WO1998031820A1 - A CTLA4-Ig FUSION PROTEIN HAVING HIGH TITER - Google Patents

A CTLA4-Ig FUSION PROTEIN HAVING HIGH TITER Download PDF

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WO1998031820A1
WO1998031820A1 PCT/KR1998/000009 KR9800009W WO9831820A1 WO 1998031820 A1 WO1998031820 A1 WO 1998031820A1 KR 9800009 W KR9800009 W KR 9800009W WO 9831820 A1 WO9831820 A1 WO 9831820A1
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ctla4
fusion protein
iggl
cys
igm
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PCT/KR1998/000009
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French (fr)
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Yong-Hoon Chung
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Boryung Pharmaceutical Co., Ltd.
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Priority to AU56814/98A priority Critical patent/AU5681498A/en
Publication of WO1998031820A1 publication Critical patent/WO1998031820A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70521—CD28, CD152
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to a CTLA4-Ig fusion protein having high titer, and more particularly, to a fusion protein connecting an extracellular region of CTLA4 and C ⁇ of IgM or C ⁇ 1 region of IgG.
  • T-cell plays an important role.
  • the reaction of T-cell starts with two kinds of signals, an antigen- sensitive stimulatory and a costimulatory signals.
  • a large number of ligand/receptor bonds including ICAM-l/LFA-1, B7/CD28 and CTLA4 and LFA-3/CD2 participate in the costimulatoion.
  • CD28 plays an important role in the reaction of the T-cell, making stable mRNA of a T-cell cytokinin by binding to the B7.1 and B7.2(June, C. H. et al, Mol.
  • interleukin-2 interleukin-2
  • IFN- ⁇ interferon- ⁇
  • TNF- a tumor necrosis factor- a
  • GM-CSF granulocyte macrophage-colony stimulating factor
  • IL-3 interleukin-3
  • CTLA4 has 67% homology with the CD28, binding to the B7(B7.1 and B7.2) of an antigen presenting cell(APC) like CD28.
  • APC antigen presenting cell
  • Yamada et al. recently reported that they manufactured a pentameric CTLA4-IgM fusion protein and that the protein extended lives of patients after the organ transplant (Yamada, A. et al, Microbio. Immunol, 40, 513-518, 1996)
  • CTLA4-Ig fusion protein since its too much dosage of 600 mg per once for a 60 kg adult and high manufacturing cost, is hardly commercially viable.
  • a CTLA4-Ig fusion protein in which an extracellular region is connected with CH 2 , CH 3 , and CH 4 region of IgM, or with a hinge, CH 2 , and CH 3 of IgGl Cys 30 8(IgGl having Cys 3 os), and which has a hexameric structure.
  • the hexameric structure of the CTLA4-Ig fusion protein is caused by forming multimer between adjoining IgMs or between IgGl Cys 3 osS forced by disulfide bonds of cy steins.
  • Cys 4 j and Cys 5 67 ⁇ f the IgM make a disulfide bond and, in case of IgGl Cys 3 osS of IgG Is make a disulfide bond.
  • the IgGl Cys 308 ⁇ f the present invention is the one that Leu 3 os of the IgGl CH 2 region, the correspondent site of Cys ⁇ of IgM, is converted to cystein in order to form polymeric IgGl like IgM.
  • DNA base sequence coding amino acid sequence correspondent to the CTLA4-Ig fusion protein is provided.
  • the expression vectors pHIGH3neo and pHIGHgpt manufactured by inserting to vectors of pSV2neo and pSV2gpt an enhancer, a promoter, CTLA4 leader sequence of which N-terminal is cut, and DNA sequence coding amino acid sequence correspondent to the CTLA4-Ig fusion protein.
  • the CTLA4 leader sequence of which N- terminal is cut makes the CTLA-Ig fusion protein secreted to the outside of cell.
  • a transformed body manufactured by inserting to a mouse SP2/0-Agl4 cell the expression vectors pHIGH3neo and pHIGH3gpt which is manufactured by inserting to the vectors pSV2neo and pSV2gpt an enhancer, a promoter, CTAL4 leader sequence of which N-terminal is cut, and the DNA sequence coding amino acid sequence correspondent to the CTLA4-Ig fusion protein.
  • an immunosuppressant containing the CTLA4-Ig fusion protein.
  • the CTLA4-Ig fusion protein of the present invention a soluble protein, binds to the B7 of the antigen presenting cell to inhibit binding of the CTLA4 and the CD28 of T-cell at the B7, to block costimulatory signal needed for the activation of T-cell and, in the result, the immunoreaction is suppressed.
  • the titer of the CTLA4-Ig fusion protein according to the present invention is 32-356 times of an existing CTLA4-Ig fusion protein.
  • the dosage of the CTLA4-Ig fusion protein according to the present invention is 2—13 mg per once for a 60 kg adult, and it's effective titer is 45-260 times of the existing CTLA4-Ig fusion protein's.
  • Fig.l is a structure of a CTLA4 gene cloned by a reverse transcription-polymerase chain reaction(RT-PCR) of example 1.
  • Fig.2 is a expression ratio of a fusion protein of example 2.
  • Fig. 3a, 3b are base sequences of a CTLA4-IgM fusion gene of example 2 and an correspondent amino acid sequence thereof.
  • Fig. 4a, 4b are base sequence of a CTLA4-IgGl Cys 3 o 8 fusion gene of example 3 and a correspondent amino acid sequence thereof.
  • Fig. 5a, 5b are a manufacturing method for the expression vectors of p QGH3neo and pFQGH3gpt of the CTLA4-IgM fusion gene and the CTLA4-IgGl Cys 3 o8 fusion gene.
  • Fig. 6a, 6b are western blots of the CTLA4-IgM fusion protein and the CTLA4-IgGl Cys 30 8 fusion protein.
  • Fig. 7 is a structure of 600kD of the CTLA4-IgM fusion protein or the CTLA4-IgG Cys 30 8 fusion protein.
  • Fig. 8 is a graph showing the immunosuppression effect of the CTLA4-IgM fusion protein and the CTLA4-IgGl Cys 30 s fusion protein.
  • Example 1 Cloning of human CTLA4, IgGl, and IgM genes
  • CTLA4, IgGl , and IgM genes were cloned respectively by the method of a reverse transcription-polymerase chain reaction(RT-PCR).
  • RT-PCR reverse transcription-polymerase chain reaction
  • the polymerase used in the reverse transcription-polymerase chain reaction was pfu(Stratagene Corp.).
  • the primers used in the reverse transcription-polymerase chain reaction are five forward primers(Ll ⁇ 5) and a reverseward primer, as follows;
  • CTLA4 without cutting, for the L2 primer as a form that 6 amino acids of it were cut from N-terminal, 11 amino acids cut for the L3, 16 amino acids cut for the L4, and 22 amino acids cut for the L5 from N-terminal
  • Inventing the forward primers to be expressed as cutting form of amino acids from N-terminal is for a part of leader sequence to be cut and expressed , and for the CTLA4 protein to be secreted to an extracellular region.
  • 5 primers were invented in order that the leader sequence is cut and expressed one by one for the determination of a leader sequence which makes the most CTLA4 proteins secreted to extracellular region.
  • CTLA4 gene obtained by the reverse transcription-polymerase reaction was cloned to pUC 18.
  • the cloned CTLA4 gene has confirmed which base No.49 was converted from adenine to guanine, and base No.331 was converted from guanine to adenine.
  • an amino acid No.17 of CTLA4 protein was converted from threonine to alanine
  • an amino acid No.111 of CTLA4 protein was converted from alanine to threonine. 2.
  • the cloning method was same with the method of the above 1 of the example 1 except template and primer.
  • the template used here was mRNA of B-cell at peripheral blood lymph node obtained from a recovering ill-defined fever patient.
  • the primer was invented in order to clone a counterbalancing of IgGl as follows;
  • the primer was invented in order to clone a counterbalancing of the IgM as follows;
  • the five CTLA4 genes obtained by serial deletion of N-terminal amino acids were fused with IgGl respectively, inserted to a vector pHIGH3, and transfected to a mouse bone marrow SP2/0-Agl4 cell(ATCC#: CRL 1581) to be expressed. And after an incubation for 48 hours, the expression ratio was analyzed by a cell circulation assay.
  • IgGl Cys 3 o8 was manufactured by converting Leu 3 08 of IgGl to cysteine using a polymerase chain reaction.
  • the primers used in the polymerase chain reaction are as follows;
  • the primary polymerase chain reaction using the forward primer and reverseward primer was performed, and then using the product of the above reaction and reverseward primer, secondary polymerase chain reaction was performed.
  • the amplified product of the secondary polymerase chain reaction was cloned in pUC 18 vector.
  • Genome DNA of SP2/0-Agl4 cell was extracted, cut with restriction enzymes of BamH I and Hind HI, transferred to a nitrocellulose membrane, and performed Southern blot with 5 -ATT TGC ATA TTT GCA TAT TTG CAT-3 ' fragment and 5 -CTC ATG ACT CAT GAC TCA-3 fragment marked with isotope to clone 5.3kb promoter.
  • genome DNA of SP2/0-Agl4 cell was cut by restriction enzymes of EcoR I and BamH I and performed the southern blot with 5 -TGA ATT GAG CAA TGT TGA ATT GAG CAA TGT-3' fragment and 5 -TAT TTG GGG AAG GGT ATT TGG GGA AGG-3 ' fragment marked with isotope to clone lkb enhancer.
  • Ig fusion gene was cloned to pUC 18 by fusing the lkb enhancer and 5.3 kb promoter in pUC 19, and inserting the fused product to the site of Sal I, the front part of CTLA4- Ig fusion gene cloned in pUC 18(CTLA4-
  • Example 5 Expression of CTLA4-Ig fusion gene and purification of CTLA4-Ig fusion protein
  • SP2/0-Agl4 cell of mouse was incubated in 10%> FCS-DMEM medium, and diluted to 5X10 6 ceWslmi by adding PBS.
  • the above suspension 0.2ml was put to cuvette(BioRad Corp.) for electroporation and the purified expression vector 15 g of the CTLA4-Ig fusion gene of example 4 was added. And then electroporation (BT 820) was performed under the condition of 480V, 99 ⁇ sec, 2cycle.
  • the above cells were incubated for 3 weeks in the FCS-DMEM medium containing 1500 g/m# of geneticin G418(Gibco Corp.). And then colonies were separated, collected, and incubated for amplifying.
  • the CTLA4-Ig fusion gene expression was examined by the a cell circulation analyzer and enzyme linked immunosorbent assay (ELI S A) method.
  • CTLA4-Ig fusion protein was purified.
  • Ig fusion protein of 600kD is 6 times as large as the existing CTL
  • Ig fusion protein(lOOkD) is a hexamer which was six of CTL
  • the existing CTLA4-Ig fusion protein is a comparative example 1
  • the pentameric CTLA4-Ig fusion protein is a comparative example 2
  • the hexameric CTLA4-Ig fusion protein is an example, and the Immunosuppression effects of them were examined as follows;
  • peripheral blood lymphocytes were separated, and on the cells of the one person 300 rad of 60 Co radiation was irradiated.
  • the cells of the two persons were spread into a 96-well plate with
  • the incubated cells were adsorbed to a glass filter by using titertek(Flow lab), put into a test tube, and after adding 5 ⁇ & of Scintillation cocktail a radioactivity was measured by using ⁇ -liquid scintillation counter. The all tests were performed three for every times under the same condition and an average of them was determined.
  • the percent value gained by adding the fusion protein of the present invention was calculated on the basis of the radiation value(100%>) gained without an addition. And when the value reaches to 50%>, the value was defined as a line of 50% division suppression and the titer between fusion proteins was compared on the basis of the concentration of the adding fusion protein.
  • the 50%> division suppression concentration of the CTLA4-Ig fusion protein of this example is 0.009-0.022 gM(the average is 0.016 ⁇ g Imi). This value is lower than 0.7-3.2 g/m£(the average is 1.4 ⁇ g/ l) of the comparative example 1 and lower than 0.031-0.056 ⁇ glml (the average is 0.44 ⁇ g/ml) the comparative example 2 (Fig.8).
  • CTLA4-Ig fusion protein of this example has high titer, 32-356 times (the average is 88 times) comparing to the existing CTLA4-Ig fusion protein of the comparative example 1 .

Abstract

The present invention relates to a CTLA4-Ig fusion protein, in which an extracellular region of the CTLA4 is connected to CH2, CH3, and CH4 of IgM or to hinge, CH2 and CH3 of IgG1 Cys308, and six monomers of which are polymerized to be a hexameric structure. According to the present invention, it is provided a CTLA4-Ig fusion protein having a decreased dosage and high titer.

Description

A CTLA-4 Ig FUSION PROTEIN HAVING HIGH TITER
Technical Field
The present invention relates to a CTLA4-Ig fusion protein having high titer, and more particularly, to a fusion protein connecting an extracellular region of CTLA4 and C μ of IgM or C γ 1 region of IgG.
Background Art
In organ transplant, fatal to a patient is the rejection by immunoreaction which occurs by discriminating self and non-self.
In the rejection of the organ transplant, T-cell plays an important role. The reaction of T-cell starts with two kinds of signals, an antigen- sensitive stimulatory and a costimulatory signals. A large number of ligand/receptor bonds including ICAM-l/LFA-1, B7/CD28 and CTLA4 and LFA-3/CD2 participate in the costimulatoion. Especially, CD28 plays an important role in the reaction of the T-cell, making stable mRNA of a T-cell cytokinin by binding to the B7.1 and B7.2(June, C. H. et al, Mol. Cell Biol., 7, 4472, 1987/Lindstent, et al, Science, 244, 339, 1989), and increasing the productivity of interleukin-2(IL-2), interferon- γ (IFN- γ ), tumor necrosis factor- a (TNF- a ), lymphotoxin, granulocyte macrophage-colony stimulating factor(GM-CSF), and interleukin-3(IL-
3).
Thus if the costimulation by the CD28 is blocked by inhibiting binding of the CD28 and the B7.1 and B7.2, the rejection of the organ transplant can be suppressed.
CTLA4 has 67% homology with the CD28, binding to the B7(B7.1 and B7.2) of an antigen presenting cell(APC) like CD28. Linsley et al. reported that a monomeric CTLA4-Ig fusion protein was prepared by fusing the CTLA4 and an IgG, and that the protein has the immunosuppression effect (Linsley, P. S. et al, J. Exp. Med. 174, 561,
1991). Yamada et al. recently reported that they manufactured a pentameric CTLA4-IgM fusion protein and that the protein extended lives of patients after the organ transplant (Yamada, A. et al, Microbio. Immunol, 40, 513-518, 1996)
However, the CTLA4-Ig fusion protein, since its too much dosage of 600 mg per once for a 60 kg adult and high manufacturing cost, is hardly commercially viable.
Disclosure of the Invention
According to one aspect of the present invention, there is provided a CTLA4-Ig fusion protein in which an extracellular region is connected with CH2, CH3, and CH4 region of IgM, or with a hinge, CH2, and CH3 of IgGl Cys308(IgGl having Cys3os), and which has a hexameric structure.
The hexameric structure of the CTLA4-Ig fusion protein is caused by forming multimer between adjoining IgMs or between IgGl Cys3osS forced by disulfide bonds of cy steins. To put it concretely, Cys4j and Cys567 θf the IgM make a disulfide bond and, in case of IgGl Cys3osS of IgG Is make a disulfide bond. The IgGl Cys308 θf the present invention is the one that Leu3os of the IgGl CH2 region, the correspondent site of Cys ι of IgM, is converted to cystein in order to form polymeric IgGl like IgM.
According to another aspect of the present invention, there is provided DNA base sequence coding amino acid sequence correspondent to the CTLA4-Ig fusion protein.
According to still another aspect of the present invention, there is provided the expression vectors pHIGH3neo and pHIGHgpt manufactured by inserting to vectors of pSV2neo and pSV2gpt an enhancer, a promoter, CTLA4 leader sequence of which N-terminal is cut, and DNA sequence coding amino acid sequence correspondent to the CTLA4-Ig fusion protein. The CTLA4 leader sequence of which N- terminal is cut makes the CTLA-Ig fusion protein secreted to the outside of cell. According to still another aspect to the present invention, there is provided a transformed body manufactured by inserting to a mouse SP2/0-Agl4 cell the expression vectors pHIGH3neo and pHIGH3gpt which is manufactured by inserting to the vectors pSV2neo and pSV2gpt an enhancer, a promoter, CTAL4 leader sequence of which N-terminal is cut, and the DNA sequence coding amino acid sequence correspondent to the CTLA4-Ig fusion protein.
According to still another aspect of the present invention, there is provided an immunosuppressant containing the CTLA4-Ig fusion protein. The CTLA4-Ig fusion protein of the present invention, a soluble protein, binds to the B7 of the antigen presenting cell to inhibit binding of the CTLA4 and the CD28 of T-cell at the B7, to block costimulatory signal needed for the activation of T-cell and, in the result, the immunoreaction is suppressed. By the features of the present invention, the titer of the CTLA4-Ig fusion protein according to the present invention is 32-356 times of an existing CTLA4-Ig fusion protein. The dosage of the CTLA4-Ig fusion protein according to the present invention is 2—13 mg per once for a 60 kg adult, and it's effective titer is 45-260 times of the existing CTLA4-Ig fusion protein's.
Brief Description of the Drawings
The above objects, and other features and advantages of the present invention will become more apparent after a reading of the following detailed description taken in conjunction with the drawings, in witch:
Fig.l is a structure of a CTLA4 gene cloned by a reverse transcription-polymerase chain reaction(RT-PCR) of example 1. Fig.2 is a expression ratio of a fusion protein of example 2. Fig. 3a, 3b are base sequences of a CTLA4-IgM fusion gene of example 2 and an correspondent amino acid sequence thereof.
Fig. 4a, 4b are base sequence of a CTLA4-IgGl Cys3o8 fusion gene of example 3 and a correspondent amino acid sequence thereof. Fig. 5a, 5b are a manufacturing method for the expression vectors of p QGH3neo and pFQGH3gpt of the CTLA4-IgM fusion gene and the CTLA4-IgGl Cys3o8 fusion gene.
Fig. 6a, 6b are western blots of the CTLA4-IgM fusion protein and the CTLA4-IgGl Cys308 fusion protein.
Fig. 7 is a structure of 600kD of the CTLA4-IgM fusion protein or the CTLA4-IgG Cys308 fusion protein.
Fig. 8 is a graph showing the immunosuppression effect of the CTLA4-IgM fusion protein and the CTLA4-IgGl Cys30s fusion protein.
Best Mode for Carrying out the Invention
The present invention is further illustrated in the following example, which should not be taken to limit the scope of the invention.
Example 1: Cloning of human CTLA4, IgGl, and IgM genes
CTLA4, IgGl , and IgM genes were cloned respectively by the method of a reverse transcription-polymerase chain reaction(RT-PCR). 1. Cloning of the CTLA4 gene A template used for the cloning of the CTLA4 gene by the reverse transcription-polymerase chain reaction was mononucleocyte mRNA of a healthy adult. The mRNA was separated as follows:
Blood taken from a healthy adult was density-gradient centrifuged using Ficoll-Hypaque to obtain monocyte cell layer. By adding RPMI- 1640 medium containing 10% bovine fetus to the above monocytes 5X105 monocytes/ml was made and here leukoagglutinin(Pharmacia Corp.) added to be 3.5 /zg/ml. The mixture was incubated 36-48 hours under the condition of 5% C02, 37 °C in order to separate mRNA.
The polymerase used in the reverse transcription-polymerase chain reaction was pfu(Stratagene Corp.).
The primers used in the reverse transcription-polymerase chain reaction are five forward primers(Ll~5) and a reverseward primer, as follows;
Forward primers
LI 5 -ATG GCT TGC CTT GGA TTT CAG-3' L2 5 -ATG CGG CAC AAG GCT CAG CTG AAC-3'
L3 5 -ATG CAG CTG AAC CTG GCT GCC AGG-3' L4 5 -ATG AGG ACC TGG CCC TGC ACT CTC-3' L5 5 -ATG CTC CTG TTT TTT CTT CTC TTC-3' Reverseward primer 5 -CTC TGC AGA ATC TGG GCA CGG TTC AGG ATC-3'
It is invented for the LI primer to be expressed as an original
CTLA4 without cutting, for the L2 primer as a form that 6 amino acids of it were cut from N-terminal, 11 amino acids cut for the L3, 16 amino acids cut for the L4, and 22 amino acids cut for the L5 from N-terminal
(Fig.l).
Inventing the forward primers to be expressed as cutting form of amino acids from N-terminal is for a part of leader sequence to be cut and expressed , and for the CTLA4 protein to be secreted to an extracellular region. And 5 primers were invented in order that the leader sequence is cut and expressed one by one for the determination of a leader sequence which makes the most CTLA4 proteins secreted to extracellular region.
CTLA4 gene obtained by the reverse transcription-polymerase reaction was cloned to pUC 18. The cloned CTLA4 gene has confirmed which base No.49 was converted from adenine to guanine, and base No.331 was converted from guanine to adenine. In the result, an amino acid No.17 of CTLA4 protein was converted from threonine to alanine, and an amino acid No.111 of CTLA4 protein was converted from alanine to threonine. 2. Cloning of IgGl gene
The cloning method was same with the method of the above 1 of the example 1 except template and primer. The template used here was mRNA of B-cell at peripheral blood lymph node obtained from a recovering ill-defined fever patient. The primer was invented in order to clone a counterbalancing of IgGl as follows;
Forward primer
5 -A TCT GCA GAG CCC AAA TCT TGT GAC-3' Reverseward primer 5 -TT CTC GAG TCA TTT ACC CGG AGA CAG GGA-3
3. Cloning of IgM gene
Same with the method of the above 2 of the example 1 except primer. The primer was invented in order to clone a counterbalancing of the IgM as follows;
Forward primer
5-GAC TGC AGA GCT GCC TCC CAA AGT G-3'
Reverseward primer
5-GTA GCA GGT GCC AGC TGT GTC TGA-3
Example 2: Determination of the optimum leader sequence for extracellular secretion
The five CTLA4 genes obtained by serial deletion of N-terminal amino acids were fused with IgGl respectively, inserted to a vector pHIGH3, and transfected to a mouse bone marrow SP2/0-Agl4 cell(ATCC#: CRL 1581) to be expressed. And after an incubation for 48 hours, the expression ratio was analyzed by a cell circulation assay.
The result of the above analysis shows that in case of L 1 primer 4.9%) of the fusion protein, 3.1%» for L2 primer, 0% for L3 primer, 7.8% for L4 primer and 6%> for L5 primer are expressed (Fig.2). It confirms that the leader sequence deleted of 16 amino acids from N-terminal, obtained by using L-4 primer, makes the most fusion proteins secreted most to an extracellular region.
Example 3: Manufacturing of IgGl Cys3os
IgGl Cys3o8 was manufactured by converting Leu308 of IgGl to cysteine using a polymerase chain reaction. The primers used in the polymerase chain reaction are as follows;
Forward primer
5 -A TCT GCA GAG CCC AAA TCT TGT GAC-3' Reverseward primer
5 -TT CTC GAG TCA TTT ACC CGG AGA CAG GGA-3'
Converting primer
5-CCAGTC CTG GTGACA GAC GGT GAG GAC-3'
First, the primary polymerase chain reaction using the forward primer and reverseward primer was performed, and then using the product of the above reaction and reverseward primer, secondary polymerase chain reaction was performed. The amplified product of the secondary polymerase chain reaction was cloned in pUC 18 vector.
Example 4: Construction of the expression vector of the CTLA4-Ig fusion gene
Genome DNA of SP2/0-Agl4 cell was extracted, cut with restriction enzymes of BamH I and Hind HI, transferred to a nitrocellulose membrane, and performed Southern blot with 5 -ATT TGC ATA TTT GCA TAT TTG CAT-3' fragment and 5 -CTC ATG ACT CAT GAC TCA-3 fragment marked with isotope to clone 5.3kb promoter.
On the other hand genome DNA of SP2/0-Agl4 cell was cut by restriction enzymes of EcoR I and BamH I and performed the southern blot with 5 -TGA ATT GAG CAA TGT TGA ATT GAG CAA TGT-3' fragment and 5 -TAT TTG GGG AAG GGT ATT TGG GGA AGG-3' fragment marked with isotope to clone lkb enhancer. An enhancer-promoter-CTLA4-
Ig fusion gene was cloned to pUC 18 by fusing the lkb enhancer and 5.3 kb promoter in pUC 19, and inserting the fused product to the site of Sal I, the front part of CTLA4- Ig fusion gene cloned in pUC 18(CTLA4-
IgM fusion gene of the example 2 and CTLA4-
IgGl Cys3o8 fusion gene of the example 3, Fig.3a,3b and Fig.4a, 4b
). By cutting only enhancer-promoter-CTLA4-
Ig fusion gene by treating EcoR I and Hind III to the above clone and then by inserting the clone to pSV2neo and pSV2gpt, the exp ression vectors of pHIGH3neo and pHIGH3gpt was constructed (Fig
. 5a, 5b).
Example 5: Expression of CTLA4-Ig fusion gene and purification of CTLA4-Ig fusion protein
SP2/0-Agl4 cell of mouse was incubated in 10%> FCS-DMEM medium, and diluted to 5X106 ceWslmi by adding PBS. The above suspension 0.2ml was put to cuvette(BioRad Corp.) for electroporation and the purified expression vector 15 g of the CTLA4-Ig fusion gene of example 4 was added. And then electroporation (BT 820) was performed under the condition of 480V, 99 μ sec, 2cycle.
The above cells were incubated for 3 weeks in the FCS-DMEM medium containing 1500 g/m# of geneticin G418(Gibco Corp.). And then colonies were separated, collected, and incubated for amplifying. The CTLA4-Ig fusion gene expression was examined by the a cell circulation analyzer and enzyme linked immunosorbent assay (ELI S A) method.
These cells were incubated in large quantities in FCS-medium and the CTLA4-Ig fusion protein was precipitated by ammonium sulfate addition. And then by an affinity chromatography using protein A, the
CTLA4-Ig fusion protein was purified.
In order to fine out the biochemical properties of the CTLA4-
Ig fusion protein, electrophoresis and western blot were performed(F ig. 6a, 6b). The result shows that there are two kinds of the CTL
A4-IgM fusion protein and six kinds of the CTLA4-
IgGl fusion protein. Among these, CTLA4-
Ig fusion protein of 600kD was separated and purified. The CTLA4
Ig fusion protein of 600kD is 6 times as large as the existing CTL
A4-
Ig fusion protein(lOOkD), and is a hexamer which was six of CTL
A4-Ig fusion protein polymerized (Fig.7).
Example 6: Immunosuppression effect of the CTLA4-Ig fusion protein
The existing CTLA4-Ig fusion protein is a comparative example 1 , the pentameric CTLA4-Ig fusion protein is a comparative example 2, and the hexameric CTLA4-Ig fusion protein is an example, and the Immunosuppression effects of them were examined as follows;
From two healthy adults peripheral blood lymphocytes were separated, and on the cells of the one person 300 rad of 60Co radiation was irradiated. The cells of the two persons were spread into a 96-well plate with
2.5X104 cells/ml, respectively. And after incubating for 88-96 hours under the condition of 37 °C , 5% C02, added 0.5 μ C\ 3H- thymidine(NEN Research product) per well and incubated 5 hours again.
The incubated cells were adsorbed to a glass filter by using titertek(Flow lab), put into a test tube, and after adding 5μ& of Scintillation cocktail a radioactivity was measured by using β -liquid scintillation counter. The all tests were performed three for every times under the same condition and an average of them was determined. The percent value gained by adding the fusion protein of the present invention was calculated on the basis of the radiation value(100%>) gained without an addition. And when the value reaches to 50%>, the value was defined as a line of 50% division suppression and the titer between fusion proteins was compared on the basis of the concentration of the adding fusion protein.
As a result, the 50%> division suppression concentration of the CTLA4-Ig fusion protein of this example is 0.009-0.022 gM(the average is 0.016 μg Imi). This value is lower than 0.7-3.2 g/m£(the average is 1.4 βg/ l) of the comparative example 1 and lower than 0.031-0.056 μglml (the average is 0.44 βg/ml) the comparative example 2 (Fig.8). CTLA4-Ig fusion protein of this example has high titer, 32-356 times (the average is 88 times) comparing to the existing CTLA4-Ig fusion protein of the comparative example 1 .

Claims

1. A CTLA4-IgM fusion protein, wherein an extracellular region of a CTLA4 is connected with CH2, CH3, and CH4 of IgM, and which has a hexameric structure by polymerization of 6 monomers thereof.
2. A DNA sequence of Fig.4a, 4b coding the amino acid sequence corresponding to the CTLA4- IgM fusion protein as claimed in Claim 1.
3. A expression vector pFQGH3neo which is constructed by connecting an enhancer, a promoter and a CTLA4 of which N-terminal is cut, and DNA sequence coding amino acid sequence correspondent to the CTLA4-IgM fusion protein of Claim 1 , and then by inserting the DNA sequence into vector pSV2neo.
4. A expression vector pHIGH3neo as claimed in Claim 3, wherein 16 amino acids of a leader sequence are cut from N-terminal.
5. A transformed body manufactured by inserting to a mouse
SP2/0-Agl4 the expression vector pFGGH3neo constructed by connecting an enhancer, a promoter and a CTLA4 of which N-terminal is cut, and DNA sequences coding amino acid sequence correspondent to the CTLA4-IgM fusion protein of Claim 1 , and then by inserting them into vector pSV2neo.
6. An immunosuppression medicine containing the CTLA4-IgM fusion protein in Claim 1.
7. A CTLA4-IgGl Cys3os fusion protein, wherein the extracellular region of CTLA4 is connected with a hinge, CH2 and CH3 of the IgGl Cys308 (IgGl having Cys3os) and which has a hexameric structure by polymerization of 6 fusion protein monomers thereof.
8. A DNA sequence of Fig.3a, 3b coding amino acid sequence c orrespondent to the CTLA4-IgGl Cys308 fusion protein in Claim 7.
9. An expression vector pHIGH3neo which is constructed by connecting an enhancer, a promoter, and CTLA4 of which N-terminal is cut, and DNA sequence coding amino acid sequences correspondent to the CTLA4-IgGl Cys308 fusion protein in Claim 7, and then by inserting them into a vector pSV2neo.
10. An expression vector pHIGH3neo as claimed in Claim 9, which 16 amino acids of a leader sequence are cut from N-terminal.
11. A transformed body which is manufactured by inserting to a mouse SP2/0-Agl4 cell the expression vector pHIGH3neo constructed by connecting an enhancer, a promoter, and CTLA4 of which N-terminal is cut, and DNA sequence coding amino acid sequences correspondent to the CTLA4-IgGl Cys3os fusion protein in Claim 7, and then by inserting them into a vector pSV2neo.
12. An immunosuppressant containing the CTLA4-IgGl Cys3os fusion pr otein in Claim 7.
PCT/KR1998/000009 1997-01-18 1998-01-19 A CTLA4-Ig FUSION PROTEIN HAVING HIGH TITER WO1998031820A1 (en)

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