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Complex of Fab fragment of C2H7 and CD20 antigen epitope polypeptide

An antigenic epitope, CD20 technology, applied in receptor/cell surface antigen/cell surface determinant, peptide preparation method, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, etc. Functional differences, etc.

Inactive Publication Date: 2009-07-29
SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The epitope of 2H7 has not been clearly reported so far. Some existing experimental data show that its epitope is roughly located in the extracellular region of the human B cell surface antigen CD20, from asparagine 163 to glutamine 187 (Polyak, M.J. and J.P.Deans(2002).Blood 99(9):3256-62; Teeling, J.L., W.J.Mackus, et al.(2006).J Immunol 177(1):362-71)
However, these antibodies still differ functionally
For example, most antibody binding causes CD20 to accumulate in detergent-insoluble lipid rafts, but B1 does not

Method used

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  • Complex of Fab fragment of C2H7 and CD20 antigen epitope polypeptide
  • Complex of Fab fragment of C2H7 and CD20 antigen epitope polypeptide
  • Complex of Fab fragment of C2H7 and CD20 antigen epitope polypeptide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 , Antibody preparation

[0024]Refer to the method published by Liu, A.Y., Robinson, R.R. et al. on pages 3521-3526 of "Journal of Immunology" No. 139 to prepare antibodies, and digest C2H7 with papain. Add papain (Sigma-Aldrich) to a final concentration of 0.1mg / ml; then digest at 37°C for 18 hours, buffer: 0.1M Tris-HCl+2mM EDTA+1mM dithiothreitol, pH8.0 . The digested product was firstly subjected to cation exchange chromatography through a chromatography column SP-Sepharose FF, and then subjected to hydrophobic interaction chromatography through a chromatography column Phenyl-Sepharose HP to obtain the Fab fragment of C2H7.

[0025] SDS-PAGE was used to verify the purity and homogeneity of the Fab fragments, and the results were as follows figure 1 shown, according to figure 1 As a result, the purified Fab fragment of C2H7 has a purity greater than 95% and good homogeneity.

[0026] The purified Fab fragment was concentrated to 7 mg / ml, and the buffe...

Embodiment 2

[0027] Example 2 , Preparation of CD20 epitope peptide

[0028] Corresponding to the peptide segment of residues 163 to 187 in the extracellular region of human CD20, an epitope peptide fragment containing 25 amino acids was designed and synthesized. The sequence of the fragment is as follows:

[0029] NIYNCEPANPSEKNSSPSTQYCYSIQ.

[0030] At the same time, an intrachain disulfide bond was introduced between the two cysteines (underlined in the sequence) corresponding to residues 167 and 183 of the extracellular region of human CD20 in this fragment, thereby simulating Out of the natural state of CD20.

[0031] With reference to the method described in the operation manual, by analytical reversed-phase chromatography ( figure 2 ) and mass spectrometry ( image 3 ), confirming that the purity of the prepared amino acid fragments is greater than 95%.

Embodiment 3-5

[0032] Example 3-5 , Antibody and epitope peptide co-crystallization

[0033] According to the molar ratio of 1:1, 1:5 and 1:10, respectively, the purified C2H7Fab and CD20 epitope peptide were mixed at 4° C. for 12 hours to obtain a protein-peptide mixed solution.

[0034] Using the hanging drop diffusion method, each 1 microliter of the obtained protein-peptide mixed solution was mixed with the crystallization pool liquid (0.2M diammonium hydrogen phosphate, 20% polyethylene glycol 3350) to form a hanging drop, and placed in a Crystal growth was carried out in a crystallization tank with 500 μl of crystallization bath liquid.

[0035] After growing for about two months at 4°C, square co-crystal complex crystals were harvested.

[0036] The crystals were stored frozen in the crystallization pool solution added with 18% polyethylene glycol 500, and then rapidly cooled to -170°C.

[0037] On the BL6A ray source (Japan Photon Factory), carry out the diffraction experiment of...

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Abstract

The invention provides a composite between Fab segment of the monoclonal antibody C2H7 of an anti-human B cell surface CD 20 molecule and CD 20 antigen epitope polypeptide, a method for obtaining the composite, and a three-dimensional crystal structure and application of the composite. The analysis result on the three-dimensional crystal structure of the composite provided by the invention not only reveals the key residue of the interaction between C2H7 and antigen epitope peptide, but also provides a theoretical basis for designing and modifying the C2H7 and other antibodies derived from theC2H7 to obtain higher-affinity and higher-specificity antibodies; and the analysis result can be used for virtual modification of a drug based on the structure of the composite. In addition, in the composite provided by the invention, CD 20 antigen epitope peptide segment takes a unique annular structure and can be used for screening new antibodies based on the epitope.

Description

technical field [0001] The present invention belongs to the technical field of antibody drugs, and in particular relates to a complex of a Fab fragment of a monoclonal antibody C2H7 anti-CD20 molecule on the surface of human B cells and a CD20 epitope polypeptide, and a method for obtaining the complex, And the three-dimensional crystal structure of the complex and its application. Background technique [0002] Human CD20 is a marker molecule expressed on the surface of B cells (Stashenko, P., L.M. Nadler, et al. (1980). J. Immunol. 125(4): 1678-85). It is a tetraspanning protein that is predicted to have a large extracellular loop (from lysine 142 to tyrosine 182) and can oligomerize on the surface of B cells (Bubien, J.K., L.J.Zhou, et al.(1993).J.Cell Biol.121(5):1121-32; Teeling, J.L., W.J.Mackus, et al.(2006).J.Immunol.177(1):362-71 .). Although the exact function of CD20 is not very clear, a lot of experimental evidence shows that the function of CD20 is likely to f...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C07K14/705C07K1/02G01N33/68
Inventor 丁建平杜嘉木钟琛
Owner SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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