Walkersville, MD) for protein production, as previously described. 14,20 All sortagged proteins also had a C-terminal triple FLAG and were purified using anti-FLAG (M2) affinity resin (Sigma-Aldrich), with the exception of αICAM mAb-ss, which was purified using protein G sepharose fast flow (GE Healthcare Life Sciences, Pittsburgh, PA). The purity of recombinant and hybridoma produced proteins was confirmed by SDS-PAGE and/or analytical SEC HPLC, using a Yarra 3 µm SEC-2000 LC Column 150 × 7.5 mm size exclusion column with a flow rate of 1.0 mL/ min or a Yarra 3 µm SEC-2000 LC Column 300 × 4.6 mm size exclusion column with a flow rate of 0.35 mL/min.…”

“…To synthesize scFv-sTM and mAb-sTM conjugates, sTM-LPETGG was first site-specifically modified with azide via sortase transpeptidation, as described previously. 20 Briefly, the protein (10 μM) was mixed with SrtA (2 μM) and a fivefold excess (50 μM) of a fluorescent peptide with a C-terminal azidolysine (GGGK[FAM]GSK-azide) in 1 mM CaCl 2 containing Tris-buffered saline (TBS), pH 7.0. The reaction was left at RT overnight and the product, sTM-azide, was purified in two steps: (1) Incubation with Ni-NTA resin to remove SrtA, and (2) centrifugation using a 30 kDa MWCO Amicon centrifugal filter (Millipore Sigma, Burlington MA) to remove unreacted peptide.…”

“…Since our prior efforts to directly label αICAM and αPECAM scFv and scFv/TM fusion proteins 14,18 had resulted in loss of binding, we utilized recently reported "sortagged" scFv, which allow site-specific transpeptidation by sortase A (SrtA) and C-terminal addition of fluorophores or functional groups for oriented conjugation to protein cargo. 20 We extended this here to a two-step radiolabeling technique, in which a small peptide incorporating the radiometal chelating group, DOTA (1,4,7,10-tetrazacyclododecane-N,N′,N′′,N′′′-tetraacetic acid) was labeled with 111 In and then attached by SrtA to the C-terminal end of the scFv ( Figure S2). We compared the affinity of labeled scFv with corresponding parental mAb via RIAs on cells stably expressing ICAM or PECAM vs. control cells.…”