Rapid and reliable cloning of antibody variable regions and generation of recombinant single chain antibody fragments

Abstract

Single chain antibody variable region fragments (sFv), by virtue of their size and method of construction are potentially useful as therapeutic reagents and as tools for exploring cell surface receptor function. sFv offer several advantages over the intact immunoglobulin molecule. For instance, they are expressed from a single transcript and can be molecularly linked to other proteins to generate bispecific sFv molecules or single-chain immunotoxins. The relatively small size of sFv is an advantage in allowing for easier penetrance into tissue spaces, and their clearance rate is exceedingly rapid. sFv are useful for gene therapy since they can be directed to a specific cellular localization and can be fused to retroviral env genes to control viral host range. To prepare sFv to murine and human leukocyte CD antigens, we devised a method for rapid cloning and expression that can yield functional protein within 2-3 weeks of RNA isolation from hybridoma cells. The variable regions were cloned by poly-G tailing the first strand cDNA followed by anchor PCR with a forward poly-C anchor primer and a reverse primer specific for constant region sequence. Both primers contain flanking restriction sites for insertion into PUC19. Sets of PCR primers for isolation of murine, hamster and rat VL and VH genes were generated. Following determination of consensus sequences for a specific VL and VH pair, the VL and VH genes were linked by DNA encoding an intervening peptide linker [usually (Gly4Ser)3] and the VL-link-VH gene cassettes were transferred into the pCDM8 mammalian expression vector. The constructs were transfected into COS cells and sFvs were recovered from spent culture supernatant. We have used this method to generate functional sFv to human CD2, CD3, CD4, CD8, CD28, CD40, CD45 and to murine CD3 and gp39, from hybridomas producing murine, rat, or hamster antibodies. Initially, the sFvs were expressed as fusion proteins with the hinge-CH2-CH3 domains of human IgG1 to facilitate rapid characterization and purification using goat anti-human IgG reagents or protein A. We also found that active sFv could be expressed with a small peptide > or = tag > or = or in a tail-less form. Expression of CD3 (G19-4) sFv tail-less or Ig tailed forms demonstrated increased cellular signalling activity and suggested that sFv have potential for activating receptors.

Lack of a role for transforming growth factor-β in cytotoxic T lymphocyte antigen-4-mediated inhibition of T cell activation

“…After ethanol purification, a poly(G) tail was added to the 5Ј end of the cDNA according to the manufacturer's instructions (GIBCO). The tailed cDNA then was amplified by lowstringency PCR by using degenerate primers designed for hamster antibodies (34). The resulting PCR products were cloned into a TA vector (Invitrogen) according to the manufacturer's instructions.…”

Blockade of T Cell Activation Using a Surface-Linked Single-Chain Antibody to CTLA-4 (CD152)

“…The modified constructs (mem-scFvs) were ligated into a mammalian expression vector for subsequent transfections of eukaryotic cell lines. Using described techniques (31,(33)(34)(35), basic scFv constructs were generated from RNA isolated from the hybridomas 145-2C11 (hamster anti-murine CD3⑀; Ref. 36), PV1 (hamster anti-murine CD28; Ref.…”

The Expression and Characterization of a Bifunctional Protein in E. Coli for Autologous Erythrocyte Agglutination Test

“…Originally, the variable region gene cloning of antibody was performed by using mixtures of degenerate oligonucleotides. Although the degenerate oligonucleotide primers were designed to anneal to the conserved residues at the beginning of framework one, the use of degenerate oligonucleotides could produce sequencing mistakes because of the random mismatching (19). As for the primers designed from the leader peptide sequence of antibodies might avoid random mismatching in the conserved region, the putative primers might not be consistent with that of the wild-type gene since the signal peptide were different in the sequence of antibodies (20).…”