Antibody Humanization

The development of monoclonal antibodies for use as human therapeutics represents one of the fastest growing segments of the biopharmaceutical industry. Monoclonal antibodies are usually first produced in mice challenged by a specific antigen. These monoclonal mouse antibodies are frequently immunogenic when administered to humans because of the differences in antibody backbone sequences between mice and humans. Therefore, it is often necessary to convert non-human antibodies into ones with human-like sequences. This humanization process can greatly improve characteristics of the antibodies when administered to humans and can significantly increase their success rate in clinical studies. However, it is essential that the antibodies retain their affinity and specificity throughout the humanization process. LakePharma has developed a novel and validated process for humanization of antibodies, which produces highly active humanized antibodies in a short time frame.

LakePharma's antibody humanization platform combines the advantages of both rational and empirical approaches. It starts with a thorough analysis of sequence and structural information related to the parent monoclonal antibody that is to be humanized. A framework region (FR) is selected from the most homologous human mature antibody and germline sequences, and often multiple FR choices are used in the design to avoid potential limitation posed by a specific framework. This is followed by sequence homology-based CDR (complementarity determining region) and SDR (specificity determining residues) analysis, as well as transfer of parent CDR/SDR sequences into the selected framework. LakePharma then uses a proprietary 3-D structure modeling based approach to identify positions in the human FR, Vernier zone, and canonical structure regions that need to be modified to restore CDR conformation and optimal antigen binding. The structural algorithm generates multiple possibilities for each position to allow maximal flexibility at this design stage, and all of these residue combinations will be put through a molecular evolution process to identify the best antibody sequence to achieve the highest activity and specificity.

Taking advantage of the capability of quick and precise gene synthesis at LakePharma, a human antibody library containing all combinations of potential residue choices is constructed. This library is presented on the surface of phage, and antibodies with the highest affinity toward the desired antigen are identified through multiple screening/amplification steps with increasing stringency. These humanized antibodies are further evaluated for their binding affinity, specificity, and production yield in transiently transfected CHO cells (which is a good indicator of antibody stability). Several antibody molecules with balanced favorable properties are selected for further sequence analysis and characterization. These optimized antibodies can be further tested for their suitability in large-scale production if needed.

By combining sequence homology/3-D structure analysis and molecular evolution approaches, LakePharma is able to generate humanized antibodies that have equivalent or higher affinity than the parent antibodies in as short as 3 months. Furthermore, multiple antibody solutions are provided based on the different frameworks for further evaluation. In addition, LakePharma's expertise in stable cell line generation can be used to develop high yield cell lines for large-scale production of humanized antibodies, which minimizes uncertainties in downstream development and production.