RAPID AND RELIABLE CLONING OF ANTIBODY VARIABLE REGIONS AND GENERATION OF RECOMBINANT SINGLE-CHAIN ANTIBODY FRAGMENTS

Single chain antibody variable region fragments (sFv), by virtue of th eir size and method of construction are potentially useful as therapeu tic 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 molec ules or single-chain immunotoxins. The relatively small size of sFv is an advantage in allowing for easier penetrance into tissue spaces, an d their clearance rate is exceedingly rapid. sFv are useful for gene t herapy 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 pro tein within 2-3 weeks of RNA isolation from hybridoma cells. The varia ble regions were cloned by poly-G tailing the first strand cDNA follow ed by anchor PCR with a forward poly-C anchor primer and a reverse pri mer specific for constant region sequence. Both primers contain flanki ng 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 V H 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 cons tructs were transfected into COS cells and sFvs were recovered from sp ent culture supernatant. We have used this method to generate function al sFv to human CD2, CD3, CD4, CD8, CD28, CD40, CD45 and to murine CD3 and gp3, 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 an d purification using goat anti-human IgG reagents or protein A. We als o found that active sFv could be expressed with a small peptide greate r than or equal to tag greater than or equal to or in a tail-less form . Expression of CD3 (G19-4) sFv tail-less or Ig tailed forms demonstra ted increased cellular signalling activity and suggested that sFv have potential for activating receptors.