VIB-RUG Department of Molecular Biomedical Research

Molecular ImmunoBiotechnology Unit









Models Used to Create Bispecific Antibodies


It is possible to create BsAb starting from monoclonal antibodies, or from recombinant antibodies

Recombinant antibodies can either contain an Fc part or not contain an Fc part.

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.


BsAb derived from moAB

Two methods have been exploited: 

  1. the hybrid hybridoma technique fuses two hybridoma's in order to combine their specificity. However, the combinations in a single cell occur at random so a range of products with nearly the same biophysical properties is produced, where the desired BsAb is only a fraction of the molecules formed.

  2. Chemical crosslinking can be used with complete antibodies (creating a large molecule with a bivalent binding for both specificities), or for Fab' fragments derived from moAb. This method requires several post-production synthesis steps which lower the overall efficiency of the process.  

Engineering bispecificity into recombinant antibodies

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. 

bulletThe Fc part increases the serum persistence time of the molecule by protecting the antibody from metabolism in the liver. Longer serum persistence times improve binding to the target but do not improve the specificity of the antibody accumulation. If the reagent is toxic or active in some way (as a BsAb is), it is usually not desirable to have it around in tissues where it does not belong for too long. 
bulletFurthermore, the Fc part can crosslink other cells via its interaction with the Fc receptor, and this can give rise to toxic side effects due to systemic triggering of immune effector cells.

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.

Recombinant BsAb including an Fc part

Some elegant methods have been devised in order to create bispecificity in recombinant molecules while pertaining the Fc part:

engineering knobs-into-holes: in one CH3 domain (part of the Fc molecule) a bulky mutation is introduced, a complementing mutation in a second CH3 domain then avoids homodimer formation and promotes the bispecificity. The result is a single IgG molecule with two monovalent, bispecific binding specificities.


Fusing the Fc part to building blocks with two specificities. The building blocks can be scFv, one Fab and a scFv, or a single chain diabody. In this way each specificity binds the target in a bivalent way since the Fc part still promotes the formation of homodimers.


Engineering BsAb not containing an 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.

These small molecules are excellent in diagnostic applications. But when aiming for a therapeutic affect, one usually attempts to have sufficient accumulation at the target site to increase the effectiveness of the molecule. A somewhat tempered clearance rate could improve the accumulation at the target cells.

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 BsAb.


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. 

Bibodies and Tribodies

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).

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