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Tumor-specific recombinant hirudin, preparation method and application thereof

A recombinant hirudin, tumor-specific technology, applied in the direction of leech inhibitors, antineoplastic drugs, chemical instruments and methods, etc., can solve the problems of time increase, bleeding risk, etc., and achieve the effect of avoiding allergic reactions

Inactive Publication Date: 2017-08-29
新钥(南京)生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, hirudin can lead to a significant increase in coagulation-related parameters, such as activated partial thromboplastin time (APTT), thrombin time (TT) and prothrombin time (PT), which may cause systemic or systemic bleeding risk

Method used

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Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0038] Correspondingly, the present invention also provides a method for preparing the above-mentioned tumor-specific recombinant hirudin, which includes the following steps: chemically synthesizing the above-mentioned tumor-specific recombinant hirudin gene fragment, connecting a signal peptide at its N-terminus, and adding Add a restriction enzyme site at the end, insert the recombinant coding gene into the gene vector to prepare a recombinant vector, use the recombinant vector to transform or transfect the host cell, cultivate the host cell, and recover and purify the tumor-specific recombinant hirudin therefrom, The host cells are Escherichia coli, lactic acid bacteria, yeast, insect cells or mammalian cells.

[0039] The vector used for recombination is preferably a eukaryotic expression vector, preferably pPIC9K. The host cell is preferably methanolotrophic yeast strain GS115.

[0040] Correspondingly, the present invention also provides the application of the above-men...

Embodiment 1

[0051] In this example, the recombinant hirudin is modified with the oligopeptide PLG at the N-terminal of the hirudin isomer HV2, which together with the N-terminal sequence I of the hirudin isomer HV2 forms the substrate peptide sequence PLGI recognized and cleaved by MMP9. Specific recombinant hirudin PLG-HV2, the amino acid sequence is: SEQ ID NO.1, the gene sequence is: SEQ ID NO.2.

[0052] Among them, PLGI can be replaced by other MMP9-recognized and enzymatically cleaved substrate peptide sequences, and its common feature is that it contains a GI sequence to ensure that the amino acid at the N-terminal of the released hirudin is I after being recognized and enzymatically cleaved by MMP9.

[0053] The application of the tumor-specific recombinant hirudin in the preparation of drugs for preventing and treating tumors, especially the application of drugs for preventing and treating tumors accompanied by hypercoagulable state and thrombotic lesions.

Embodiment 2

[0055] In this example, the recombinant hirudin is modified with oligopeptide PLG at the N-terminal of the hirudin isomer HV3, and together with the N-terminal sequence I of the hirudin isomer HV3, constitutes the substrate peptide sequence PLGI recognized and digested by MMP9. The amino acid sequence of the recombinant hirudin PLG-HV3 is: SEQ ID NO.3, and the gene sequence is: SEQ ID NO.4.

[0056] Among them, PLGI can be replaced by other MMP9-recognized and enzyme-cleaved substrate peptide sequences, and its common feature is that it contains a GI sequence to ensure that the amino acid at the N-terminal of the released hirudin is I after enzyme-cleaved by MMP9.

[0057] The application of the tumor-specific recombinant hirudin in the preparation of drugs for preventing and treating tumors, especially the application of drugs for preventing and treating tumors accompanied by hypercoagulable state and thrombotic lesions.

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Abstract

The invention discloses tumor-specific recombinant hirudin, of which an N-terminal contains an oligopeptide sequence PLGI, which can be recognized and cleaved by matrix metalloproteinase 9 (MMP9), wherein hirudin used for recombination may be a hirudin isomer, a hirudin mutant, truncated hirudin peptide, gene-modified hirudin or hirudin fusion protein. Correspondingly, the invention provides a preparation method of the tumor-specific recombinant hirudin and applications thereof in preparation of antitumor medicines, and also provides a preparation method and an application of adoptive immune-cells for expression of the tumor-specific recombinant hirudin. The recombinant hirudin herein allows anticoagulation activity of the hirudin is released in a tumor targeted manner, thus forming a microenvironment which is not liable to form thrombus, and even dissolving formed small thrombus to achieve the functions of anticoagulation, thrombolysis and prevention of tumor cell metastasis, thereby overcoming the risk of systematic hemorrhage due to systemic administration of wild-type hirudin.

Description

technical field [0001] The invention relates to the fields of biotherapy and biomedicine, in particular to a tumor-specific recombinant hirudin and its preparation method and application. Background technique [0002] Hirudin is the most active natural thrombin-specific inhibitor found so far. It was originally isolated from the salivary gland of medical leeches. It consists of 65-66 amino acids and can directly bind thrombin in a 1:1 (molar ratio) manner. Combined to form a non-covalent complex, so that thrombin loses the ability to crack fibrinogen and inhibits the formation of thrombus. There are more than ten kinds of variants of natural hirudin, mainly three kinds of highly homologous isomers (Hirudin Variant) referred to as HV1, HV2 and HV3. [0003] At present, two recombinant hirudin products have been approved for marketing abroad: 1. Desirudin (trade name: Revasc, product of Novartis, Switzerland), 2. Lepirudin (trade name: Refludan, product of British Pharmion an...

Claims

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Application Information

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IPC IPC(8): C07K14/815C07K5/10A61K38/58A61P7/02A61P35/00A23L33/17
CPCC07K14/815A23L33/17A23V2002/00A61K38/00A23V2200/308A23V2200/326
Inventor 何向锋施文
Owner 新钥(南京)生物科技有限公司
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