Affinity peptide specific to herceptin and segment thereof

A technology of trastuzumab and affinity peptides, which is applied in the field of affinity peptides, can solve the problems of inability to separate antibodies, harsh elution conditions, and poor chemical stability, and achieve biodegradable, low-cost, and small molecular weight Effect

Inactive Publication Date: 2011-01-26
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It is mainly expressed and produced by Chinese hamster ovary cells (CHO), and the current expression level is less than 10g / L
Although the current affinity ligands such as dye molecules and transition metal ions are low in cost, they usually have certain toxicity and cannot be used for the separation of therapeutic antibodies; while the commonly used natural ligands such as protein A and protein B are expensive, Small adsorption capacity, poor chemical stability, easy biodegradation, harsh elution conditions, easy to fall off, possible immune response and other defects

Method used

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  • Affinity peptide specific to herceptin and segment thereof
  • Affinity peptide specific to herceptin and segment thereof
  • Affinity peptide specific to herceptin and segment thereof

Examples

Experimental program
Comparison scheme
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Embodiment 1

[0031] Embodiment 1: the design of trastuzumab affinity peptide

[0032] The peptide of the present invention is rationally designed based on the complex configuration of the antigen-binding fragment of trastuzumab and HER2 by using molecular dynamics method, and then characterized.

[0033] The three-dimensional structure data (PDB ID: 1N8Z) of the complex of the antigen-binding fragment of trastuzumab and HER2 was determined by Hyun-Soo Cho et al. (Hyun-Soo Cho et al. (2003) Nature, 421: 756-760) . The interaction sites between the two include three loop structures in HER2, loop1: 557-561, loop2: 570-573, and loop3: 593-603. Residues 557-574 of HER2 were intercepted from the structure of the complex to form a structure as follows:

[0034] Pro-Glu-Ala-Asp-Gln-Cys-Val-Ala-Cys-Ala-His-Typ-Lys-Asp-Pro-Pro-Phe-Cys

[0035] The above-mentioned octadecapeptide and the Fab segment of trastuzumab were equilibrated in a water box with periodic boundaries for 4 ns, and the simulati...

Embodiment 2

[0045] The affinity peptide specific to the trastuzumab fragment with the structure shown in SEQ ID Nos.11-14 is placed near the binding site on the heavy chain of the trastuzumab Fab segment (residue numbers 33, 50, 55, 59, 99-105), by manipulative molecular dynamics (SMD) method along the centroid of the affinity peptide-trastuzumab Fab segment centroid direction at 0.25 Compress the ligand at a constant speed to make the two tightly combined, and then perform molecular dynamics simulation according to the method and conditions in "Design of Trastuzumab Affinity Peptide", and obtain an optimized affinity peptide-trastuzumab with a relaxation of 4ns The equilibrium configuration of the Fab fragment complex is found by calculating the interaction energy between the two during the relaxation process and reaching the equilibrium state. These structures are specific to the trastuzumab fragment as shown in SEQ ID Nos.8-19 The interaction energy between the affinity peptide and th...

Embodiment 3

[0046] Embodiment 3: the affinity of polypeptide and trastuzumab

[0047] The complete trastuzumab was constructed, the PDB ID of its FC segment was 3D6G, and the amino acid sequence of the humanized part (greater than 95%) of the complete trastuzumab came from Taxonomy ID 9606. Investigate the affinity peptide and trastuzumab specific for the trastuzumab fragment as shown in SEQ ID Nos.14 in the above-mentioned preference according to the method in "the affinity of the polypeptide and the Fab fragment of trastuzumab" Anti-affinity, its interaction energy size is 210kJ / mol.

[0048] The above shows that the affinity peptide specific to trastuzumab fragments obtained through rational design and interaction energy screening of the present invention can be well combined with trastuzumab or its fragments, using this affinity The purpose of separating, purifying or detecting it can be achieved.

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Abstract

The invention discloses an affinity peptide specific to herceptin and a segment thereof, belonging to the technical field of biology. The structure of the peptide is R1-X-R2, wherein R1 is NH2, R2 is COOH, and X is a peptide segment from 3 peptides to 18 peptides and is preferably 4 peptides or 6 peptides. The affinity peptide is coupled to a carrier so as to separate and purify herceptin and the segment thereof. The invention can overcome the defects of high cost, poor stability and insufficient safety of the traditional ligand, and has the advantages of high affinity, high specificity, quick herceptin capture, low production cost, low immunogenicity, high chemical stability and the like. The affinity peptide has excellent application prospects and can produce great social benefits and economical benefits.

Description

technical field [0001] The invention discloses an affinity peptide specific to trastuzumab and its fragments, belonging to the field of biotechnology. Background technique [0002] Trastuzumab (trade name: Herceptin) is a recombinant DNA-derived humanized monoclonal antibody that selectively acts on human epidermal growth factor receptor-2 (Human Epidermal growth factor Receptor 2, HER2) extracellular sites. At present, it is mainly used to treat metastatic breast cancer with overexpression of HER2, and it also has potential therapeutic effects in the treatment of other cancers such as gastric cancer, lung cancer, and ovarian cancer caused by overexpression of HER2. It is mainly expressed and produced by Chinese hamster ovary cells (CHO), and the current expression level is less than 10g / L. Affinity chromatography is the most powerful method to quickly obtain high-purity active antibodies from low-concentration biological products, and has been widely used in engineering o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K5/08C07K5/113C07K7/06C07K7/08C07K16/28C07K1/22G01N33/577
Inventor 王琦孙天杨吴韬
Owner ZHEJIANG UNIV
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