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antibody humanization

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Abstract or Description:

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, 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 were developed. The first approach is CDR grafting and the second approach is chain shuffling. These two methods are all based on phage display of humanized scFv variants and selection of high-affinity 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, a computer modeling method has been developed  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 anti-idiotypic responses might be avoided by administration of alternative antibodies.

 
In this method, a mammalian cell surface display library is made to display full-size humanized IgG variants that are to be further selected suing FACS. First, we will select an acceptor human VH and a receptor VL from a subgroup of human antibodies based on consensus sequences. Next, CDR grafting is conducted as described above. In order to increase [or keep] the affinity of the humanized antibodies, a mammalian cell surface display IgG library is created to display all possible variants of the humanized IgG. Since the size limit for the mammalian cell surface display IgG library, decisions must be made as to which amino acids to diversify and to what extent so that there are fewer nonsense antibody mutants that waste the capacity of the library. Back mutations in framework regions are designed based on computer modeling or antigen/antibody structure information. Also, we will distinguish between residues with solvent accessible side chains from those with buried side chains. 

 

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Posted By: Candy Ton on 5/26/2015 5:49:08 AM
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