"Diabodies": small bivalent and bispecific antibody fragments.

TLDR

Bivalent and bispecific antibody fragments hold great promise for practical applications. The study aims to design small antibody fragments that present two antigen‑binding sites, enabling bivalent or bispecific interactions. The authors engineered VH–VL polypeptides with a short linker that prevents intrachain pairing, causing the domains to pair with complementary chains to form dimers; they constructed bivalent and bispecific diabodies from NQ11 and D1.3 antibodies. Expressed in bacteria, the diabodies bound their targets with affinities comparable to the parent antibodies, and a VH–VL fusion showed slower dissociation and higher hapten affinity, demonstrating that diabodies enable efficient production of small bivalent and bispecific fragments.

Abstract

Bivalent and bispecific antibodies and their fragments have immense potential for practical application. Here we describe the design of small antibody fragments with two antigen-binding sites. The fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) on the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. As indicated by a computer graphic model of the dimers, the two pairs of domains can pack together with the antigen-binding sites pointing in opposite directions. The dimeric antibody fragments, or "diabodies," can be designed for bivalent or bispecific interactions. Starting from the monoclonal antibodies NQ11.7.22 (NQ11) and D1.3 directed against the hapten phenyloxazolone and hen egg lysozyme, respectively, we built bivalent fragments (VHNQ11-VLNQ11)2 and (VHD1.3-VLD1.3)2 and bispecific fragments VHNQ11-VLD1.3 and VHD1.3-VLNQ11. The fragments were expressed by secretion from bacteria and shown to bind specifically to the hapten and/or antigen. Those with 5- and 15-residue linkers had similar binding affinities to the parent antibodies, but a fragment with the VH domain joined directly to the VL domain was found to have slower dissociation kinetics and an improved affinity for hapten. Diabodies offer a ready means of constructing small bivalent and bispecific antibody fragments in bacteria.