Antibody Humanization

Creative Biolabs has extensive experience in antibody humanization. Humanisation 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 optimized two 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. 

CDR 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.

Chain shuffling

We have also optimized a chain shuffling strategy that is an entirely selective humanization strategy based on construction and screening of two chimeric phage display libraries. In this approach, the light chain of the rodent antibody is first replaced by light chains in one of our well-tested human antibody libraries; the resulting hybrid antibody library is then screened by panning against the particular antigen. After that, the heavy chain of the selected hybrid antibodies is replaced by heavy chains of the human antibody library. Subsequent screening of this secondary chimeric library will produce fully humanized antibodies. Since phage display library screening mimics in vivo antibody selection and evolution procedure, chain shuffling can result in humanized antibodies whose affinities are higher than that of the original antibody. Also, this sequential chain shuffling procedure can generate several versions of humanized antibodies with different sequences. The production of multiple humanized antibodies retaining the same epitope specificity is important in therapeutic regimens that call for long-term treatment with antibodies in which antiidiotypic responses might be avoided by administration of alternative antibodies.

There are two features in our antibody humanization service that single out us from our peers. First of all, antibody affinity maturation is an integrated step in our humanization procedure, thus there is no need to improve the affinity after humanization. Secondly, in addition to the common computational and biochemical methods, we have developed a proprietary in vivo approach to evaluate the immunogenicity of the humanized antibodies in primates. The immunogenicity measured in primates is the closest one that may mimic the true immunogenicity of the humanized antibodies in humans.