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Antibody Humanization

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Antibody Humanization

Mouse antibodies can cause immunogenicity and reduced efficacy in human body, which limits their clinical applications. Antibody humanization is an important method for reducing the immunogenicity of mouse antibodies and improving their activation in human immune system. Genekine has extensive experience to offer the service of antibody humanization for therapeutic development. We provide humanization service to mouse antibodies and antibodies derived from other species, such as non-human primate (NHP), rabbit, dog, chicken, llama and etc.
 Genekine has developed three antibody humanization strategies, including: 1/ CDR and SDR grafting, 2/ CDR3-H grafting, 3/ Chain shuffling and guided selection. They can preserve the specificity and affinity of antibodies while significantly or completely eliminate the immunogenicity of murine antibodies in humans.

1/ CDR Grafting & SDR Grafting

We have established a CDR (complementarity-determining region) and SDR (specificity-determining residues) grafting platform for antibody humanization. After analysis of sequence and structural information related to mouse monoclonal antibody to be humanized, several framework regions (FR) are selected from the most homologous human mature antibodies and germline sequences. Six mouse CDRs are grafted into selected human framework regions. Since framework residues are involved in antigen binding, either indirectly, by supporting the conformation of the CDRs, or directly, by contacting the antigen, simple grafting of mouse CDRs into human frameworks does not always reconstitute the binding affinity and specificity of the original mouse antibody. Some residues from the mouse framework sequences need to be retained to replace the human residues at the corresponding positions (back mutations) in order to restore some of the lost affinity. For this reason, we have developed a computer modeling method to make back mutations in framework residues in addition to CDR grafting. The grafted CDRs together with the back mutations are expressed and screened to select for humanized antibodies with the best affinity. This approach allows the epitope specificity of the original antibody to be retained. Of note, humanization by this approach is not 100% due to mouse CDR.
In order 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 some amino acids that are directly interact with antigen, i.e., specificity-determining residues (SDRs), whereas there are some conserved residues that maintain the conformations of CDRs. 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 selected human framework regions.

2/ CDR3-H Grafting

In addition, Genekine has also developed a new antibody humanization method using its proprietary full-length IgG yeast surface display technology. Using this technology, the heavy chain CDR3 region (CDR3-H) alone from mouse antibody is grafted into selected human heavy chain framework regions, then the grafted heavy chain is introduced into a naïve human light chain library. The resulting CDR3-grafted human antibody library is displayed on the yeast surface in full-length IgG format. The high affinity antibodies can be selected from this library by fluorescence-activated cell sorting (FACS) against the target antigen. The resulting humanized antibodies will have fully human antibody sequence, except for heavy chain CDR3 which is from the original non-human antibody. These humanized antibodies can significantly reduce potential immunogenicity, and maintain or improve affinity of the original antibody.

Schematic illustration of CDR3-H grafting for antibody humanization

Mouse heavy chain CDR3 region (CDR3-H) is grafted into human heavy chain framework regions, then the grafted heavy chain is introduced into human light chain library. The resulting CDR3-grafted IgG library is displayed on yeast cell surface and selected by FACS against the target antigen. The high affinity humanized antibodies will have fully human antibody sequence, except for heavy chain CDR3 which is from mouse antibody

3/ Chain Shuffling and Guided Selection

Genekine has developed an approach of guided selection to convert a mouse antibody into a completely human antibody, based on chain shuffling of V-genes by using full-length IgG yeast surface display technology. In this approach, the chimeric light chain, which contains original mouse variable (VL) domain and human constant (CL) domain, is introduced into human naïve heavy chain library, and the resulting semi-human antibody library is displayed on the yeast cell surface in full-length IgG format. Using fluorescence-activated cell sorting (FACS) against the target antigen, it can select human heavy chains that, in combination with the mouse VL domain, shows binding specificity for the antigen. Once such semi-human antibodies had been selected, the selected human heavy chains are introduced into human naïve light chain library, and the resulting human antibody library is displayed on the yeast cell surface in full-length IgG form. Using FACS selection against the target antigen, it will produce fully humanized antibodies with the best affinity.
Since full-length IgG yeast display library screening mimics in vivo antibody selection and evolution procedure, chain shuffling can produce fully humanized antibodies, and hence, better therapeutic efficacy can be expected by removing problems related to the immunogenicity of mouse residues. In addition, the guided selection method using chain shuffling can generate diverse human antibodies with better properties compared with the original mouse antibody. The production of multiple humanized antibodies retaining the same epitope specificity is important in therapeutic regimens that need long-term treatment with antibodies, in which anti-idiotypic responses might be avoided by administration of alternative antibodies. While it is common to select antibodies with related sequences to the original mouse antibody, it is also possible to select antibodies with distinct sequences, which show enhanced affinity, expression, and/or stability.

Schematic illustration of chain shuffling and guide selection for antibody humanization

The chimeric light chain, which contains original mouse variable (mVL) domain and human constant (hCL) domain, is introduced into human heavy chain library to display semi-human antibody library on yeast cell surface. FACS can select human heavy chain that, in combination with the mouse VL domain, shows binding specificity for the antigen. Next, the selected human heavy chain is introduced into human light chain library to display human antibody library. FACS can select fully humanized antibodies with the best affinity.
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