Generation of Recombinant Multimeric Antibody Fragments for Tumor Diagnosis and Therapy

Single chain recombinant antibody fragments can multimerise to provide high binding avidity and unique specificity for target antigens and can be used to replace the parent antibody or Fab derivatives (1). A unique advantage in using bacterial expression systems is the high yield and low cost, especially when the VH and VL domains are tethered by a single-chain linker (scFv). This chapter describes expression systems for production of new types of scFv molecules with size, flexibility, and valency suited to in vivo cell-targeting, tumor imaging, and therapy. Further, we review the design of expression cassettes that create multi-specific scFv dimers suited to crosslinking target antigens for T-cell recruitment, viral delivery, and immunodiagnostics. Choice of the linker length that joins VH and VL domains dictates precisely whether the (scFv) product is a soluble monomer or a high-avidity multimer (dimer, trimer, etc.). Based on the Kabat numbering system (2) and X-ray structures of Fv domains (3,4), we define the C-terminal end of the VH domain as SerH112 or ArgL107 of VL. In either VH-VL or VL-VH orientation, single chain Fv fragments (scFvs, ≈30 kDa) are predominantly monomeric when the Fv domains are joined by polypeptide linkers of at least 12 residues (5). An scFv molecule with a linker of 3–12 residues cannot fold into a functional Fv domain and instead associates with a second scFv molecule to form a bivalent dimer (diabody, ≈60 kDa) (5,6). Reducing the linker length below three residues can force scFv association into trimers (triabodies, ≈90 kDa) or tetramers (≈120 kDa) depending on linker length, composition, and Fv domain orientation (7–10). The increased binding valency in these scFv multimers results in high avidity (long off-rates). A particular advantage for tumor targeting is that molecules of ≈60–100 kDa have increased tumor penetration and fast clearance rates compared to the parent Ig (150 kDa) (1,11–14). A number of cancer-targeting scFv multimers have recently undergone preclinical evaluation for in vivo stability and efficacy (11–14). Bi- and tri-specific multimers can be formed by association of different scFv molecules and, in the first examples, have been designed as crosslinking reagents for T-cell recruitment into tumors (immunotherapy) and as red blood cell agglutination reagents (immunodiagnostics) (1,15,16).