Models Used to Create Bispecific Antibodies
Summary:
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.
Two methods have been exploited:
- 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.
- 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.
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:
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.
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.
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
