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Protein derivatives of human granzyme B, and use thereof in targeted therapy on adenocarcinoma

A granzyme and derivative technology, applied in the direction of microorganism-based methods, microorganisms, peptide/protein components, etc., can solve the problems of low targeting accuracy, inability to successfully apply clinically, immunogenicity and side effects, etc.

Inactive Publication Date: 2010-03-31
THE INST OF BASIC MEDICAL SCI OF CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, a variety of targeted protein drugs fused with human exogenous toxins have not been successfully applied in clinical practice.
After medication, the body will produce neutralizing antibodies in a short period of time, resulting in repeated medication failure; in severe cases, even an immune reaction, resulting in shock and death; accompanied by side effects such as vascular leakage syndrome and liver and kidney damage
The reason is mainly the low targeting accuracy of this class of drugs and serious immunogenicity and side effects.

Method used

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  • Protein derivatives of human granzyme B, and use thereof in targeted therapy on adenocarcinoma
  • Protein derivatives of human granzyme B, and use thereof in targeted therapy on adenocarcinoma
  • Protein derivatives of human granzyme B, and use thereof in targeted therapy on adenocarcinoma

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0115] Example 1 Obtaining the GrB gene fragment encoding the targeted fusion protein

[0116] The present invention uses the Trizol method to extract total RNA of human skin T lymphoma cells HuT-78. The method is: centrifugation to collect about 10% of cultured cells 6 Resuspend them in 100μl PBS, put them in a 1.5ml Eppendorf tube treated with DEPC, add 1.1ml Trizol, shake well and mix well, and ice bath at 0°C for 5 minutes. Add 350μl of chloroform, shake well, let it stand for a while until stratification occurs, and centrifuge at 4°C at 12000rpm for 10 minutes. Transfer the supernatant to another 1.5ml Eppendorf tube treated with DEPC, add an equal volume of isopropanol pre-cooled at 4°C, and mix well. Place it at -20°C for 1 hour, and centrifuge at 4°C at 12000 rpm for 20 minutes. Add 0.25ml of 75% ethanol to the precipitate, centrifuge at 12000rpm at 4°C for 5min, and dry the precipitate at 37°C or vacuum dry. Dissolve the precipitate with 20μl-50μl DEPC-treated deioniz...

Embodiment 2

[0119] Example 2 Construction of yeast eukaryotic expression recombinant pPIC9K-GrB

[0120] Use primers NK266 and CK266 (primer sequences as shown in PRIMER SEQ ID NO. 3 and 4) to amplify the α-factor signal sequence (SP) in the original pPIC9K expression plasmid upstream of the BamH I cleavage site to the Kex2 break site (GAGAAAAGA↓) 270bp DNA fragment. Using the pBSSK-GrB transition plasmid in Example 1 as a template, primers NKB and CKB (primer sequences are shown in PRIMER SEQ ID NO. 5 and 6) were used to amplify a 739 bp DNA fragment. Use OVERLAP-PCR to connect the above two amplified products. Amplification reaction system I has a total volume of 50μl, consisting of: 1μl each of PCR amplification products, 0.01μmol dNTPs, 1U Pyrobest DNA polymerase, 10×PCR Buffer (20mM Mg 2+ ) 5μl, ddH 2 O make up to 50μl. The total volume of reaction system II is 10μl, the composition is: dNTPs 0.01μmol, PyrobestDNA polymerase 1U, 10×PCR Buffer (20mM Mg 2+ ) 1μl, 10μM primer each 1μl, d...

Embodiment 3

[0121] Example 3 Construction of yeast eukaryotic expression recombinant pPIC9K-mGrB

[0122] The present invention combines the two cationic sequences of human mature GrB protein CS1 ( 96 RKAKRTRA, 96 Arginine-lysine-alanine-lysine-arginine-threonine-arginine-alanine) and CS2 ( 221 KKTMKRY, 221 Lysine-lysine-threonine-methionine-lysine-arginine-tyrosine) part of the basic amino acid coding sequence with cell binding function is replaced with non-polar alanine ( The coding sequence of A) was mutated to 96 A KAK A T A A( 96 Alanine -Lysine-Alanine-Lysine- Alanine -Threonine- Alanine -Alanine) and 221 AA TM AA Y( 221 Alanine - Alanine -Threonine-Methionine- Alanine - Alanine -Tyrosine) to obtain the mutant gene mGrB.

[0123] Using pPIC9K-GrB in Example 2 as a template, primers NK266 and Ncs1 (primer sequences shown in PRIMER SEQ ID NO. 7) were used to amplify a 558 bp DNA fragment. Using pBSSK-GrB in Example 1 as a template, primers cs1 and cs2 (primer sequences shown in PR...

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PUM

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Abstract

The invention belongs to the pharmacological field of gene engineering, and provides a group of protein derivatives of human granzyme B by adopting gene engineering technology. The group of protein derivatives of the human granzyme B comprises GrB-G4S-GnRH (GrBLG) and mGrB-C4S-GnRH (mGrBLG) which are targeted fusion proteins formed by connecting human matured granzyme B (GrB) and mutagenic human matured granzyme B (mGrB) with human gonadotropin-releasing hormone (GnRH) through flexible connecting short peptide GlyGlyGlyGlySer (G4S) respectively and which can be combined with human GnRH receptors (GnRHR) on the surfaces of cells through ligand human GnRH. The mGrB eliminates the functions of the combination through a personal 'electrostatic exchange-absorption mode' and the entering into target cells of the prior GrB, and simultaneously reserves the enzymatic activity and the cytocidal function of the GrB. The invention provides experimental evidences for peculiarly targeted-killing GnRHR positive cells and minimizing toxic side effect by the fusion proteins, and shows the use of the group of protein derivatives of the human granzyme B in the targeted therapy on the positive adenocarcinoma of a type of gonadotropin-releasing hormone receptors.

Description

Technical field [0001] The invention belongs to the field of genetic engineering drugs. Using genetic engineering technology, the present invention provides a group of human granzyme B protein derivatives GrB-G4S-GnRH (GrBLG) and mGrB-G4S-GnRH (mGrBLG). The two are human mature granzyme B (GrB) and mutated human mature granzyme B (mGrB), respectively, through the flexible link peptide GlyGlyGlyGlySer (G4S) and human gonadotropin releasing hormone (GnRH) linked by targeted fusion proteins, Both can bind to the human GnRH receptor (GnRHR) on the cell surface through the ligand human GnRH. mGrB eliminates the function of original GrB to bind and enter target cells through its own "electrostatic exchange-adsorption mode (that is, the basic amino acid in the specific cationic sequence of the GrB protein binds to the negatively charged mucopolysaccharide on the cell surface)", while retaining the function of GrB Enzymatic activity and cell killing function. The present invention pr...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/81C12N15/70C12N9/00C12N1/19C12N1/21A61K38/43A61P35/00C12R1/84C12R1/19
Inventor 卢圣栋李一叶李涛陈伟京路金芝
Owner THE INST OF BASIC MEDICAL SCI OF CHINESE ACAD OF MEDICAL SCI
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