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Engineered binding proteins of EpCAM and uses thereof

A technology of binding protein, pet22b-aep3d4-aep4g2, applied in the field of binding protein, can solve the problems of restricting tumor tissue penetration, affecting treatment effect, low immunogenicity, etc., achieving low immunogenicity, high specificity and immunogenicity , the effect of strong tissue penetration

Active Publication Date: 2021-08-10
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Monoclonal antibodies have the advantages of less side effects and strong pertinence. They can specifically target tumor cell surface antigens and activate classic ADCC and CDC activities, but their large molecular weight (about 150kDa) greatly limits their ability to Infiltration of tumor tissue, which in turn seriously affects the therapeutic effect
Single-chain antibodies and single-domain antibodies have the advantages of low immunogenicity, low immune rejection, small molecular weight, and easy penetration into tumor tissues. Therefore, there is an urgent clinical need for antibody drugs that can not only specifically target tumor cells but also easily penetrate into tumor tissues and have a long half-life

Method used

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  • Engineered binding proteins of EpCAM and uses thereof
  • Engineered binding proteins of EpCAM and uses thereof
  • Engineered binding proteins of EpCAM and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Embodiment 1 obtains negative control binding protein

[0047] The previous experiments in our laboratory used phage display technology to use synthetic peptides of human epidermal growth factor receptor 2 (Her2) and vascular endothelial growth factor (VEGF) as antigens. The above method was used to screen the phage library of human binding proteins, and two were selected by ELISA. For the clones that do not bind to the corresponding antigen, the negative control binding proteins pET22b-aHER2-13C1 and pET22b-aVE201 were respectively obtained. Negative control binding proteins pET22b-aHER2-13C1 and pET22b-aVE201 have the amino acid sequences shown in SEQ ID NO.7 and SEQ ID NO.8 respectively; the nucleotide sequences encoding pET22b-aHER2-13C1 and pET22b-aVE201 are shown in SEQ ID NO .15 and SEQ ID NO.16.

Embodiment 2

[0048] The construction of the binding protein particle of embodiment 2 transformation

[0049] In the early stage, our laboratory screened out 2 fully human EpCAM binding proteins pET22b-aEP3D4 and pET22b-aEP4G2 by screening phage library, and obtained their nucleotide sequences by gene sequencing. This example uses (G 4 S) 3 The linker connects the nucleotide sequences of the above two human EpCAM binding proteins, and submits it to Jinweizhi Company to synthesize the nucleotide sequence of the pET22b-aEP3D4-aEP4G2 modified binding protein; by using (G 4 S) 3 The linker connects the nucleotide sequence of the above two human EpCAM binding proteins with the nucleotide sequence of the anti-HSA protein, and sends it to Jinweizhi Company to synthesize the nucleoside of the binding protein modified by pET22b-aEP3D4-anti-HSA-aEP4G2 acid sequence. And the above two nucleotide sequences were connected to the prokaryotic expression plasmid pET22b(+) by enzyme digestion to construc...

Embodiment 3

[0050] Prokaryotic expression of the binding protein of embodiment 3 transformation

[0051] 1. Extraction of plasmid: according to the laboratory purchased Operate with the enclosed manual of the Plasmid Mini Kit.

[0052] 2. Transform the prokaryotic expression plasmid into BL21(DE3)

[0053] (1) Take out the prepared BL21(DE3) competent cells from the -80°C ultra-low temperature refrigerator, and put them on ice to thaw.

[0054] (2) After the competent cells are thawed, add 0.1 μg of the prokaryotic expression plasmid constructed above, flick the EP tube with your fingers to mix well, and put it on ice again for half an hour.

[0055] (3) Place in a 42°C water bath, heat shock for 2 minutes, and then quickly place on ice for 2 minutes.

[0056] (4) Add 900 μL of autoclaved LB medium, place in a constant temperature shaker at 37°C, shake the bacteria for 1 hour, then centrifuge, discard 900 μL of the supernatant and mix the bacterial pellet.

[0057] (5) Using a steril...

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Abstract

The invention discloses a modified binding protein of EpCAM and application of the modified binding protein. The modified binding protein is at least one of a modified binding protein of which the amino acid sequence is shown as SEQ ID NO.1, a modified binding protein of which the amino acid sequence is shown as SEQ ID NO.2, a modified binding protein of which the amino acid sequence is shown as SEQ ID NO.3 and a modified binding protein of which the amino acid sequence is shown as SEQ ID NO.4. The invention also discloses an application of the binding protein modified by the EpCAM in preparation of antitumor drugs. The binding protein has high structural stability and strong tissue permeability, is human-derived, has no immunogenicity in a human body, and can be better applied to development of antitumor drugs.

Description

technical field [0001] The invention belongs to the field of binding proteins, in particular to a modified binding protein of EpCAM and its application. Background technique [0002] Tumor cells have the characteristics of self-renewal, unlimited proliferation, and drug resistance. Currently, the main ways to treat tumors include surgical resection, radiotherapy or chemotherapy, but these treatments have relatively large side effects for patients. Therefore, it is urgent to develop new treatment methods in clinical practice. Epithelial cell adhesion molecule (EpCAM) is one of the earliest tumor markers discovered, EpCAM is a type I transmembrane glycoprotein composed of 314 amino acids, its expression level is low in normal tissues, but in human tumor cells Lines, especially squamous cell carcinoma and adenocarcinoma, are widely expressed. For example, EpCAM is overexpressed in colorectal cancer, lung cancer, gastric cancer, pancreatic cancer, ovarian cancer, breast cancer a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/30C12N15/13C12N15/70C12N15/85A61K39/395A61P35/00
CPCC07K16/30C12N15/70C12N15/85A61P35/00C07K2317/34C07K2317/73A61K2039/505
Inventor 魏星张衡
Owner JINAN UNIVERSITY
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