Humanization Techniques

Humanization is important for reducing the immunogenicity of monoclonal

antibodies derived from xenogeneic sources (commonly rodent) and for
improving their activation of the human immune system. Since the
development of the hybridoma technology, a large number of rodent
monoclonal antibodies with specificity for antigens of therapeutic interest
have been generated and characterized. Rodent antibodies are highly
immunogenic in humans, which limits their clinical applications, especially
when repeated administration is required. Importantly, they are rapidly
removed from circulation and can cause systemic inflammatory effects as
well. As a means of circumventing these problems, we have developed three
antibody humanization strategies that can preserve the specificity and affinity
of the antibody toward the antigen whereas significantly or completely
eliminate the immunogenicity of the antibody in humans. The first approach is
CDR grafting and the second is chain shuffling. These two methods are all
based on phage display of humanized scFv variants and selection of highaffinity humanized binders through bio-panning. The third method, humanized
IgG library screening, is somehow unique. We will make a library of humanized
whole IgG to be displayed on the surface of mammalian cells and then high
affinity binders will be sorted by FACS.
CDR Grafting & SDR Grafting
We have established a CDR (complementarity-determining region) grafting
platform, which is featured with randomization of a small set of framework
residues using phage display technology and computer modeling. In this
platform, six CDR loops comprising the antigen-binding site are grafted into
corresponding human framework regions. Unfortunately, simple grafting of
the rodent CDRs into human frameworks does not always reconstitute the
binding affinity and specificity of the original antibody because framework
residues are involved in antigen binding, either indirectly, by supporting the
conformation of the CDR loops, or directly, by contacting the antigen. For this
reason, we have developed a computer modeling method to randomize
certain framework residues in addition to CDR grafting. The grafted CDRs
together with the randomized residues are cloned into a phage display library
and the humanized antibodies with the best affinity are selected by screening
of the library. This approach allows the epitope specificity of the original
antibody to be retained. Of note, humanization by this approach is not 100%
since the CDR regions are still of a rodent origin.
To reduce the immunogenicity of CDR-grafted humanized antibodies, the
murine content in the CDR-grafted humanized antibodies is minimized
through SDR grafting. Within each CDR, there are more variable positions that
are directly involved in the interaction with antigen, i.e., specificitydetermining residues (SDRs), whereas there are more conserved residues that
maintain the conformations of CDRs loops. SDRs may be identified from the
3D structure of the antigen antibody complex and/or the mutational analysis
of the CDRs. An SDR-grafted humanized antibody is constructed by grafting
the SDRs and the residues maintaining the conformations of the CDRs onto
human template, and its immunogenic potential is evaluated by measuring
the reactivity to the sera from patients who had been immunized with the
parental antibody.