Design and Generation of Humanized Single-chain Fv Derived from Mouse Hybridoma for Potential Targeting Application

Abstract

Single-chain variable antibody fragments (scFvs) are attractive candidates for targeted immunotherapy in several human diseases. In this study, a concise humanization strategy combined with an optimized production method for humanizing scFvs was successfully employed. Two antibody clones, one directed against the hemagglutinin of H5N1 influenza virus, the other against EpCAM, a cancer biomarker, were used to demonstrate the validity of the method. Heavy chain (VH) and light chain (VL) variable regions of immunoglobulin genes from mouse hybridoma cells were sequenced and subjected to the construction of mouse scFv 3-D structure. Based on in silico modeling, the humanized version of the scFv was designed via complementarity-determining region (CDR) grafting with the retention of mouse framework region (FR) residues identified by primary sequence analysis. Root-mean-square deviation (RMSD) value between mouse and humanized scFv structures was calculated to evaluate the preservation of CDR conformation. Mouse and humanized scFv genes were then constructed and expressed in Escherichia coli. Using this method, we successfully generated humanized scFvs that retained the targeting activity of their respective mouse scFv counterparts. In addition, the humanized scFvs were engineered with a C-terminal cysteine residue (hscFv-C) for site-directed conjugation for use in future targeting applications. The hscFv-C expression was extensively optimized to improve protein production yield. The protocol yielded a 20-fold increase in production of hscFv-Cs in E. coli periplasm. The strategy described in this study may be applicable in the humanization of other antibodies derived from mouse hybridoma.

Chimeric antigen receptor preparation from hybridoma to T-cell expression

“…2B ). The primers were designed in-house except pGI15_TOPO [ 5 ], mIgG2bCH1, mIgGKCrev, mIgG1CH1–2, mIgG2bCH1–2 [ 6 ], mIgG2aCH1–2 [ 7 ] and mIgGKCrev-2 [ 8 ]. Each PCR was run in these conditions: 4′ 94°C for the initial denaturation and 25 cycles (1′ 94°C, 1′ 53°C, 1′ 72°C) with the following mix: 5 μL 10X TAQ Buffer, 1.5 μL of primer (10 mM stock), 1 μL dC-tailed cDNA, 1 μL Taq DNA polymerase (Hospital collection) and dH2O added to a V final = 50 μL.…”

A comprehensive comparison between camelid nanobodies and single chain variable fragments

“…Another approach to lower scFv aggregation promoted by inter-domain hydrophobic interaction is the humanization of scFvs by replacing hydrophobic amino acids with hydrophilic residues to prevent accumulation. Although protein solubility has improved by this method, these replacements can also have minor effects on the antigen-binding affinity of the final scFv [ 65 ]. The single entity nature of VHH makes their production much easier besides scFvs.…”

“…Also, scFvs’ engineering for reducing human anti-mouse antibody (HAMA) responses [ 78 ] will inactivate [ 79 ] injected scFvs and lessen their clinical effectiveness [ 80 , 81 ], and allergic reactions will arise in repeated administration [ 78 , 79 ]. Furthermore, humanization reduces the binding affinity of these fragments [ 10 , 65 ], and CDR grafting may represent new immunogenic epitopes [ 75 – 77 , 82 ]. In general, the humanization of murine-derived scFvs can overcome these immunogenicity problems to some extent but not entirely [ 59 ].…”

Immunogenicity of CAR-T Cell Therapeutics: Evidence, Mechanism and Mitigation

“…Under such circumstances, humanizing murine sequences could be a feasible alternative. In this process, murine CDR sequences are grafted onto human framework region, thus reducing the foreignness in CAR design without loss of its binding properties ( 82 ). Humanizing murine scFvs could help reduce immunogenicity.…”