Anti-tumor bispecific miniaturized antibody with double functions of targeting therapy and detection

An anti-tumor and anti-tumor drug technology, applied in the field of bioengineering, can solve the problems of high accumulation toxicity, low treatment efficiency, lack of target specificity, etc., and achieve high tumor treatment and detection efficiency, significant curative effect, and rapid clearance effect

Active Publication Date: 2014-07-30
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The molecular weight of monoclonal antibody drugs is usually about 150kD. Its too large molecular weight makes it less efficient to enter tumor tissue cells, and the metabolism time in the body is too long, which leads to its disadvantages such as low therapeutic efficiency and high accumulation toxicity in the body; On the one hand, the monoclonal antibodies currently on the market are all single-targeting EGFR1 and HER2. Due to the synergistic effect of EGFR1 and HER2, these antibodies do not show good curative effect on EGFR1/HER2 double positive tumors
Although small-molecule ...

Method used

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  • Anti-tumor bispecific miniaturized antibody with double functions of targeting therapy and detection
  • Anti-tumor bispecific miniaturized antibody with double functions of targeting therapy and detection
  • Anti-tumor bispecific miniaturized antibody with double functions of targeting therapy and detection

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Experimental program
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Effect test

Embodiment 1

[0050] Construction of recombinant expression plasmid pET22b-MaAbNA:

[0051] (1) The protein sequences of the specific domains of EGFR1 (gene coding sequence 1-372) and HER2 (gene coding sequence 420-804) were obtained from the literature, through (G 4 S) 3 The flexible peptide connects the HER2-specific domain and the EGFR1-specific domain in sequence, deduces the DNA sequence based on its overall amino acid sequence, and optimizes it according to the preferred codons of Escherichia coli, which is the MaAbNA gene coding sequence shown in SEQ ID No:2 . The recombinant gene sequence was synthesized by Nanjing GenScript Biotechnology Co., Ltd. after introducing restriction endonuclease (BamHI and NcoI) restriction sites at the 5' end and 3' end respectively. The gene synthesis adopts the chemical synthesis method, and its synthesis starts from the 3' end base of the DNA sequence shown in SEQ ID No: 2. The specific reaction steps are as follows:

[0052] ①Deprotection group ...

Embodiment 2

[0065] Expression, purification and renaturation of fusion protein:

[0066] (1) Transform the recombinant plasmid pET22b-MaAbNA into Escherichia coli BL21star TM (DE3), positive clones were screened with Amp-resistant SOB plates. After the transformed strain was cultured overnight at 37°C and 200 rpm, it was transferred to fresh LB medium at a ratio of 1:100, and continued to culture until the OD value of the bacterial solution was about 600, and the final concentration of 1 mmol / L IPTG was added to induce protein expression for 5 hours After the induction was terminated, the bacterial liquid was centrifuged and the bacterial cells were collected. After the induced recombinant bacteria were resuspended and ultrasonically lysed, the precipitate was dissolved in 8 mol / L urea solution.

[0067] (2) Use HisTrap Ni 2+ The column was used to purify the MaAbNA recombinant protein inclusion body. After loading the inclusion body solution, equilibration buffer, 5mmol / L, 10mmol / L, 2...

Embodiment 3

[0069] Synthesis of MaAbNA-rhodamine B in vitro diagnostic probe:

[0070] (1) Take 4.79mg of rhodamine B and 2.11mg of (3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and dissolve them in 1ml of dimethylformamide (DMF) Afterwards, the mixture was stirred and reacted for 2 hours at room temperature in the dark. After the reaction, 1.38 mg of N-hydroxysuccinimide (NHS) and 6 mg of MaAbNA were added to the solution to continue the reaction with stirring in the dark, and the reaction was stopped after 12 hours.

[0071] (2) The reaction product was applied to a Sephadex G-75 molecular sieve column, and washed with PBS buffer (pH 8.0) to collect red aggregated color bands. The collected product is the MaAbNA-rhodamine B in vitro diagnostic probe.

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Abstract

The present invention belongs to the field of bio-engineering pharmacy, protein polypeptide drugs and biomedical engineering, and relates to design and construction of a miniaturized fusion antibody providing high affinity for human epidermal growth factor receptors EGFR1 and HER2, and applications of the miniaturized fusion antibody in tumor targeting diagnosis and therapy, wherein the bispecific antibody is obtained by connecting a HER2 specific domain and a EGFR1 specific domain in series through a (G4S)3 connecting peptide and carrying out gene recombinant expression, the full-length of the fusion protein coding gene is 804 bp, 268 amino acids are coded, the molecular weight of the fusion protein is 29 kD, and the amino acid sequence is the sequence represented by SEQ ID No:1. Compared with combination of the EGFR1 and HER2 antibodies, the bispecific miniaturized antibody of the present invention provides significant in vitro and in vivo activity advantages, has advantages of low toxicity, being rapidly cleared in non-tumor sites in vivo, and the like, has high efficiency in targeting diagnosis and therapy, and can be used for tumor diagnosis and therapy.

Description

technical field [0001] The invention belongs to the field of bioengineering, and specifically relates to a miniaturized fusion antibody with high affinity for human epidermal growth factor receptors EGFR1 and HER2 and application thereof, which can be used for cancer diagnosis and treatment. Background technique [0002] The receptor tyrosine kinase ErbB family plays an important role in physiological processes such as cell growth, proliferation and differentiation. This receptor family includes four members: epidermal growth factor receptor (EGFR1, ErbB1 or HER1), HER2 (ErbB2), HER3 (ErbB3) and HER4 (ErbB4). Members of this family are composed of extracellular ligand-binding domain, intracellular kinase domain and transmembrane domain, and are closely related to the biological process of tumor cells. [0003] Epidermal growth factor (EGF) is the natural ligand of EGFR1. After it binds to EGFR1, it activates the receptor tyrosine kinase activity of EGFR1, and then activate...

Claims

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Application Information

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IPC IPC(8): C07K16/28G01N33/574A61K47/42A61P35/00
Inventor 顾月清丁笠
Owner CHINA PHARM UNIV
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