Reinhilde Schoonjans

Fab chains as an efficient heterodimerization scaffold for the production of recombinant bispecific and trispecific antibody derivatives.

Due to their multispecificity and versatility, bispecific Abs (BsAbs) are promising therapeutic tools in tomorrow’s medicine. Especially intermediate-sized BsAbs that combine body retention with tissue penetration are valuable for therapy but necessitate expression systems that favor heterodimerization of the binding sites for large-scale application. To identify heterodimerization domains to which single-chain variable fragments (scFv) can be fused, we compared the efficiency of heterodimerization of CL and CH1 constant domains with complete L and Fd chains in mammalian cells. We found that the isolated CL:CH1 domain interaction was inefficient for secretion of heterodimers. However, when the complete L and Fd chains were used, secretion of L:Fd heterodimers was highly successful. Because these Fab chains contribute a binding moiety, C-terminal fusion of a scFv molecule to the L and/or Fd chains generated BsAbs or trispecific Abs (TsAbs) of intermediate size (75–100 kDa). These disulfide-stabilized bispecific Fab-scFv (“bibody”) and trispecific Fab-(scFv)2 (“tribody”) heterodimers represent up to 90% of all secreted Ab fragments in the mammalian expression system and possess fully functional binding moieties. Furthermore, both molecules recruit and activate T cells in a tumor cell-dependent way, whereby the trispecific derivative can exert this activity to two different tumor cells. Thus we propose the use of the disulfide-stabilized L:Fd heterodimer as an efficient platform for production of intermediate-sized BsAbs and TsAbs in mammalian expression systems.

A new model for intermediate molecular weight recombinant bispecific and trispecific antibodies by efficient heterodimerization of single chain variable domains through fusion to a Fab-chain

Due to their specificity and versatility in use, bispecific antibodies (BsAbs) are promising therapeutic tools in tomorrows medicine, provided sufficient BsAb can be produced. Expression systems favoring efficient heterodimerization of intermediate-sized bispecific antibodies will significantly improve existing production methods. Recombinant BsAb can be made by fusing single chain variable fragments (scFv) to a heterodimerization domain. We compare the efficiency of the isolated CL and CH1 constant domains with complete Fab chains to drive heterodimerization of BsAbs in mammalian cells. We found that the isolated CL:CH1 domain interaction was inefficient for secretion of heterodimers. However, when the complete Fab chains were used, secretion of a heterodimerized bispecific antibody was successful. Since the Fab chain encodes a binding specificity on its own, bispecific (BsAb) or trispecific (TsAb) antibodies can be made by C-terminal fusion of scFv molecules to the L or Fd Fab chains. This gave rise to disulphide stabilized Fab-scFv BsAb (Bibody)or Fab-(scFv)2 TsAb (Tribody) of intermediate molecular size. Heterodimerization of the L and Fd-containing fusion proteins was very efficient, and up to 90% of all secreted antibody fragments was in the desired heterodimerized format. All building blocks remained functional in the fusion product, and the bispecific character of the molecules as well as the immunological functionality was demonstrated.

Efficient heterodimerization of recombinant bi- and trispecific antibodies

Bispecific antibodies (BsAb) are promising therapeutic tools in tomorrows medicine. Expression systems favoring efficient heterodimerization of intermediate-sized bispecific antibodies will significantly improve existing production methods. By C-terminal fusion of scFv molecules to the Fd- and the L-chains efficient heterodimerization in mammalian cells was obtained and a novel intermediate sized, disulfide stabilized BsAb could be efficiently produced. This type of antibody derivative easily allows for the production of trispecific antibodies, BsAb with bivalent binding for one antigen, or immunoconjugates.

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:CH1 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.