A Novel Antibody Humanization Method By Epitope Mapping Based On MALDI-TOF MS Combined With Antibody-antigen Molecular Docking

As is known to all, cancer is the most lethal illness in the world. The number of cancer patients has been soaring in China since the 1970s. About 80 percent of cancer patients died from common cancers of the lungs, liver and stomach, according to previously released figures. The usual treatments include surgery, radiation therapy, chemotherapy and biological therapy. These treatment methods are sometimes used alone and sometimes in combination. Biological therapy is to stimulate or restore the ability of the immune system to fight infection and disease. It is thus any form of treatment that uses the body's natural abilities that constitute the immune system to fight infection and disease or to protect the body from some of the side effects of treatment.Antibody therapeutics is of growing importance as medical treatments in different types of biological therapies. It continue to be one of the bright spots in pharmaceutical drug development, with 20 monoclonal antibodies approved for the treatment of various diseases, and many more in clinical trials with promising results. Antibody drugs developed based on genetic engineering, cell engineering technology and antibody engineering technologies for the preparation of the main drug. Widespread use of the antibody may be limited, however, by the immune response typically elicited by murine mAbs. Such an immune response may cause allergic reactions and reduce the effectiveness of re-treatment by immune complex formation. In order to reduce immunogenicity, different strategies have been developed based on protein structural information. With the biology development, people can humanize mouse antibody with DNA recombination and antibody library which developed antibody techniques from chimeric and reshaped to human antibody, and now variety of monoclonal antibody derivatives. However, these methods are often accompanied by loss of antibody affinity.This work described a novel antibody humanization method by epitope mapping based on MALDI-TOF MS of its antibody bound epitope peptides combined with antibody-antigen molecular docking by computer modeling. Studies were undertaken employing a mAb of clinical utility, HAb18, to define those CDRs that are essential for antigen binding and those that may be immunogenic in humans. We fabricated a humanized version of HAb18-scFv, HAb18-huscFv, and detected its biological activity.Part I: identification of HAb18G/CD147 epitope by MALDI-TOF MS analysisObjective: to ascertain the sequence of HAb18G/CD147 epitopeMethod: first, the Dynabeads should be washed before use to remove preservatives. The washing procedure was facilitated by using a magnet. After calculating the required amount of Dynabeads and HAb18, we incubated the beads and HAb18 at room temperature for 1~2 hours with gentle rotation of the tube. Then, we separated the beads coated with HAb18 using a magnet and washed the beads for 4~5 times in Phosphate buffered saline (0.1% BSA, pH=7.4) by a magnet. When the purified monoclonal antibody HAb18 was bound to the magnetic beads, HAb18G/CD147 was added to the affinity matrix and proteolytic digestion performed by trypsin. The released supernatant, non-epitope, peptides and the protease were removed by washing with PBS buffer. For unequivocal identification of epitope peptides, the antibody-bound peptide fragment mixture was eluted by 0.1﹪TFA(pH=2.5). After freeze-drying, the combined sample solution was ready for MALDI-TOF MS analysis.Results: HAb18G/CD147 epitope peptide was obtained by limited proteolysis of antigens bound to an immobilized monoclonal antibody. The epitope sequence was identified by MALDI-TOF MS analysis.Part II: humanization design of HAb18 by antibody-antigen molecular docking combined with epitope mapping by mass spectrometric.Objective: determine the residues that need to be mutated to their human counterpart.Method: the sequence of HAb18G/CD147 epitope was determined by using the procedure of epitope excision in combination with MALDI-TOF MS analysis. After that, HAb18 structure modeling was constructed by homology modeling. Then, the docking of HAb18G/CD147 and HAb18 was performed by InsightII. Furthermore, we identified the mutated amino acid residues for humanization based on analyzing of antibody spatial structure and interaction among residues by means of computer assisted molecule design.Results: we proposed a method of verifying the epitope and the key residues based on analyzing the complex model of HAb18G/CD147 extracellular portion and its mAb based on computer assisted molecule docking. Besides, we identified the mutated amino acid residues for humanization and determined the candidate mutation sites were that H42E, H90A and L43S were replaced by H42G, H90T and L43P.Part III: expression and characterization of HAb18-huscFvObjective: confirms the feasibility of the method of humanization.Method: the humanized gene sequences of HAb18-huscFv were synthesized by overlapping PCR technique after computer aided CDR grafted humanization design of the variable region of HAb18. The genetic fragment of HAb18-huscFv was cloned to construct pCANTAB-6His-HAb18-huscFv. The recombinant plasmids containing HAb18-huscFv were identified by restriction enzyme analysis and PCR method. After that, the expression vector pCANTAB-6His-HAb18-huscFv was expressed in E. coli. Expression proteins were purified by affinity chromatography and detected by SDS-PAGE and Western blot. Indirect competitive ELISA was performed to analyze HAb18-huscFv binding activity to HAb18G/CD147 and its immunogenicity compared with HAb18-scFv. Cell immunofluorescence was used to compare binding activity of HAb18-huscFv and HAb18-scFv to antigen HAb18G/CD147 of liver cancer cells SMMC-7721. SPR biochemical analysis was used to detect association constant (Kon) and dissociation constant (Koff) of HAb18-huscFv and HAb18-scFv.Results: on the basis of humanization design, we fabricated a humanized version of HAb18scFv that H42E, H90A and L43S were replaced by H42G, H90T and L43P. After PCR and sequencing, the expression plasmid pCANTAB-6His-HAb18-huscFv was expressed in E. coli. The expressed products were briefly purified and analyzed by SDS-PAGE, western blot, cell immunofluorescence, indirect competitive ELISA and SPR biochemical analysis. These results suggest HAb18-huscFv had similar biological functions compared with HAb18-scFv, but a lower immunogenicity. Conclusion: here, we describe a novel antibody humanization method by epitope mapping based on MALDI-TOF MS analysis combined with antibody-antigen molecular docking. By this means, the functional conformation of the antibody-combining site was preserved for the retention of ligand-binding property, which requires maintenance of the CDRs and their interaction with each other and with the rest of the structure of the antibody-combining site.