CD73 enzyme activity-related antigen epitope and preparation method of specific antibody targeting to epitope

An antigen epitope and specific technology, applied in the field of medicine, can solve the problems of reducing the possibility of becoming a drug, lack of blocking its enzyme activity, etc., to shorten the time of antigen preparation, wide application prospects and practical value, and reduce the concentration of adenosine Effect

Active Publication Date: 2021-07-02
ZHEJIANG UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patented technology provides an improved way to study how certain parts of DNA called histones help regulate gene expressions during cell division. These modifications make it easier to break up cells into smaller units like chromatids that carry important functions within them. By measuring these changes through Western blotting techniques, researchers may predict what types of genes they might express at different stages of development. They also found out if other factors could affect this process differently - including whether some chemical compounds were added to enhance its ability to bind specifically against another molecule on top of the targeted part being studied. Overall, this new technique allows scientists to better identify targets involved in cancer biology and develop effective therapies based upon understanding those targets' role in controlling disease progression.

Problems solved by technology

This patented technical problem addressed in previous works relates to how certain factors affect the expression levels (activity) of specific types of genes called CD74 family members like cytoskeleton associated gene-1/cytotoxic shock pathway). These factors include changes made during inflammations caused by chemotherapons used against breast cancer patients, increased intravasculature pressure due to blood flow disorders, and alterments from receptors involved in regulating Toll Like Receptor signal transduction. Overall, understanding the relationship between these factors will help identify new treatable targets for developing drugs that could potentially lead to better clinically relevant responses.

Method used

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  • CD73 enzyme activity-related antigen epitope and preparation method of specific antibody targeting to epitope
  • CD73 enzyme activity-related antigen epitope and preparation method of specific antibody targeting to epitope
  • CD73 enzyme activity-related antigen epitope and preparation method of specific antibody targeting to epitope

Examples

Experimental program
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Effect test

Embodiment 1

[0051] Example 1: Identification of antigenic epitopes related to CD73 enzymatic activity

[0052] We simulated the spatial conformation of the CD73 enzymatic activity-related epitope binding to the antibody in a 3D environment through the biological modeling Docking Model method. (1) Obtain the crystal structure of NT5E (CD73) from the Program Database, and the protein IDs are 4H2F and 1CLY, respectively. (2) With the help of Schrodingger 2015 software, the following processes were performed on the two protein structures: adding the side chain of amino acid residues; adding the missing loop part in the crystal structure; assigning the protonation state of amino acid residues and the situation of applying the OPLS2005 force field Next, optimize the entire protein structure. (3) Use ZDOCK online calculation (http: / / zdock.umassmed.edu / ) for the docking of IgG Fab and NT5E. During the docking process, NT5E is selected as the receptor, and 1CLY is set as the ligand. The result i...

Embodiment 2

[0053] Example 2: Preparation of monoclonal antibodies against CD73 enzymatic activity-related epitopes

[0054] 1 Synthesis of peptide-carrier protein conjugates

[0055] Two polypeptides shown as SEQ ID NO: 1 and SEQ ID NO: 2 were respectively synthesized by peptide synthesis technology, and the HPLC detection purity was above 95%. Using the bifunctional coupling agent EDC, the -COOH on the above-mentioned synthetic peptide was directly combined with the -NH on the bovine serum albumin (BSA) and hemocyanin (KLH) respectively. 2 Coupling was carried out at 100:1 (molar ratio), and the coupling efficiency was identified by HPLC after removing the free polypeptide, that is, a polypeptide-BSA and polypeptide-KLH conjugate was obtained.

[0056] 2 Polypeptide-BSA conjugate immunized mice

[0057] Five Balb / c mice weighing 18-22 g and 4 weeks old were selected. Use the polypeptide-BSA conjugate as the antigen, the first immunization concentration is 1mg / mL, the mouse immunizati...

Embodiment 3

[0067] Example 3: Anti-CD73 enzymatic activity-related epitope-specific monoclonal antibody recognition amino acid sequence identification

[0068] (1) Coating plate: Dilute the two polypeptide-KLH conjugates of SEQ ID NO:1 or SEQ ID NO:2 to 2.5 μg / mL with coating buffer, add 100 μL / well, overnight at 4°C, the next day, discard the solution in the well, wash 3 times with 1×TBST washing buffer, and pat dry;

[0069] (2) Blocking: add 100 μL of 1% BSA to each well for blocking, incubate at 37°C for 1 hour, discard the blocking solution;

[0070] (3) Adding samples: add 50 μL / well of the above-mentioned 5A4 or 8-5 hybridoma culture supernatant+50 μL / well of 1% BSA or 50 μL / well of the above-mentioned 5A4 or 8-5 hybridoma culture supernatant+50 μL / well 1 mg / mL naked peptide in each well (Table 2 is the amino acid sequence of different naked peptides), each set up 3 duplicate wells, incubate at 37°C for 120 min, pat dry, wash 3 times with 1×TBST washing buffer, and pat dry;

[00...

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Abstract

The invention relates to the field of medicines, and discloses an epitope related to CD73 enzyme activity and a preparation method of a specific antibody targeting to the epitope. The antigen epitope related to the CD73 enzyme activity comprises an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2, or an amino acid sequence 90% or more homologous with the amino acid sequences as shown in SEQ ID NO: 1 and SEQ ID NO: 2. The amino acid sequence of the antigen epitope has very strong irreplaceability, enough polypeptide can be obtained by utilizing a polypeptide synthesis method, and a large amount of recombinant protein does not need to be produced, so experimental materials are saved, and antigen preparation time is shortened; and the epitope specific antibody prepared by using the antigen epitope can effectively inhibit CD73 enzyme activity and stimulate lymphocyte to release interferon, plays an anti-tumor role, and has wide application prospect and practical value.

Description

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Claims

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Application Information

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Owner ZHEJIANG UNIV
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