Identify Putative Backmutations:
This is the most difficult and unpredictable procedure in the humanisation of
monoclonal antibodies. It is also the area which throws much light on
protein structure and function. A solid body of data for helping to
identify strategic alterations (using the
sequence search form) is available in
the list of previously humanised antibodies.
- Analysis of Donor and Acceptor Sequences
Putative point backmutations from the human framework residue back
to the original source residues will already have been identified
from the analysis of the donor and acceptor sequences for canonical
residues, interchain packing residues, unusual residues and glycoslyation
sites. Experience has shown that it is especially important to
retain the source's canonical and interchain packing residues, though
not in all cases (see antibodies 60.3
- Vernier Zone and CDR-H3
Consider residues in the Vernier zone
which form a "platform" on which the CDRs rest and therefore potentially
affect their conformation. Due to their extreme variability, there are
no canonical residues defined for CDR-H3 (although see antibody
RSV19 for interaction between position 94
and residue 101 in CDR-H3). Therefore, special attention
should be paid to this loop, analysing the structural model for residues
which may potentially affect its conformation.
- Proximity to Binding Site
Using the structural model, analyse residues within 5 angstroms of any
CDR residue (this was increased to 6 angstroms for antibody
anti-B4). These residues are likely to bind
to antigen especially if they are unusual as classified by the analysis
of the source sequence.
- Glycosylation Sites
Consider removal of any potential glycosylation sites, especially if
they are on the surface of the structural model, since in the case
of antibody M195, removal enhanced the avidity
of the humanised molecule. However, antibody SK2
has shown that glycosylation, even when affecting a canonical residue and
close to the antigen binding site, does not adversely affect binding.
Having decided on the residues to backmutate, return to the question of
human acceptor frameworks. It is not unlikely that an overlooked human
framework may actually contain the backmutations which are to be retained.
If this is the case, then there is no need to introduce residues from the
source sequence, thus making the humanised antibody more "human".