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WO1994009812A1 - PROCEDE DE PRODUCTION DE COMPLEXES DE GRANDS FACTEURS β TRANSFORMANTS DE CROISSANCE LATENTS ET GRAND PEPTIDE DE LATENCE ASSOCIE - Google Patents

PROCEDE DE PRODUCTION DE COMPLEXES DE GRANDS FACTEURS β TRANSFORMANTS DE CROISSANCE LATENTS ET GRAND PEPTIDE DE LATENCE ASSOCIE Download PDF

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WO1994009812A1
WO1994009812A1 PCT/US1993/010230 US9310230W WO9409812A1 WO 1994009812 A1 WO1994009812 A1 WO 1994009812A1 US 9310230 W US9310230 W US 9310230W WO 9409812 A1 WO9409812 A1 WO 9409812A1
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tgf
ltbp
cell
codes
eukaryotic
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PCT/US1993/010230
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English (en)
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Carl-Henrik Heldin
Kohei Miyazono
Pascal Colosetti
Ulf Helmann
Yasuyuki Ishii
Hideya Ohashi
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Kirin Brewery Company, Limited
Ludwig Institute For Cancer Research
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Priority to AU55870/94A priority Critical patent/AU5587094A/en
Publication of WO1994009812A1 publication Critical patent/WO1994009812A1/fr

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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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—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
    • 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
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/495—Transforming growth factor [TGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates to transforming growth factor- ⁇ (TGF- ⁇ ) .
  • TGF- ⁇ transforming growth factor- ⁇
  • the invention relates to the expression of large latent TGF- ⁇ complexes in eukaryotic cells, such as Chinese Hamster Ovary (CHO) cells.
  • eukaryotic cells such as Chinese Hamster Ovary (CHO) cells.
  • LL-TGF- ⁇ large latent TGF- ⁇
  • the invention further relates to a method for isolating large latency associated peptide (L-LAP) from rLL-TGF- ⁇ as described herein.
  • Transforming growth factor- ⁇ s are a family of proteins with potent cellular modulating activities on many types of cells. See, e.g., Roberts and Sporn, Peptide Growth Factors and Their Receptors I, 419-472, 1990. Three human isoforms of TGF- ⁇ s have been identified and characterized, TGF- ⁇ l, - ⁇ 2, - ⁇ 3. TGF- ⁇ l was first identified as a growth factor which stimulated some rodent fibroblasts to grow in semi-solid agar. It is becoming clear, however, that TGF- ⁇ l is also a potent growth inhibitor for many different cell types, a modulator of cellular differentiation, and an inducer of extracellular matrix production and deposition.
  • TGF- ⁇ l is produced by a wide variety of normal and malignant cells as a latent complex of high molecular weight.
  • the structure of latent TGF- ⁇ l has been determined after purification from human platelets, Miyazono et al., J. Biol. Chem. 263, 6407-6415, 1988; Wakefield et al. , J. Biol. Chem. 263, 7646-7654, 1988, and rat platelets, Okada et al., J. Biochem. 106, 304-310, 1989. Its biosynthesis has been characterized by using a human erythroleukemia cell line, Miyazono et al., EMBO J. 10, 1091-1101, 1991.
  • the latent form of TGF- ⁇ l consists of three distinct components: 1) mature TGF- ⁇ l; 2) an N-terminal remnant of the TGF- ⁇ l precursor; and 3) the latent TGF- ⁇ l binding protein (hereinafter referred to as "LTBP") .
  • the N-terminal remnant of the TGF- ⁇ l precursor is important for TGF- ⁇ l latency, so it has been denoted TGF- ⁇ l latency associated peptide ( ⁇ l-LAP).
  • TGF- ⁇ l latency associated peptide ⁇ l-LAP
  • the large latency associated peptide (rL- ⁇ l-LAP) as designated herein, consists of two components; the TGF- ⁇ l latency associated peptide ( ⁇ l-LAP) and LTBP.
  • Mature TGF- ⁇ l is a disulfide-bonded dimer which has been proteolytically cleaved from ⁇ l-LAP, which forms a disulfide-bonded dimer linked to a single molecule of LTBP.
  • the latent TGF- ⁇ l complex which includes LTBP is referred to as the "large latent complex (LL-TGF- ⁇ l) " whereas the complex without LTBP is the "small latent complex”.
  • LTBP was first purified from human platelets as a free form and as a component of the LL-TGF- ⁇ l complex.
  • a cDNA clone coding for LTBP was recently isolated from human foreskin fibroblasts. See U.S. Serial No. 07/487,343, U.S. Patent No. 5,177,197, the contents of which are, incorporated herein by reference in its entirety.
  • the open reading frame of the cDNA sequence predicted that LTBP is a 1394 amino acid protein containing two different types of repeat sequences; sixteen epidermal growth factor (EGF)- like repeats and three copies of a repeat sequence containing eight cysteins in one motif.
  • EGF epidermal growth factor
  • LTBP is not directly associated with TGF- ⁇ l latency, but it plays a role in the assembly and secretion of latent TGF- ⁇ l molecules by producer cells.
  • TGF- ⁇ l binding glycoproteins have been found. These molecules are characterized by molecular masses of 160 kD, 70-80 kD, and 30-40 kD as determined by SDS-PAGE and the ability to bind the TGF- ⁇ l molecule. See U.S. Serial No. 07/717,316, U.S. Patent No. 5,229,495, the contents of which are incorporated herein by reference in its entirety.
  • This invention generally provides for a method of producing recombinant large latent TGF- ⁇ by introducing DNA sequences coding for LTBP and pro-TGF- ⁇ into eukaryotic cells. These sequences are introduced to the eukaryotic cells and co-expressed.
  • co- expression and “co-expressed” mean expression of the two components of the complex, i.e., pro-TGF ⁇ and LTBP, by any of the means known to a skilled artisan. These means may include, e.g., sequential transformation using two expression vectors, or transforming a single vector capable of expressing both components.
  • This invention further provides for a method for producing recombinant large, latent TGF- ⁇ by introducing DNA sequence for LTBP into a eukaryotic cell which expresses pro-TGF- ⁇ .
  • the invention provides for the construction of a LTBP expression plasmid, pDSVE2- BP, its subsequent transfection into CHO cells, the CHO cell, T23-7-11, is particularly preferred.
  • the clone with the highest LL-TGF- ⁇ complex expression was chosen for purification (LT3-1 clone) .
  • the invention also provides for the construction of a pME-TGF- ⁇ 2 plasmid and its co-transfection with pRSVneo plasmid into BP-1-1 CHO cells which contains an amplified LTBP cDNA sequence in the genome.
  • LL-TGF-B2 activity into culture medium is assayed.
  • Also provided herein is a method for isolating L- LAP by degrading LL-TGF- ⁇ and separating L-LAP therefrom.
  • Figure 2A Detection of LL-TGF- ⁇ l secreted from cell clones transfected by plasmid pDSVE2-BP into culture medium using Ab-39 polyclonal antibody.
  • Figure 4A Analysis of purified LL-TGF- ⁇ l complex on SDS- PAGE.
  • Figure 4B Analysis of purified LL-TGF- ⁇ l complex after SDS- PAGE by immunoblotting.
  • Figure 6 Inhibition of CCL-64 cell DNA synthesis.
  • Figure 7 Analysis of recombinant LTBP eluted from C4 reversed phase column by SDS-PAGE
  • Figure 8 Detection of LL-TGF- ⁇ 2 secreted from BP-1-1 cell clones transfected with plasmid DNA containing a prepro-TGF- ⁇ 2 cDNA.
  • Figure 9 45 Ca 2+ binding to LTBP.
  • Figure 10B Effect of Ca 2+ on the susceptibility to trypsin of the recombinant LL-TGF- ⁇ l.
  • Figure 11A Effects of Ca 2+ on LTBP anion exchange chromatography on LTBP in the presence of 5mM EDTA.
  • pDSVE2-BP An expression plasmid for LTBP, named pDSVE2-BP, was constructed utilizing the standard recombinant methods such as described in Maniatis et al., Molecular Cloning: A Laboratory Manual, 1982. Specifically, the plasmid pDSVE2- BP was constructed as follows: pUC19-A and pUC19-B are plasmids carrying the 5'-half EcoRI fragment of an LTBP cDNA with the 5' untranslated region (Kanzaki et al., Cell 61, 1051-1061, 1990) and the 3'-half EcoRI fragment of the LTBP cDNA with the 3' untranslated region, respectively.
  • Dral-EcoRI fragment carrying the 5' half of LTBP cDNA was isolated from pUC19-A and subcloned into the Pstl- EcoRI site of SR ⁇ -296 plasmid (Takabe et al., Mol. Cell. Biol. 8, 466-469, 1988). The 3'-half EcoRI fragment was then isolated from pUC19-B and introduced into the EcoRI site of this SR ⁇ -296 plasmid to construct SR ⁇ -BP, a plasmid carrying the full-length cDNA of LTBP.
  • LTBP cDNA was isolated from SR ⁇ -BP by Xhol cleavage and inserted into the Sail site of a mammalian expression plasmid pDSVE2 containing a mouse dihydrofolate reductase (dhfr) minigene for gene amplification.
  • pDSVE2-BP mouse dihydrofolate reductase
  • T23-7-11 is a CHO cell line which produces a large amount of recombinant human pro-TGF- ⁇ l complex.
  • the construction and the properties of this cell line is described in detail in a laid-open Japanese patent application (KOKAI 3-180192, 1981) which is hereby incorporated by reference.
  • the essential elements describing the construction of T23-7-11 cell line, however, are briefly described below.
  • Human prepro-TGF- ⁇ l cDNA clone was isolated from a ⁇ gtlO cDNA library of human placenta cells. Total RNA was prepared from human placental tissue and poly(A) RNA was isolated by oligo(dT)- cellulose column chromatography using methods well known in the art. Double-stranded cDNA was synthesized by the method of Gluber and Hoffmann (Gluber et al.. Gene 25, 263, 1983) and cloned into the EcoRI site of a ⁇ gtlO vector. An oligo DNA probe was synthesized according to the published nucleotide sequence of human TGF- ⁇ l cDNA (Derynck et al.
  • Clones carrying the cDNA sequence for human prepro-TGF- ⁇ l protein were screened from the human placenta library using this probe. Using plaque hybridization and DNA sequencing, one cDNA clone, Tl, carrying a full-length cDNA sequence for human prepro-TGF- ⁇ l was identified from 3 x 10 5 independent plaques. This cDNA clone was then subcloned into the EcoRI site of pUC19.
  • a mammalian expression plasmid for human prepro-TGF- ⁇ l cDNA, pEC ⁇ was constructed as follows. First, the pUC-19 plasmid carrying the human prepro-TGF- ⁇ l cDNA was digested with EcoRI. An isolated EcoRI fragment containing the cDNA was made blunt-ended and then inserted into the Smal site of vector pSVL to obtain pSVL-TGF- ⁇ l. Second, the mammalian expression plasmid CDM8 (Seed, B., Nature 329, 840-842, 1987) was digested with SacII and BamHI.
  • the isolated SacII-BamHI fragment containing an amber suppressor supF tRNA gene, the cytomegalovirus (CMV) immediate early promoter DNA sequence and the Simian Virus 40 (SV40) polyadenylation DNA sequence was made blunt-ended and then introduced into the PvuII site of vector pSV2dhfr (Subramani, S. et al, Mol. Cell. Biol. 1, 854-864, 1981) to obtain pCMV-dhfr.
  • CMV cytomegalovirus
  • SV40 Simian Virus 40
  • the Xbal-EcoRI fragment of pSVL-TGF- ⁇ l (containing the human prepro-TGF- ⁇ l cDNA, the SV40 polyadenylation sequence, an ampicillin-resistant gene and the ori derived from pBR322 DNA) and the Xbal-EcoRI fragment of pCMV-dhfr (containing the CMV promoter sequence and a gene cassette consisting of the SV40 early promoter, mouse dihydrofolate reductase (dhfr) coding sequence and the SV40 polyadenylation sequence) were ligated together.
  • the resulting expression plasmid, pEC ⁇ carries the CMV promoter, the SV40 polyadenylation signal for human prepro-TGF- ⁇ l expression, the SV40 early promoter and the SV40 polyadenylation signal for mouse dhfr expression.
  • a dhfr " cell line i.e. CHO-DUKX Bll (Chasin, L.A. and Urlaub, G. , PNAS USA 77, 4216-4220, (1980) was used.
  • An equivalent dhfr " CHO cell line is publicly available from the ATCC as ATCC CRL 9096 and could be used in place of CHO-DUKX Bll.
  • Cells were cultured in MEM ⁇ (-) supplemented with 10% fetal calf serum (FCS) .
  • FCS fetal calf serum
  • the cells were transfected with a calcium-phosphate co-precipitate of pEC ⁇ plasmid DNA and selected in the same medium containing 15% dialyzed fetal calf serum (FCS) .
  • FCS dialyzed fetal calf serum
  • the presence of human pro-TGF- ⁇ l in the conditioned media of transformants was detected by the growth inhibition assay of CCL-64 cells as described in detail in Example 6 and immunoblotting using a rabbit polyclonal antibody raised against pro-region of TGF- ⁇ l purified from human platelets (Kanzaki, et al., supra.).
  • the cell clone with the highest TGF- ⁇ l activity in the conditioned medium was selected and named T23-7-11.
  • the T23-7-11 cell line has been deposited under the Budapest Treaty in Fermentation Research Institute (Japan) at 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, 305 Japan, as FERM BP-4024 on October 7, 1992.
  • the T23-7-11 cells described in Example 2, supra. were cultured in Ham's F-12 and Dulbecco's Modified Eagle's medium (1:1) supplemented with 15% FCS and 500nM methotrexate.
  • the cells were transfected with the plasmid pDSVE2-BP constructed as described above and plasmid pSV2neo, using standard electroporation methods.
  • a high concentration of plasmid DNA containing a cloned gene is added to a suspension of cells and the mixture is shocked with an electrical field of 200-600 V/cm.
  • the brief electric pulse is discharged across the electrodes, transiently opening holes in cell membranes.
  • Figure 2 shows the results of the incubation with the Ab39 and LT-1 polyclonal antibody conjugates. These results show that all seven cell clones secreted LL-TGF- ⁇ l complex and pro-TGF- ⁇ l into serum-free culture medium.
  • the LT3-1 clone secreted the highest levels of LL-TGF- ⁇ l into the culture medium and was selected as a candidate cell clone for the purification of LL-TGF- ⁇ l complex from conditioned medium.
  • Cell line LT3-1 has been deposited under the Budapest Treaty in Fermentation Research Institute (Japan) as FERM BP-4015 on September 29, 1992.
  • the LT3-1 cells were grown to confluence in roller bottles containing 200ml selective medium.
  • the cultures were rinsed with phosphate-buffered saline (PBS) and incubated for 7 days in serum-free F-12/DMEM supplemented with insulin, transferrin, monoethanolamine, sodium selenite, aprotinin and polyethylene glycol 6000 as growth-promoting supplements (200ml/bottle) . After 7 days, 80 liters of conditioned medium were collected.
  • PBS phosphate-buffered saline
  • the eighty liters of conditioned medium collected were concentrated, and desalted using Millipore ultrafiltration membranes with a molecular cutoff range of lOOkD.
  • the concentrated conditioned medium was then fractionated on a Q-Sepharose Fast Flow cation- exchange chromatography column equilibrated with lOmM sodium phosphate buffer at pH 7.2. Bound protein was eluted using a 200-660mM NaCl gradient with a flow rate of 8.0 l/min.
  • the presence of the LL-TGF- ⁇ l complex was monitored by SDS-PAGE and immunoblotting using the rabbit polyclonal antibody Ab39.
  • Fractions containing the LL-TGF- ⁇ l complex were further processed on an HP-10 hydroxyapatite column (50x100mm) equilibrated with lOmM sodium phosphate buffer at pH 7.2.
  • the unbound fraction was collected and loaded on a Sulfated Cellulofine column (30x100mm) equilibrated with lOmM sodium phosphate butter at pH 7.2.
  • the bound proteins were eluted using a 0-200 mM NaCl gradient with a flow rate of 4.0ml/min.
  • the fractions containing the LL- TGF- ⁇ l complex were pooled and dialyzed with 40mM Tris-HCl buffer at pH 8.0.
  • the dialyzed sample was applied to a DEAE-Toyopearl anion-exchange column (15x100mm) equilibrated with 40mM Tris-HCl buffer at pH 8.0, and then eluted using a 0-250mM NaCl gradient with a flow rate of 2.0ml/min.
  • the fractions containing high concentrations of the LL- TGF- ⁇ l complex were pooled, concentrated using an Amicon YM100 ultrafiltration membrane, and then applied to a Sephacryl S-300HR gel filtration column (20xl000mm) equilibrated with PBS. The column was eluted in the same buffer at a flow rate of 0.5ml/min.
  • the purified protein was analyzed by SDS-PAGE followed by silver staining and immunoblotting as shown in Figure 4, which shows the purified protein from S-300HR was analyzed by SDS-PAGE on 4-12% gradient gel and silver staining in the presence or absence of dithiothreitol (panel A) , and the purified protein also analyzed by immunoblotting using Ab39 (A) or LT-1 (B) antibodies in the presence or absence of dithiothreitol (panel B) .
  • purified LL-TGF- ⁇ l complex revealed two protein bands with an apparent molecular mass of 220 kD and 270 kD. Both bands are recognized by Ab39 and LT-1.
  • these proteins were separated into four bands with apparent molecular masses of 12.5kD, 40kD, 53kD and 150-190kD.
  • Protein bands of 40kD and 53kD were recognized by LT-1 indicating that these may contain ⁇ l-LAP.
  • Protein bands of 12.5kD and 53kD were recognized by rabbit polyclonal antibody Ab57 against the partial polypeptide of mature TGF- ⁇ l, suggesting that these may contain a mature TGF- ⁇ l sequence.
  • the protein band of 150-190 kD was recognized by Ab39.
  • test samples were subjected to SDS-PAGE and then transferred to a PVDF membrane. After electroblotting, proteins on the membrane were detected with Ponceau.S dye. The stained spots were cut out and the proteins purified from PVDF membrane in the presence of dithiothreitol and electroblotting.
  • 12.5kD component revealed the sequence: Ala-Leu-Asp-Thr- Asn-Tyr-X-Phe-Ser-Ser, which was identical to the sequence of mature TGF- ⁇ l.
  • the N-terminal amino acid sequence analysis of the 40kD and 53kD components revealed the sequence: Leu-Ser-Thr-X-Lys-Thr-Ile-Asp-Met-Glu, which was identical to that of the precursor sequence of TGF- ⁇ l.
  • the attempt to determine the N-terminal amino acid sequence of the 160-190kD component was unsuccessful, indicating that the N-terminal may be blocked. Therefore purified LTBP was digested with Endoproteinase Asp-N and separated on a narrow-bore, reversed-phase HPLC column eluted with linear gradients of acetonitrile/2-propanol. The effluents were monitored at 215 nm. Amino acid sequence analyses were performed on the materials under the numbered peaks by using a SHIMADZU PSQ-1 protein sequencer. The sequences obtained were comparable to those described by Kanzaki et al., Cell 61, 1051-1061 (1990).
  • the purified LL-TGF- ⁇ l complex was found to be composed of four different subunits with molecular masses of 12.5 kD, 40 kD, 53 kD, and 150-190 kD, which were identified as mature TGF- ⁇ l, ⁇ l-LAP, pro-TGF- ⁇ l and LTBP, respectively.
  • TGF- ⁇ l activity was determined using a 3 H- thymidine incorporation assay of mink lung epithelial
  • CCL-64 cells and the test samples were transferred into a 96 well tissue culture plate in DMEM supplemented with 10% FCS and antibiotics. After a 48 hour incubation, cells were pulsed with 0.5 ⁇ Ci of 3 H-thymidine for four hours. The 3 H radioactivity incorporated into DNA was determined with a liquid scintillation counter. Purified protein was quantitated using a Bio-Rad protein assay kit. Figure 6 shows the biological activity profiles. Recombinant mature TGF- ⁇ l was a potent growth inhibitor of CCL-64 cells.
  • Recombinant LL-TGF- ⁇ l complex was much less effective in inhibiting CCL-64 cells with half maximal inhibition at approximately 200 ng/ml and its inhibition curve appeared to have a slightly altered slope when compared with recombinant TGF- ⁇ l and acid-activated LL-TGF- ⁇ l complex. It was not previously reported that the natural LL-TGF- ⁇ l complex inhibited the proliferation of CCL-64 cells.
  • the dhfr " cell line CHO-DUKX-B11, as described in Example 2, supra.. was transfected with the LTBP expression plasmid pDSVE2-BP of Example 1.
  • Cells were maintained in MEM ⁇ (-) supplemented with 5% FCS and transfected with 20 mg of pDSVE2-BP plasmid in a calcium phosphate co-precipitate.
  • Dhfr " expressing transformants were selected by replacing the medium with MEM ⁇ (-) supplemented with 5% dialyzed FCS.
  • Cell clones producing LTBP in the conditioned medium were selected and further cultured in the medium containing an increasing concentration of methotrexate up to 50 nM to amplify the introduced LTBP cDNA.
  • methotrexate up to 50 nM
  • BP1-1 was chosen for further study.
  • BP1-1 cells were grown to confluence in roller bottles containing 200 ml selective medium. The cells were rinsed with phosphate-buffered saline (PBS) and further incubated for 7 days in serum-free F-12/DMEM supplemented with insulin, transferrin, monoethanolamine, sodium selenite, aprotinin and polyethylene glycol 6000 as growth-promoting supplements (200 ml/bottle) . After 7 days, twenty liters of conditioned medium were collected.
  • PBS phosphate-buffered saline
  • the collected conditioned medium was concentrated and desalted using Millipore ultrafiltration membranes with a molecular cut off range of 10 kD.
  • Concentrated conditioned medium was dialyzed against 25 mM sodium phosphate buffer containing 0.5 M ammonium sulfate at pH 7.2 and then applied to a Phenyl-Toyopearl 650M column (50 x 100 mm, Tosoh) equilibrated with the same buffer.
  • the bound proteins were eluted using a 0.5-0 M ammonium sulfate gradient at a flow rate of 5.0 ml/min.
  • LTBP LTBP-polyacrylamide gel electrophoresis
  • SDS-PAGE SDS-polyacrylamide gel electrophoresis
  • immunoblotting immunoblotting and/or enzyme-immunoassay using the rabbit polyclonal antibody Ab-39.
  • Fractions containing LTBP were pooled, dialyzed against 10 M Tris-HCl buffer at pH 8.0 and further processed on a Q-Sepharose FF anion exchange column (26 x 100 mm) equilibrated with the same buffer.
  • the bound proteins were eluted using a 100-600 mM NaCl gradient at a flow rate of 3.0 ml/min.
  • Fractions containing LTBP were pooled, concentrated and loaded on Superose 12 prep grade column (26 x 500 mm) equilibrated with PBS. The loaded samples were eluted with PBS at a flow rate of 1.0 ml/min. Fractions containing LTBP were pooled and applied to a reversed-phase C4 HPLC column (11 x 250 mm) equilibrated with 10% acetonitrile containing 0.1% TFA, and then eluted using a 10-50% acetonitrile gradient at a flow rate of 2.0 ml/min.
  • Purified protein was separated by SDS-PAGE and analyzed by silver staining, immunoblotting using the anti- LTBP antibody Ab-39 as described above, and amino acid sequencing. Under both reducing and non-reducing conditions, the purified protein revealed a single broad band with an apparent molecular weight of 110-130 kD by silver staining. As shown in Figure 7, SDS-PAGE revealed a diffuse protein band with an apparent weight of about 120 to about 140 kDa. This band was recognized by the rabbit anti-LTBP Ab-39 antibody, indicating that it was LTBP. The identity of the purified protein was further confirmed by amino acid sequencing under reducing conditions.
  • the protein was digested with an endopeptidase Asp-N, subjected to a reversed-phase HPLC and the separated polypeptides were sequenced.
  • the obtained amino acid sequences were identical to those found in the reported amino acid sequence of LTBP purified from human platelets (Kanzaki et al., Cell 61, 1051-1061, 1990).
  • Cell line BP1-1 was deposited under the Budapest
  • LL-TGF- ⁇ 2 The production of LL-TGF- ⁇ 2 was achieved by co- expressing the LTBP cDNA with a prepro-TGF- ⁇ 2 cDNA in CHO cells.
  • a prepro-TGF- ⁇ 2 expression plasmid was introduced to the BP1-1 cell line described above.
  • TGF- ⁇ 2 The complete nucleotide sequence of human TGF- ⁇ 2 has been published (De Martin et al., EMBO J. 6, 3673-3677, 1987) .
  • a cDNA clone for human TGF-B2 could be obtained essentially as described for the TGF- ⁇ l cDNA in Example 2.
  • a mammalian expression plasmid pME-TGF- ⁇ 2 was constructed using the obtained prepro-TGF- ⁇ 2 cDNA. This plasmid contains the human TGF- ⁇ 2 cDNA under the control of a modified SV40 promoter and the SV40 early region for transcription termination and polyadenylation.
  • pME-TGF- ⁇ 2 plasmid was co-transfected with pRSVneo plasmid, which allows for neomycin resistance selection, into the BP1-1 cell line by electroporation. G418 resistant transformants were selected and assayed for the production of TGF- ⁇ 2 activity by the growth inhibition assay of CCL-64 cells (see Example 6) . Several cell clones were isolated which secreted TGF- ⁇ 2 activity into culture medium. The methodology by which these clones were identified is described in Example 9.
  • the 200-220 kDa represents LTBP associated with pro-TGF- ⁇ 2
  • the 100-110 kDA, 120-130 kDA and 150- 160 kDA species may represent free LTBP protein because these species are found in conditioned medium of BP-1-1 cells.
  • This result shows that BP-1-1 transfected with a plasmid containing cDNA coding for human prepro-TGF-B2 protein secrete pro-TGF- ⁇ 2 associated with a LTBP, yielding a LL-TGF- ⁇ 2 complex.
  • association of LTBP and pro-TGF- ⁇ 2 co-expressed in CHO cells occurs during synthesis of each protein.
  • a cell line producing LL-TGF- ⁇ 2 was chosen and named LT2-14.
  • Cell line LT2-14 was deposited under the Budapest Treaty in Fermentation Research Institute (Japan) as FERM BP-4016 on September 29, 1992.
  • LL-TGF- ⁇ l and LTBP are capable of binding Ca + , leading to methodologies for purifying and stabilizing LL-TGF- ⁇ via combination with the cation.
  • the methods disclosed below are further described in Colosetti et al., FEBS Letters 320: 140-144, (1993), which is incorporated by reference herein in its entirety.
  • LTBP (lO ⁇ g) (isolated from a side fraction from the purification of LL-TGF- ⁇ l from human platelets, Kanzaki et al., Cell 61:1051-1061, 1990), recombinant small latent TGF- ⁇ l complex (SLC; lO ⁇ g) , and human blood (B; 2 ⁇ l) , were subjected to SDS-PAGE (4-10% polyacrylamide gradient) , and then transferred to a nitrocellulose membrane. After overnight incubation with 45 CaCl 2 (l ⁇ Ci/ml) , the membrane was rinsed, dried and subjected to autoradiography.
  • SLC small latent TGF- ⁇ l complex
  • B human blood
  • free LTBP isolated from the conditioned medium of human prostate cell line PC-3 labelled with [ 35 S] cysteine were immunoprecipitated by Ab39 antibodies and subjected to proteolytic digestion using trypsin over different time periods at constant temperature of 37°C, in the presence of 2 mM CaCl 2 or 2mM EDTA. The incubations were quenched by the addition of
  • the effect of Ca 2+ on the LTBP molecule was also investigated by anion exchange chromatography in the absence or presence of EDTA or CaCl 2 .
  • Free LTBP obtained from PC-3 conditioned medium in the presence of 5 mM EDTA or 5mM CaCl 2 was subjected to Q-Sepharose chromatography.
  • LTBP mainly eluted at about 460 mM NaCl. In the presence of 5 mM EDTA, LTBP eluted later i.e., at about 500 mM NaCl. An earlier elution at about 420 mM NaCl was observed if Ca 2+ was present.
  • the lower affinity of LTBP for the anionic ion exchanger in the presence of Ca 2+ could be due to a changed charge on LTBP after Ca 2+ binding, or to a Ca + induced change in conformation of the molecule.
  • Example 13 Large quantities of rL- ⁇ l-LAP can be isolated from rLL-TGF- ⁇ , which has been prepared according the procedure set forth in Example 3 above.
  • the LL-TGF- ⁇ complex can be treated with a conventional denaturing agent, e.g. urea, to dissociate L-LAP from the LL-TGF- ⁇ complex.
  • L- ⁇ l-LAP was prepared by first dialyzing one mg of rLL-TGF ⁇ with 20mM sodium phosphate buffer (pH of 7.2) containing 8M urea. The dialyzed protein was concentrated and then applied to a Superose 12 prep grade gel filtration column.
  • FIG. 12 shows the purification of rL- ⁇ l-LAP by gel filtration on Superose 12. The separation proceeds based on the relative size of the two populations.
  • the first peak represents L- ⁇ l-LAP fractions which eluted with sample 8-16 and the second peak represents the mature TGF- ⁇ fraction.
  • the fractions containing rL- ⁇ l-LAP were collected and then dialyzed against phosphate-buffered saline and finally filtered with a 0.22 ⁇ m membrane filter.
  • the purified protein was then analyzed by SDS-PAGE. As shown in Figure 13, the purified rL-LAP migrated to a molecular weight of about 180 to about 200 kDa.
  • Antibodies specific to the LL-TGF- ⁇ complex can be produced by introducing the complex to laboratory mice and screening the blood for response to the complex. Monoclonal antibodies may be produced by standard techniques.
  • the antibodies are useful for detection of the abnormal production of the LL-TGF- ⁇ complex.
  • TGF- ⁇ Mature TGF- ⁇ is currently under study for use in the treatment of many diseases. Sporn et al., JAMA, 262: 938-941 (August 16, 1989) ; however mature TGF- ⁇ l has potent side effects such as body weight loss, anemia and thrombocytopenia. It is assumed that the use of LL-TGF- ⁇ l may be able to overcome some of these side effects.
  • TGF- ⁇ l is currently under study for use in the treatment of certain bone diseases, such as osteoporosis and bone fracture healing because TGF- ⁇ is known to affect bone formation.
  • TGF- ⁇ is a potent inducer of type II collagen and proteoglycans, which form the extracellular matrix of cartilage.
  • Sporn et al., supra. It has been found that TGF- ⁇ is one of the critical peptide growth factors that acts on chondrocytes or osteocytes and related cell types. Pfeilschifter et al., PNAS 84:2024-2028 (1987) . It has been found to be involved in the embryonic formation of cartilage and bone and is present in the growth plates of long bones. Large amounts are also found in adult bone. In fact, TGF- ⁇ 2 was isolated from adult bovine bone.
  • LL-TGF- ⁇ can also be useful as an immunosuppressant, in cases of autoimmune diseases or organ transplantation.
  • TGF- ⁇ is the most potent known endogenous suppressant of lymphocyte proliferation and function, and serves as an autocrine "stop" signal inhibiting the action of the interleukins and other cytokines, such as tumor necrosis factor, which stimulate lymphocyte function. Therefore, TGF- ⁇ inhibits proliferation of T cells stimulated by interleukin 1 or 2, inhibits proliferation and antibody production in B cells stimulated by any of several activating factors, depresses cytolytic activity of natural killer cells, and inhibits the generation of cytotoxic T cells and lymphokine-activated killer cells. Sporn et al., supra.
  • TGF- ⁇ may also provide hematoprotection from cytostatic drugs by stem cell inhibition. Taking advantage of the immunosuppressive properties of LL-TGF- ⁇ , patients, suffering from conditions for which chemotherapy is the treatment of choice, may be administered LL-TGF- ⁇ prior to chemotherapy in order to protect cells of the bone marrow. TGF- ⁇ also has important actions on fibroblasts involved in tissue repair. TGF- ⁇ stimulates the production of critical components of extracellular matrix, such as collagen, fibronectin and proteoglycans, and inhibits the action of proteolytic enzymes that destroy newly formed connective tissue. Sporn et al., J.Cell Biol.
  • TGF- ⁇ has been shown to enhance gene transcription of collagen, Heine et al., J.Cell Biol. 105:2861-2876 (1987), and to play a critical role in providing the structural strength of healing wounds as well as serving as an essential part of the matrix of bone and cartilage. TGF- ⁇ has been shown to enhance wound healing in animals. Roberts et al., Recent Prog. Horm. Res. 44:157-197 (1988).
  • LL-TGF- ⁇ l may also be used to stimulate soft tissue healing, i.e. skin ulcers or peptic ulcers.
  • TGF- ⁇ has been shown to be a potent antiproliferative agent for most epithelial cells and this may be of use as an antiproliferative agent in neoplastic diseases.
  • L-LAP The large latency associated peptide
  • L-LAP can be utilized as an antagonist for mature TGF- ⁇ molecules.
  • L-LAP can be employed i.e., in therapeutic or diagnostic modalities, to control, modulate or regulate the harmful or undesired effects of TGF- ⁇ treatment or presence.
  • the use of L-LAP can overcome the drawbacks of TGF- ⁇ therapy and can decrease or diminish the potent side effects of TGF- ⁇ .
  • the administration of L-LAP can also provide for a protective effect during treatment of other TGF- ⁇ related diseases, i.e., fibrotic disorders, glomerulonephritis, liver fibrosis, keloid formation; and carcinoma metastasis.
  • Antibodies specific to the L-LAP can also be produced by introducing the peptide to laboratory animals and screening the blood for response to the complex. Monoclonal antibodies may be produced by standard techniques. These antibodies can be used as diagnostic aids.
  • the foregoing experiments show that it is possible to produce a recombinant LL-TGF- ⁇ l by introducing a DNA sequence for LTBP to a T23-7-11 CHO cell which expresses pro-TGF- ⁇ l.
  • a plasmid containing LTBP By constructing a plasmid containing LTBP, and transfecting CHO cells with the plasmid, it is possible to express recombinant LL-TGF- ⁇ l.
  • the experiments also show that it is possible to produce a recombinant LL- TGF- ⁇ 2 by introducing a plasmid containing a cDNA sequence for TGF-B2 into a BP-1-1 cell containing an amplified LTBP CDNA sequence.
  • L- ⁇ -LAP can be prepared from the rLL-TGF- ⁇ complex by treating the complex with a denaturing agent to dissociate L- ⁇ -LAP therefrom. It is to be expected that the above-described methods of producing the recombinant LL-TGF- ⁇ l and LL-TGF- ⁇ 2 complexes can be applied generally to producing recombinant LL-TGF- ⁇ complexes, including but not limited to recombinant TGF-B3, because it is well documented that the individual members of the family of TGF- ⁇ s are similar in peptide sequence and in biological activity. The TGF- ⁇ isofor s exhibit comparable properties and thus are expected to behave similarly.

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Abstract

L'invention concerne d'une manière générale, un procédé de production de grands facteurs β transformants de croissance TGF-β latents recombinés par l'introduction de séquences d'acides nucléiques codant pour la protéine de liaison latente (LTBP) et le pro-TGF-β dans des cellules eucaryotes. L'invention porte également sur un procédé de production de grands facteurs β transformants de croissance latents LL-TGF-β par l'introduction d'une séquence d'ADN pour la protéine de liaison latente dans une cellule eucaryote exprimant le pro-TGF-β. La construction d'un plasmide pME-TGF-β2 et sa co-transfection avec un néo-plasmide pRSV dans des cellules de protéine de liaison 1-1 d'ovaires de hamster chinois (CBO1-1CHO) lesquelles contiennent dans le génome une séquence d'ADNc amplifiée de protéine de liaison latente (LTBP) décrites. L'invention concerne également un procédé de traitement du complexe de grands facteurs transformants de croissance latents β1 LL-TGF-β1 produits pour fixer des ions Ca2+ pendant l'activité du processus de purification. Un anticorps dirigé contre le complexe de grand TGF-β1 latent est également décrit ainsi qu'une L-LAP isolée et son procédé de production.
PCT/US1993/010230 1992-10-26 1993-10-25 PROCEDE DE PRODUCTION DE COMPLEXES DE GRANDS FACTEURS β TRANSFORMANTS DE CROISSANCE LATENTS ET GRAND PEPTIDE DE LATENCE ASSOCIE WO1994009812A1 (fr)

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WO1998035695A1 (fr) * 1997-02-13 1998-08-20 The Victoria University Of Manchester Cicatrisation d'une blessure
US5972335A (en) * 1994-03-29 1999-10-26 The Victoria University Of Manchester Wound healing
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US7527791B2 (en) 2004-03-31 2009-05-05 Genentech, Inc. Humanized anti-TGF-beta antibodies
US7625410B2 (en) 2001-05-02 2009-12-01 Boston Scientific Scimed, Inc. Stent device and method
EP2230252A1 (fr) 2006-03-13 2010-09-22 The Johns Hopkins University Augmentation de la thromborésistance endothéliale
WO2012090997A1 (fr) 2010-12-27 2012-07-05 京都府公立大学法人 CELLULES SPi ET LEUR PROCÉDÉ DE PRODUCTION
WO2013014262A1 (fr) 2011-07-27 2013-01-31 INSERM (Institut National de la Santé et de la Recherche Médicale) Méthodes de diagnostic et de traitement du syndrome de myhre
WO2013100208A1 (fr) 2011-12-28 2013-07-04 京都府公立大学法人 Normalisation d'une culture de cellules endothéliales de la cornée
US8642034B2 (en) 2006-10-03 2014-02-04 Genzyme Corporation Use of TGF-β antagonists to treat infants at risk of developing bronchopulmonary dysplasia
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EP2862867A2 (fr) 2005-10-25 2015-04-22 The Johns Hopkins University Procédés et compositions pour le traitement du syndrome de Marfan et troubles associés
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US9399676B2 (en) 2013-05-06 2016-07-26 Scholar Rock, Inc. Compositions and methods for growth factor modulation
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US9468612B2 (en) 2011-10-26 2016-10-18 Seattle Children's Hospital Cysteamine in the treatment of fibrotic disease
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WO1998035695A1 (fr) * 1997-02-13 1998-08-20 The Victoria University Of Manchester Cicatrisation d'une blessure
US6319907B1 (en) 1997-02-13 2001-11-20 Renovo Limited Wound healing
US7511070B2 (en) 1997-04-11 2009-03-31 Poniard Pharmaceuticals, Inc. Compounds and therapies for the prevention of vascular and non-vascular pathologies
US7625410B2 (en) 2001-05-02 2009-12-01 Boston Scientific Scimed, Inc. Stent device and method
US7527791B2 (en) 2004-03-31 2009-05-05 Genentech, Inc. Humanized anti-TGF-beta antibodies
EP2862867A2 (fr) 2005-10-25 2015-04-22 The Johns Hopkins University Procédés et compositions pour le traitement du syndrome de Marfan et troubles associés
EP2230252A1 (fr) 2006-03-13 2010-09-22 The Johns Hopkins University Augmentation de la thromborésistance endothéliale
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US8642034B2 (en) 2006-10-03 2014-02-04 Genzyme Corporation Use of TGF-β antagonists to treat infants at risk of developing bronchopulmonary dysplasia
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EP2835053A1 (fr) 2010-03-12 2015-02-11 Genzyme Corporation Thérapie combinée pour le traitement du cancer du sein
WO2012090997A1 (fr) 2010-12-27 2012-07-05 京都府公立大学法人 CELLULES SPi ET LEUR PROCÉDÉ DE PRODUCTION
WO2013014262A1 (fr) 2011-07-27 2013-01-31 INSERM (Institut National de la Santé et de la Recherche Médicale) Méthodes de diagnostic et de traitement du syndrome de myhre
US9468612B2 (en) 2011-10-26 2016-10-18 Seattle Children's Hospital Cysteamine in the treatment of fibrotic disease
US9925154B2 (en) 2011-10-26 2018-03-27 Seattle Children's Hospital Cysteamine in the treatment of fibrotic disease
WO2013100208A1 (fr) 2011-12-28 2013-07-04 京都府公立大学法人 Normalisation d'une culture de cellules endothéliales de la cornée
EP3553169A1 (fr) 2011-12-28 2019-10-16 Kyoto Prefectural Public University Corporation Normalisation de culture de cellules endothéliales cornéennes
EP2916867A4 (fr) * 2012-11-06 2016-10-05 Scholar Rock Inc Compositions et procédés pour la modulation de la signalisation cellulaire
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