New recombinant bi- and trispecific antibody derivatives.

Bispecific antibodies (BsAb) are promising therapeutic tools in tomorrow's medicine. When constructing BsAbs, the final molecular size should be large enough to avoid rapid renal clearing, but small enough to allow efficient tissue distribution. In order to produce such intermediate sized BsAb, a good heterodimerisation technique will improve existing production methods. When considering recombinant expression of BsAbs, the heterodimerisation motif can be incorporated into the molecule. Recombinant BsAb can e.g. be made by fusing single chain variable fragments (scFv) to a heterodimerisation domain. We compared the efficiency of the isolated CL and CH1 constant domains with complete Fab chains to drive heterodimerisation of BsAbs in mammalian cells. We found that the isolated CL:CHI domain interaction was inefficient for secretion of heterodimers. However, when the complete Fab chains were used, secretion of a heterodimerised bispecific antibody was successful. By C-terminal fusion of scFv molecules to the Fd- and the L-chains efficient heterodimerisation in mammalian cells was obtained and a novel intermediate sized, disulfide stabilised BsAb could be efficiently produced. Since the Fab chain encodes a binding specificity on its own, bispecific (BsAb) or trispecific (TsAb) antibodies can be made. This gave rise to disulphide stabilised Fab-scFv BsAb (Bibody) or Fab-(scFv)2 TsAb (Tribody) of intermediate molecular size. Heterodimerisation of the L and Fd-containing fusion proteins was very efficient, and up to 90% of all secreted antibody fragments was in the desired heterodimerised format. All building blocks remained functional in the fusion product, and the bispecific character of the molecules as well as the functionality was demonstrated. Due to the high heterodimerisation efficiency, the ease of purification of the desired product from by-products and the lack of post-production processing, this method for producing bi- or trispecific antibodies in mammalian cells could become a method of choice for the production of intermediate sized trispecific antibodies, BsAb with monovalent or bivalent binding for one antigen, or immunoconjugates.