Expression and functional analysis of recombinant scFv and diabody fragments with specificity for human RhD

Abstract

In an attempt to generate recombinant anti-D reagents for possible diagnostic and therapeutic use we cloned the genes encoding the variable (V) domains of a human anti-D antibody secreted by the lymphoblastoid cell line BTSN4. A single-chain Fv (scFv) fragment was constructed using a 21 amino acid linker to join the genes encoding the variable domains of the BTSN4 heavy (VH) and light chains (VL). A diabody construct was also generated by reducing the length of the scFv linker from 21 to 10 residues. The scFv and diabody constructs were cloned into the pFLAG-CTS vector, expressed in E. coli host cells and the recombinant proteins were affinity-isolated from bacterial culture medium. Analysis of the recombinant proteins indicated that they retained the D antigen binding specificity of the parental BTSN4 IgG. Furthermore, both fragments mediated agglutination of papain-treated D positive erythrocytes in the absence of a cross-linking second antibody. While the agglutinating property of BTSN4 diabody was readily explained by the non-covalent association of this protein as a bivalent dimer, oligomeric forms of BTSN4 scFv were not detected when the protein was analysed by size exclusion chromatography. Thus, the agglutinating property of the scFv is not the result of the formation of non-covalently associated multimeric forms of the antibody fragment.

Engineered humanized diabodies for microPET imaging of prostate stem cell antigen-expressing tumors

“…This observation differs from the anti-CEA T84.66 and anti-CD20 2B8 V L -V H orientated diabodies, which form stable dimers with an eight-residue linker (Wu et al, 1999). In addition, there are reports of stable scFv dimers (V H -V L ) with linkers longer than eight residues (Kortt et al, 1997;Asvadi et al, 2002). Thus, linkerdependent dimer/oligomerization behavior of scFv fragments appears highly dependent on the specific antibody selected and the orientation of the variable domains.…”

A study of monoclonal antibody‐producing CHO cell lines: What makes a stable high producer?

“…The heavy chain expression vector phCMV-VHRhD-g1C-neo (Fig. 1A) contained the heavy chain variable domain (VH) for BTSN4 (Asvadi et al, 2002) fused to the cDNA coding for human g1 constant (g1C) region under the control of the human cytomegalovirus (hCMV) major immediate-early (MIE) enhancer and promoter for initiation of transcription of the recombinant chimaeric IgG1 heavy chain, and the coding sequence for the bacterial neomycin (neo) under control of the SV40 early promoter for drug selection of transfected cells. The g1C cDNA was PCR-cloned from a human cell line that secreted a human g1 antibody.…”

“…The SV40 early promoter/neo gene unit was derived from the neomycin expression vector pSV2-neo (Southern and Berg, 1982). The light chain expression vector phCMV-VLRhD-K R -neo contained the light chain variable domain (VL) for BTSN4 (Asvadi et al, 2002) fused to the genomic human kappa (K R ) constant region (Rabbitts et al, 1984) under the control of the hCMV-MIE enhancer and promoter for initiation of transcription of the recombinant chimaeric IgG1 light chain, and the coding sequence for neomycin under control of the SV40 early promoter. In order to facilitate selection, the neomycin resistance gene was replaced with mouse dihydrofolate reductase (dhfr) gene, which also allows for gene amplification (Fig.…”

“…In our study we generated a panel of 17 CHO-mAb subclones expressing a humanized antibody against the Rhesus D antigen (RhD) (Asvadi et al, 2002;Miescher et al, 2004) and analyzed these high-and low-producer clones regarding their growth characteristics, mAb productivity, their molecular features, and their stability in long-term culture. These extensive characterization studies could help understand the limitations to high-level, stable recombinant protein production and find ways to improving and accelerating the process for high-producer cell line generation and selection.…”