VIB-RUG Department of Molecular Biomedical Research
Models Used to Create Bispecific Antibodies
Using Fab chains to fuse binding domains (Fab-scFv bibodies or tribodies) creates a versatile model for efficient expression, high heterodimerization and choice of valency of intermediate weight recombinant bispecific antibodies not containing an Fc part.
Two methods have been exploited:
Obviously, recombinant antibodies could improve the production of molecules with controlled bispecificity. Several manifolds have been suggested in order to combine antibody derived building blocks (such as Fc, (Fab')2, Fab, scFv, diabody) with heterodimerizing motifs in order to efficiently create BsAb.
A first choice to be made is whether or not to include the Fc part of the antibody in the recombinant molecule.
Both of these mechanisms decrease the specificity of action and increase the toxicity of the molecule. For these reasons, its is wise to consider using recombinant bispecific antibodies lacking the Fc part of the antibody. I will discuss both engineering BsAb including an Fc part and engineering BsAb not containing an Fc part.
Some elegant methods have been devised in order to create bispecificity in recombinant molecules while pertaining the Fc part:
Using antibody fragments not containing an Fc part improves the specificity of binding. A simple model is to genetically fuse two antigen binding building blocks to a single polypeptide. This can be done with two scFv molecules, a scDb molecule, or two single domain binding molecules. Another elegant solution is by coupling the antigen binding domains to small heterodimerizing peptides.
This leads to molecules with a MW of 25-50 kDa. Molecules of this size
however are cleared rapidly from the body through filtration in the kidney
glomerulla. this can lead to clearance times as short as 30-60 minutes.
In order to avoid rapid filtration in the kidney, it was suggested to
increase the MW of the antibody fragment to about 80 kDa or more. This was
accomplished by including also constant domains in the recombinant manifold for
What is important in choosing a manifold, is the efficiency of production, the specific heterodimerization and the control of the valency of all the binding specificities.
Our team proposes a manifold designed to take advantage of the eukaryotic quality control system, also responsible for the specific heterodimerization of normal antibodies. Indeed, production of the Fab fragment is controlled by chaperones in a eukaryotic cell.
By fusing antigen binding domains to the C-terminus of either of the Fab
chains or to both of them, Fab-scFv or Fab-(scFv)2 are formed.
This manifold allows efficient expression in eukaryotic systems, a high degree of specific heterodimerised end product, and perfect control over valency for each of the specificities.
The antibody is of intermediate weight and the heterodimer is disulfide stabilized. The heterodimerizing moiety is not heterogenic, nor antigenic (Fab chains are normally found in the serum).
More on this model: follow the link below
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