Compound for Fab fragment of Rituximab and CD20 antigen epitope polypeptide

An antigen epitope, CD20 technology, applied in polycrystalline material growth, instrument, crystal growth, etc., can solve problems such as unclear mechanism

Inactive Publication Date: 2007-09-26
SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the specific mechanism of Rit...

Method used

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  • Compound for Fab fragment of Rituximab and CD20 antigen epitope polypeptide
  • Compound for Fab fragment of Rituximab and CD20 antigen epitope polypeptide
  • Compound for Fab fragment of Rituximab and CD20 antigen epitope polypeptide

Examples

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Embodiment 1

[0021] Embodiment 1, the preparation of antibody

[0022]Digest Rituximab (Roche) with papain, and the enzyme digestion conditions are: add papain (Sigma-Aldrich) to 10 mg / ml Rituximab to a final concentration of 0.1 mg / ml; then digest at 37°C for 18 hours, buffer Solution: 0.1M Tris-HCl+2mM EDTA+1mM Dithiothreitol, pH 8.0.

[0023] The digested product was first subjected to cation exchange chromatography using SP-Sepharose FF (GE Healthcare), and then subjected to hydrophobic interaction chromatography using Phenyl-Sepharose HP (GE Healthcare); through the above steps, the Fab fragment of Rituximab was obtained.

[0024] The purity and homogeneity of the Fab fragments were verified by SDS-PAGE and dynamic light scattering experiments, and the results are shown in Figure 1; sex is good.

[0025] The purified Fab fragment was concentrated to 8 mg / ml, and the buffer was replaced with: 100 mM NaCl+10 mM Tris-HCl, pH 8.0.

Embodiment 2

[0026] Embodiment 2, the preparation of CD20 epitope peptide

[0027] 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:

[0028] NIYN C EPANPSEKNSPSTQY C YSIQ.

[0029] At the same time, a chain was introduced between the two cysteines corresponding to residues 167 and 183 of the human CD20 extracellular region (the cysteines have been underlined in the sequence) in this fragment internal disulfide bonds to mimic the natural state of CD20.

[0030] Referring to the method described in the operation manual, the purity of the prepared amino acid fragments was determined to be greater than 95% through analytical reversed-phase chromatography ( FIG. 2 ) and mass spectrometry ( FIG. 3 ).

Embodiment 3-5

[0031] Example 3-5, Co-crystallization of Antibody and Epitope Peptide

[0032] According to the molar ratio of 1:1, 1:5 and 1:10 respectively, the purified Rituximab Fab and CD20 epitope peptide were mixed at 4° C. for 12 hours to obtain a protein-peptide mixed solution.

[0033] Using the hanging drop diffusion method, mix the obtained protein-peptide mixed solution and the crystallization pool solution (0.2M calcium acetate, 0.1M sodium cacodylate, pH 6.5, 18% polyethylene glycol 8000) each 1 microliter After mixing to form hanging drops, place them in a crystallization tank containing 500 microliters of crystallization bath liquid for crystal growth.

[0034] After about two weeks of growth at 4°C, square-shaped co-crystallized complex crystals were harvested.

[0035] Crystals were stored frozen in Paratone-N (Hampton Research) and rapidly cooled to -170°C.

[0036] On the NW12 ray source (Japan Photon Factory), carry out the diffraction experiment of the co-crystal com...

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Abstract

The invention discloses a compound of Fab segment of anti-cancer drug Rituximab and CD20 antigen epitope peptide, which also provides the preparing method of compound and three-dimensional crystal structure of compound and application, wherein the three-dimensional crystal structure of compound displays the key residue interacted between Rituximab and antigen epitope peptide, which provides theoretical base for higher affinity and specificity antibody; the CD20 antigen epitope peptide displays peculiar ring-shaped structure, which is fit for sieving new antibody of epitope.

Description

technical field [0001] The present invention belongs to the technical field of antibody drugs, and in particular relates to a complex of an anticancer drug Rituximab Fab fragment and a CD20 epitope polypeptide, a method for obtaining the complex, and a 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 form or regulate voltage-gated calcium channels (Bubien, J.K....

Claims

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Application Information

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IPC IPC(8): C30B29/54G01N33/574
Inventor 丁建平杜嘉木钟琛
Owner SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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