High avidity scFv multimers; diabodies and triabodies
Introduction
Recombinant antibodies and their fragments are still the paradigm for the design of high-affinity, protein-based binding reagents (Hudson, 1998, Hudson, 1999; Dall'Acqua and Carter, 1998). Recombinant antibodies now represent over 30% of all biological proteins undergoing clinical trials, which recently culminated in FDA approval for the first engineered cancer therapeutic antibodies (Rituxan for non-Hodgkin lymphoma, Herceptin for breast cancer). Antibodies have been reduced in size, dissected into minimal binding fragments, and rebuilt into multivalent high-avidity reagents (Fig. 1). Antibody fragments have also been fused with a range of molecules limited only by the imagination, including radioisotopes for cancer imaging, enzymes for prodrug therapy, toxins for targeted cell killing, viruses for gene therapy, lipids for improved systemic delivery and biosensors for real-time detection of target molecules. Recombinant antibodies now capture a significant share of the US$3 billion per annum immunodiagnostic market, from in vitro immunoassays to in vivo tumor and clot imaging reagents. This review describes how careful choice of linker length between V-domains creates new types of Fv modules with different size, flexibility and valency suited to in vivo imaging and therapy. Further, we review the design and expression of multi-specific Fv modules suited to cross-linking target antigens for cell-recruitment, viral delivery and immunodiagnostics.
Section snippets
Multivalent antibodies
Intact antibodies are multivalent molecules (Fig. 1) which provide a significant increase in functional affinity (avidity), particularly for pentameric IgM molecules. There is therefore a similar increase in functional affinity when Fab or single chain Fv antibody fragments (scFv) fragments are complexed into dimers, trimers or larger aggregates (Pluckthun and Pack, 1997). Flexibility between antigen binding sites is important; in intact Igs the `elbow' angle within Fab arms and `hinge' angles
Linker design for formation of scFv multimers
The antigen binding domain of an antibody can be expressed in Escherichia coli as a single chain molecule (scFv) in which the VH and VL domains of the antibody are joined by a flexible polypeptide linker (Bird et al., 1988; Huston et al., 1988). The linker is generally designed with glycine and serine residues to provide flexibility and protease resistance (and the linker sequence can be optimised by phage display, Turner et al., 1997). When the linker is greater than 12 amino acids in length,
Expression and stability of scFv multimers
ScFv molecules can be expressed into the periplasm of E. coli with efficient signal peptide excision, correct V-domain folding and disulphide bond formation (Verma et al., 1998). These antibody fragments fold efficiently in the oxidising extracellular environment and are targeted to the periplasm using secretion signals (most commonly pelB and ompA). Bacterial synthesis of scFv molecules was originally done in `primitive' periplasmic expression vectors using leaky promoters (lac, trp and tac),
Size of scFv multimers
ScFv multimers such as diabodies (∼60 kDa) and triabodies (∼90 kDa) are significantly larger than scFv monomers (∼30 kDa) and thereby have an advantage for in vivo applications by minimising the rapid clearance from the circulation. This advantage of increased size in clearance rate is offset by decreased tumor penetration, since dissemination rate through the vascular network into tumors is correlated to the size of the in vivo therapeutic molecule. Indeed, the most recent in vivo data using
Flexibility and avidity in scFv multimers
The most important advantage of multivalent scFvs over monovalent scFv and Fab fragments is the gain in functional binding affinity (avidity) to target antigens. High avidity requires that scFv multimers are capable of binding simultaneously to separate target antigens. This avidity gain also occurs from multivalent binding to cancer cell surfaces as demonstrated in the improved in vivo tumour imaging of diabodies over scFv monomers (Wu et al., 1996, Wu et al., 1999; Adams et al., 1998, Adams
Multispecific scFv multimers
The discussion above has focussed on the association of identical scFv molecules to form diabodies, triabodies and tetrabodies which comprise a number of identical Fv modules. These reagents are therefore multivalent, but monospecific. The association of two different scFv molecules, each comprising a VH and VL domain derived from different parent Ig will form a fully functional bispecific diabody (Fig. 7; Holliger et al., 1993; Atwell et al., 1996; Humphreys et al., 1998; Malby et al., 1993).
Conclusions
During the past 12 months there has been a rapid increase in both scientific and commercial interest in multivalent scFvs leading to the large number of publications covered in this review. Clinical interest is due to encouraging results from phase 3 trials which has lead to several recent FDA approvals for humanised therapeutic antibodies (Farah et al., 1998). Scientific interest has stemmed from the elucidation of the key elements required for multivalent antibody design and efficient
Acknowledgements
We thank all our colleagues in the antibody engineering program at CSIRO Molecular Science for their helpful advice on the design and expression of scFv multimers and for their agreement to cite unpublished results in this review. We also thank Airlie McCoy, currently at the MRC Laboratory of Molecular Biology, Cambridge, UK, for the modelling studies on scFv multimers.
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