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Anti-Met humanized Fab, anti-Met humanized Fab and doxorubicin conjugate and preparation method and application of anti-Met humanized Fab and doxorubicin conjugate

A doxorubicin and product technology, applied in the application field of anti-Met human Fab in the biological targeted therapy of liver cancer after coupling, can solve the problems of large toxic side effects and limited application, and achieve the effect of good affinity

Inactive Publication Date: 2011-09-07
SINOBIOWAY CELL THERAPY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Doxorubicin is one of the earliest tumor chemotherapy drugs used clinically. It has a wide range of applications and is one of the commonly used drugs for liver cancer chemotherapy. Application in Tumor Therapy

Method used

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  • Anti-Met humanized Fab, anti-Met humanized Fab and doxorubicin conjugate and preparation method and application of anti-Met humanized Fab and doxorubicin conjugate
  • Anti-Met humanized Fab, anti-Met humanized Fab and doxorubicin conjugate and preparation method and application of anti-Met humanized Fab and doxorubicin conjugate
  • Anti-Met humanized Fab, anti-Met humanized Fab and doxorubicin conjugate and preparation method and application of anti-Met humanized Fab and doxorubicin conjugate

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1 Construction of a fully human immune Fab phage antibody library

[0057] Collect 40 parts of peripheral whole blood from patients with liver cancer, separate lymphocytes, use Invitrogen Trizol to extract total RNA, and use oligo(dT) 20 As primers, cDNA was generated by reverse transcription. Then use specific primer PCR to amplify the heavy and light chain variable region genes respectively. The Fab gene was synthesized by Overlap PCR ( figure 1 ), was connected with the expression vector pComb3XSS cut with the same restriction enzymes to construct the prokaryotic recombinant expression vector of Fab; electrotransformed competent Escherichia coli XL1-Blue, and constructed a capacity of 6.5×10 8 Fully human immune Fab antibody library.

[0058] The connection of the constructed Fab gene with the pComb3xSS carrier refers to: the Fab gene after purification and quantification is subjected to double digestion with SfiI endonuclease; after purification and quant...

Embodiment 2

[0059] Example 2 Screening, expression and purification of anti-Met human Fab

[0060] (1) Enrichment screening of phage antibody library: Coat solid-phase screening ELISA plate with purified human Met recombinant protein, 1 μg per well, wash, add blocking solution, wash, add phage antibody library antibody, wash to remove unbound phage Antibody; add trypsin, elute the specifically bound phage antibody, increase the infection value, and assist phage M13K07 superinfection; repeat the above screening steps, and perform three rounds of "adsorption-elution-amplification" enrichment screening;

[0061] (2) Positive clones identified by ELISA: Dilute the phage obtained in the last round of screening and multiplication, spread on a culture plate and culture overnight, pick 564 single colonies in a cell culture plate, shake and culture overnight; from the first plate Transfer 5 μl of bacterial solution from each well to the second plate, shake culture; add helper phage M13K07 for supe...

Embodiment 3

[0073] Embodiment 3MetFab is coupled with Adriamycin

[0074] MetFab is coupled to doxorubicin by chemical bonding. First, PEG100 (polyethylene glycol 100) generates polyethylene glycol diacid (2) under the oxidation of potassium permanganate acidic solution. Then acid chloride at 80°C to obtain polyethylene glycol acid chloride (3). Polyethylene glycol acid chloride reacts with DOX to produce PEGylated DOX. Remove the organic reaction solvent, adjust the pH of the reaction solution to 7.2 with PBS, activate with EDC, and react with Met antibody for 24 hours. Dialyzed, concentrated, and separated by Sephadex G100, the target product MET-DOX was obtained. After high-performance liquid chromatography analysis, it was considered that the antibody was successfully coupled with doxorubicin. And the conjugate was named DOX-MetFab. ( Figure 27 )

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Abstract

The invention relates to anti-Met humanized Fab, an anti-Met humanized Fab and doxorubicin conjugate and a preparation method and application of the anti-Met humanized Fab and doxorubicin conjugate. The light-chain amino acid sequence is shown as the SEQ ID No.1, and the heavy-chain amino acid sequence is shown as the SEQ ID No.2. The light-chain nucleotide sequence is shown as the SEQ ID No.3, and the heavy-chain nucleotide sequence is shown as the SEQ ID No.4. The result of the in vitro cell toxicity test shows that the anti-Met humanized Fab and doxorubicin conjugate and free doxorubicin can effectively kill Met positive expression liver cancer cells, and the toxic effect of the anti-Met humanized Fab and doxorubicin conjugate on the Met positive expression cells is obviously less thanthat of the free doxorubicin. The result of the in vivo subcutaneous human liver cancer cell transplantation tumor treatment experiment on the nude mouse shows that the anti-Met humanized Fab and doxorubicin conjugate and the free doxorubicin can effectively inhibit the growth of subcutaneous human liver cancer cell transplantation tumor, and the anti-Met humanized Fab and doxorubicin conjugate can obviously alleviate side effects such as weight loss resulted from the chemotherapeutic medicament on the mouse.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals, and relates to a coupling of a human Fab anti-cell surface Met receptor and a chemotherapeutic drug doxorubicin and a preparation method thereof, and the anti-Met human Fab after coupling in biological targeted therapy of liver cancer Applications. Background technique [0002] 90% of primary liver cancer (primary hepatocellular carcinoma, PLC) is hepatocellular carcinoma (HCC). Hepatocellular carcinoma is the fifth most common tumor in the world. Due to the lack of effective treatment, it is currently the third most lethal malignant tumor, which seriously threatens human life and health. With the rapid development of modern molecular biology technology and genetic engineering technology, it has opened up a new field for the biological treatment of liver cancer. Biological treatment has become the fourth mode of tumor treatment after surgery, radiotherapy and chemotherapy, and has shown good T...

Claims

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Application Information

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IPC IPC(8): C07K16/28C07K1/113A61K31/704A61K47/48A61P35/00A61P1/16A61K47/68
Inventor 朱进冯振卿
Owner SINOBIOWAY CELL THERAPY CO LTD
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