CN1114685A - Human interleukin-2 immunotoxin - Google Patents

Human interleukin-2 immunotoxin Download PDF

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Publication number
CN1114685A
CN1114685A CN94117768A CN94117768A CN1114685A CN 1114685 A CN1114685 A CN 1114685A CN 94117768 A CN94117768 A CN 94117768A CN 94117768 A CN94117768 A CN 94117768A CN 1114685 A CN1114685 A CN 1114685A
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ang
human
fusion rotein
immunotoxin
gene
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CN1065276C (en
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马大龙
王露
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Beijing Medical University
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Beijing Medical University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/642—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the peptide or protein in the drug conjugate being a cytokine, e.g. IL2, chemokine, growth factors or interferons being the inactive part of the conjugate

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  • Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)

Abstract

The present invention discloses a new type human-source interleukin-2 immunotoxin ( abbreviation: IL2-Ang), and it is characterized by that the human angiogenin (Ang) is fused on the carboxyl terminal of human interleukin-2 (IL-2), and between those two a part of joint consisting of hydrophilic amino acid is borne, the gene engineering technique is used to make expression and purification in colibacillus so as to obtain IL-2-Ang having the immunosuppressive activity which is shown. Its advantages are that its toxin component source is human albumen, so that it is not easy to result in immune respose of mechanism, so that it has a good application praspect for curing autoimmune disease, allergic rection, transplantation rejection and T-lymphocytomatosis.

Description

Human interleukin-2 immunotoxin
The present invention relates to a kind of novel protein molecule that utilizes the genetic engineering technique preparation, relate in particular to interleukin II (IL-2) and angiogenin (angiogenin, Ang) fusion rotein (abbreviation IL-2-Ang), this kind albumen can be applied to the treatment of some diseases as immunosuppressor.
IL-2 is a kind of cytokine by T emiocytosis, most immobilized T cells are not expressed the IL-2 acceptor, must just be expressed after the special activation, and the unusual high expression level of IL-2 acceptor is particularly arranged in the patient body of leukemia, lymphoma, autoimmune disease, graft-rejection in some tumours, these cells of expressing the IL-2 acceptor can become a target of IL-2 fusion rotein treatment just, make the IL-2 toxin can kill and wound this class cell exactly.
Angiogenin (Ang) is to be present in the intravital normal composition of people, in the extracellular free of toxic effects, because having stronger promotion new vessel nucleus formation, it gains the name, this factor also has the activity of RNA enzyme, 18S RNA in cell in the cleavable 40S subunit and arrestin matter synthetic, the effect of pair cell toxigenicity (Rybak SM et al, PNAS 1992; 89:3165).
At present, existing panimmunity toxin is developed out, interleukin II (IL-2) immunotoxin particularly, comprise the ectotoxic fusion rotein of IL-2 and Pseudomonas aeruginosa, the fusion rotein of IL-2 and diphtheria toxin etc., the latter has entered the clinical I/II phase in the U.S. and has tested, and receives better curative effect, be the good immunosuppressor of new generation of a kind of application prospect (Terry B et al, Immunol Rev 1992; 129:131).But its toxin of IL-2 immunotoxin of development all is from bacterium or plant at present, to human body is foreign protein, causes the generation of neutralizing antibody easily, and curative effect is descended, and may cause serious side effects, still there is not the report of relevant humanized's IL-2 immunotoxin so far.
Because the toxin of IL-2 immunotoxin all is from bacterium or plant at present, concerning human body, has stronger antigenicity, all produced can detected neutralizing antibody for most patient in clinical trial, this just makes treatment time be restricted, also influence the performance of detoxifying function, also might cause some serious side effects.
The IL-2 immunotoxin that the purpose of this invention is to provide a kind of full-length human.
Another object of the present invention provides a kind of method that adopts biotechnology to prepare the IL-2-Ang fusion rotein.
Another purpose of the present invention is to adopt IL-2-Ang fusion rotein treatment graft-rejection, some t cell lymphoma and autoimmune disorder.
The objective of the invention is to realize by following method:
The present invention utilizes gene fusion, and the program of genetic expression and gene engineering product purifying is produced the IL-2-Ang fusion rotein in intestinal bacteria.We synthesize the upstream and downstream primer of IL-2 and Ang respectively, wherein the downstream primer of IL-2 is removed the IL-2 termination codon, through the pcr amplification goal gene, carry out cloning respectively, import 14 amino acid coding of N end of IL-3 and the dna fragmentation that restriction endonuclease digestion obtains IL-2 and Ang through pcr amplification again, purifying is after gene fusion, coli expression carrier pKpL-3a (Zhou Baohong etc. recombinate, China's microbiology and Journal of Immunology, 1992,12 (3): 174), transformed into escherichia coli, abduction delivering separates inclusion body, sex change, renaturation and purifying obtain recombinant il-2-Ang fusion rotein, and it contains 14 amino acid coding of people IL-3N end between the two as joint.
Biggest advantage of the present invention is that IL-2 and Ang all are the people source, and is more weak to human antigen's property, can prolonged and repeatedly use to patient.Simultaneously Ang is present in the intravital normal composition of people, and in the extracellular free of toxic effects, only entering in the cell can the toxigenicity effect, can be to irrelevant cell toxigenicity effect, and this has just reduced non-special toxic action in the body of IL-2 immunotoxin.
Elaborate below in conjunction with accompanying drawing:
Fig. 1 is an IL-2-Ang fusion rotein DNA base sequence (bp).Comprising IL-2 sequence (dna sequence dna 1--402bp), IL-3N-holds wetting ability intermediate head sequence (dna sequence dna 403-444bp), Ang sequence (dna sequence dna 445-813) coding.
Fig. 2 is aminoacid sequence figure.
Fig. 3 is the fusion protein expression vector synoptic diagram, and wherein a shows the PL promotor, and b shows the IL-2 gene, and c shows intermediate head, and d shows the Ang gene, and e shows the pKpL carrier DNA.
Detailed implementation step of the present invention is as follows:
One .IL-2 function area gene clone:
Arrange for obtaining best base, avoid to disturb the mRNA secondary structure of rotaring intertranslating start, increase IL-2 homogeneity and the stability expressed, when making up IL-2cDNA plasmid expression clone pKpL-h IL-2, removed its initiator codon ATG, now the SD sequence with synthetic is a upstream primer, (5 '-TCGACCTAAGGAGGTTTAAC-3 '), IL-2 gene function district 3 ' hold is downstream primer (5 '-GTTAGTGTTGAGATGATGCTTT-3 '), downstream primer is removed termination codon TAG, with pKpL-h IL-2cDNA clone is template, through pcr amplification IL-2 gene fragment, the 0.4Kb product that obtains Promega Magic reagent purifying, repurity after the SalI enzyme is cut is connected with the SmaI joint, cuts through the SmaI enzyme again, to recombinate through the PCR of above-mentioned processing product and the pKpL expression vector behind SalI and SmaI double digestion, transform the pop2136 recipient bacterium, selected clone is identified acquisition positive colony pKpL-mh IL-2 through abduction delivering and plasmid enzyme restriction.
Two. contain the Ang gene clone of intermediate head
According to the people Ang sequences Design primer of having announced, with the liver cell library is that template is through the cDNA of pcr amplification Ang fragment, be inserted into people IL-3 cloning by expression pMT-h IL-3 plasmid (the horse big dragon etc. that this chamber makes up, the hi-tech communication, 1991,11:26) the cloning by expression pMT-hAng of acquisition Ang, be template with the pMT-hAng plasmid DNA again, upstream sequence primer (5 ' GCTCCCATGACCCAGACA-3 ') with IL-3, with Ang downstream sequence primer (5 '-TTTAAGCTTACGGACGGGGAAAATGG-3 '), through pcr amplification, can obtain 5 ' end and have the gene fragment that people IL-3N-holds 14 amino acid (seeing Fig. 3 c) and people Ang encoding sequence (seeing Fig. 3 d).
The structure of three .IL-2-Ang expression plasmids
The PCR product of Ang is cut the back with the HindIII enzyme be connected, promptly constitute the IL-2-Ang cloning by expression pKpL-IL2-Ang (the IL-2-Ang nucleotide sequence is seen Fig. 1, and complete plasmid map is seen Fig. 3) of reorganization with the IL-2 cloning by expression that removes termination codon (seeing Fig. 3 b).The pL promotor (see Fig. 3 a) act under, express IL-2-Ang fusion rotein (aminoacid sequence is seen Fig. 2).
Four. the escherichia coli high-level expression fusion rotein
The pKpL-IL2-Ang clone is transformed the pop2136 recipient bacterium, be inoculated in the LA liquid nutrient medium with 2% inoculum size, 30 ℃ of water-baths were vibrated about 8 hours, adding the fresh LA liquid nutrient medium of equivalent induced 3 hours for 42 ℃, the results cellular lysate, the SDS-PAGE electrophoresis records expressing protein with thin layer chromatography scanner and accounts for tropina 25%, the molecular weight of protein band is 28KD, conforms to theoretical estimated value.
Five. purifying
Above-mentioned results thalline is put the ice-bath ultrasonic fragmentation, centrifugal 10000rpm10 minute, 4 ℃, inclusion body washs secondary with washing lotion, wash secondary with 0.15MPBS pH7.4 again, 37 ℃ of sex change of 8M urea 3 hours, ambient temperature overnight renaturation, the DEAE ion exchange column is crossed in dialysis back, can obtain purification degrees greater than 90% product.
Six. determination of activity
Warp 3The H thymus pyrimidine mixes the β liquid scintillation instrument and measures, 0.2ug the IL-2-Ang fusion rotein can obviously suppress external mixed lymphocyte reacion, activity in vivo detects 15ug/ days/only injects mouse, gets spleen after 5 days and detects the proliferative response of finding can obviously suppress ConA is activated splenocyte.

Claims (3)

1. novel fusion rotein, called after IL-2-Ang is characterized in that the N end is the human IL-2, the C end is angiogenin (Ang), contain one section polypeptide of forming by hydrophilic amino acid between the two, play joint, can prevent the intermolecular interference of IL-2 and Ang.
2. as the fusion rotein as described in the claim 1., it is characterized in that it prepares the method for IL-2-Ang, this method is to utilize gene fusion, and the program of genetic expression and gene engineering product purifying is produced the IL-2-Ang fusion rotein in intestinal bacteria.
3. as claim 1,2. described fusion rotein, the purposes of IL-2-Ang can be used as immunosuppressor and is used for transplant rejection, autoimmune disease, treatment of diseases such as some t cell lymphoma.
CN94117768A 1994-11-09 1994-11-09 Human interleukin-2 immunotoxin Expired - Fee Related CN1065276C (en)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1217070A1 (en) * 2000-12-22 2002-06-26 CellGenix Technologie Transfer GmbH Selective cytotoxic fusion proteins
WO2008006187A2 (en) * 2006-07-12 2008-01-17 Legeev, Yury V. Protein complexes for prevention and treatment of diseases with angiogenesis disorders
CN100365022C (en) * 2005-03-02 2008-01-30 中国人民解放军军事医学科学院军事兽医研究所 Fusion toxin protein and uses thereof
CN101518644B (en) * 2008-02-26 2011-07-13 上海交通大学医学院附属第九人民医院 Application of Ang-2 and genes thereof in pharmacy
WO2015110002A1 (en) * 2014-01-27 2015-07-30 刘根桃 Human interleukin ii mutant and application thereof

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1217070A1 (en) * 2000-12-22 2002-06-26 CellGenix Technologie Transfer GmbH Selective cytotoxic fusion proteins
WO2002052022A3 (en) * 2000-12-22 2002-10-31 Cellgenix Technologie Transfer Selective cytotoxic fusion proteins
CN100365022C (en) * 2005-03-02 2008-01-30 中国人民解放军军事医学科学院军事兽医研究所 Fusion toxin protein and uses thereof
WO2008006187A2 (en) * 2006-07-12 2008-01-17 Legeev, Yury V. Protein complexes for prevention and treatment of diseases with angiogenesis disorders
WO2008006187A3 (en) * 2006-07-12 2009-02-12 Legeev Yury V Protein complexes for prevention and treatment of diseases with angiogenesis disorders
CN101518644B (en) * 2008-02-26 2011-07-13 上海交通大学医学院附属第九人民医院 Application of Ang-2 and genes thereof in pharmacy
WO2015110002A1 (en) * 2014-01-27 2015-07-30 刘根桃 Human interleukin ii mutant and application thereof

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