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Combined MALDI-TOFMS identified epitope antibody technology and molecular docking for human transformation

Author: WangYu
Tutor: LiuWenChao；LiYu
School: Fourth Military Medical University
Course: Oncology
Keywords: Epitope Mass Docking Humanized antibodies HAb18 HAb18G/CD147
CLC: R392
Type: Master's thesis
Year: 2010
Downloads: 76
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Abstract

As we all know, cancer is the most deadly diseases. Since the 1970s, China's increased year by year the number of cancer patients, about eighty percent of cancer patients die from lung cancer, liver cancer, stomach cancer and other common tumors. The usual treatment methods include surgery, radiation therapy, chemotherapy, biological therapy, these treatments are sometimes used alone, sometimes in combination. Antibody therapy is a biological treatment, its importance is more obvious. Antibody therapy prospects are bright, has more than 20 kinds of antibody drug approved for the treatment of various diseases in clinical expected results have been obtained. The use of an antibody drug in cell engineering and genetic engineering techniques as the main antibody engineering technology to prepare drugs, but because of its attendant human anti-mouse antibody response (HAMA), an antibody drug applications are often subject to a certain extent. In order to reduce the HAMA-reactive antibody drug, protein structural information on the development of a series of transformation antibodies. With the development of molecular biology, chimeric antibodies and humanized antibodies emergence applications as a solution to reduce the HAMA-reactive antibodies effective way, but the transformation is often accompanied by antibody affinity dropped significantly. This study established a new method of humanized antibody, the first joint epitope identified based on mass spectrometry and molecular docking on the transformation of human antibody. Through the identification of antigenic epitopes HAb18G/CD147 HAb18 humanized antibody designed so that the antibody HAb18 in guaranteed on the basis of affinity humanized transformation, followed by the synthesis of new recombinant DNA technology and detecting the antibody antibody fragment Biological activity of fragments, respectively, the following three parts from the following description. The first part: MALDI-TOF MS to determine the epitope HAb18G/CD147 Objective: To determine the epitope sequence HAb18G/CD147 Methods: supporting new magnetic bead washing rack, wash beads preservatives. HAb18 adding an appropriate amount of antibody at room temperature under conditions combined with beads 1 to 2 hours and a slight shake, fully integrated with the antibody and beads. Followed by PBS (0.1% BSA, pH = 7.4) was washed 4-5 times beads, unbound antibody to block the active site of beads. The amount of antibody HAb18G/CD147 beads was added to the suspension prepared at room temperature, with 1 to 2 hours, the antigen binding antibody sufficient. Add trypsin solution hydrolysis HAb18G/CD147, washed off with PBS unbound peptide antigen and with 0.1% TFA (pH = 2.5) was eluted HAb18G/CD147 epitopes and rapidly lyophilized detected using MALDI-TOF MS Samples prepared. Results: Preparation of HAb18G/CD147 epitopes described hydrolysis of using immunomagnetic antigen approach is feasible, and a sample prepared in accordance with MALDI-TOF MS detection criteria. The prepared epitope peptides HAb18G/CD147 MALDI-TOF MS analysis and prediction software with trypsin digestion PeptideCutter HAb18G/CD147 comparison of the molecular weight peptides obtained HAb18G/CD147 peptide epitope sequence information. Part II: Application identified epitopes and molecular docking on HAb18 humanized antibody designed to: identify mutant amino acid sites: According to existing models and get HAb18 HAb18G/CD147 antibody epitope sequence software application DOCKING HAb18 create HAb18G/CD147 molecule docking models with and predicted epitope critical amino acid residues. In the simulation of the antibody with its HAb18 HAb18G/CD147 docking model, the method of reshaping a surface HAb18 variable region of the humanized design. Sequence analysis and structural analysis using murine antibody to determine differences in the exposed residues, selected amino acid residues to be mutated base sites to enable the synthesis of low immunogenicity and having an antigen binding activity of the humanized antibody. Results: The establishment of a new humanized antibody, for the first time will be based on MALDI-TOF MS to determine epitope technology into molecular docking, the establishment of HAb18 HAb18G/CD147 and molecular docking model, complete HAb18 variable The humanized design area to determine the amino acid mutation site is a H42E, H90A and L43S. Part III: The humanized antibody fragment HAb18-huscFv expression and identification purposes: Verify that humanized Method: According to the design of humanized scheme, with the overlapping PCR method for the synthesis of the humanized form of the gene HAb18-huscFv, cloned into the expression vector pCANTAB-6His-HAb18-huscFv in and transformed into E. coli induced to express. Purified by Ni2 expressed protein, the use of SDS-PAGE, Western blot, immunofluorescence methods such as expression products for the detection, identification, synchronization construct an expression in the form of its parental murine antibody as a control. Analysis by indirect competitive ELISA antibody HAb18-huscFv and HAb18G/CD147 binding activity, SPR HAb18-huscFv Determination of antibody affinity and HAb18G/CD147 and the synchronous form of murine antibody was expressed as a control, whether the differences of the two antibodies . Finally HAMA positive serum, compared HAb18-huscFv and murine HAb18-scFv difference in immunogenicity. Results: According to the selected mutation, synthesized using overlapping PCR method and a new gene was cloned into the expression vector, sequenced to verify correct transformation induced in E. coli, purified by Ni2 column to obtain a more pure HAb18 -huscFv. SDS-PAGE and Western blot detection of target protein molecular weight of about 27kDa. Determined by SPR, HAb18-huscFv affinity compared with HAb18-scFv no significant difference. With immunofluorescence assay HAb18-huscFv with hepatoma SMMC-7721 cells with a certain binding capacity. Indirect competitive ELISA assay showed: antibody HAb18-huscFv with HAb18-scFv compared with the low immunogenicity. Conclusion: Based on the use of the new method to create humanized antibody program, after the transformation of antibodies HAb18-huscFv with HAb18-scFv compared to reduced immunogenicity while retaining the antigen-binding capacity, proved that the method is feasible and effective for antibody humanization design provides a new way of thinking.

Combined MALDI-TOFMS identified epitope antibody technology and molecular docking for human transformation

Abstract

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