Chicken scFvs with an Artificial Cysteine for Site-Directed Conjugation

Abstract

For the site-directed conjugation of chemicals and radioisotopes to the chicken-derived single-chain variable fragment (scFv), we investigated amino acid residues replaceable with cysteine. By replacing each amino acid of the 157 chicken variable region framework residues (FR, 82 residues on VH and 75 on VL) with cysteine, 157 artificial cysteine mutants were generated and characterized. At least 27 residues on VL and 37 on VH could be replaced with cysteine while retaining the binding activity of the original scFv. We prepared three VL (L5, L6 and L7) and two VH (H13 and H16) mutants as scFv-Ckappa fusion proteins and showed that PEG-conjugation to the sulfhydryl group of the artificial cysteine was achievable in all five mutants. Because the charge around the cysteine residue affects the in vivo stability of thiol-maleimide conjugation, we prepared 16 charge-variant artificial cysteine mutants by replacing the flanking residues of H13 with charged amino acids and determined that the binding activity was not affected in any of the mutants except one. We prepared four charge-variant H13 artificial cysteine mutants (RCK, DCE, ECD and ECE) as scFv-Ckappa fusion proteins and confirmed that the reactivity of the sulfhydryl group on cysteine is active and their binding activity is retained after the conjugation process.

CD19/CD22 bispecific chimeric antigen receptor‑NK‑92 cells are developed and evaluated

“…NK-92 cells, although immortalized, have been shown to be safe in phase I clinical trials (13)(14)(15) and irradiation of NK-92 cells can further improve their safety (18)(19)(20). These results support the use of CAR-NK-92 cells as off-the-shelf therapeutics (38). However, NK cell activity is decreased in patients with MM, particularly in advanced stages, and patients with aggressive extramedullary cutaneous plasmacytoma (39,40).…”

An approach towards development of monoclonal IgY antibodies against SARS CoV-2 spike protein (S) using phage display method: A review

“…It has been proven that antibody receptors that are specifically engineered to a target antigen with combined specific sites were used in human therapy and reported to be effective with these single-chain antigen binding molecules [91]. Also, chicken immunoglobulin fragments like chFcR/L receptor molecules were identified to be the first member of this avian antibody family which plays a vital role in Fig.…”

The level of decoy epitope in PCV2 vaccine affects the neutralizing activity of sera in the immunized animals

“…After incubation with 1 mg/mL of anti-CP 169e180 antibody, horseradish peroxidase (HRP)conjugated goat anti-human C k antibodies (Chemicon-Millipore, Billerica, MA, USA) were added to each well. After washing with 0.05% (v/v) Tween 20 in PBS (PBST), 3,3 0 ,5,5 0 -tetramethyl benzidine (TMB) (GenDEPOT, Barker, TX, USA) substrate solution was added and the absorbance was measured at 650 nm with a Multiskan Ascent microplate reader (LabSystems, Helsinki, Finland) [26].…”