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WO2000060945A1 - ENDOSTATINE RECOMBINEE, SOLUBLE, ET PROCEDE DE PREPARATION A PARTIR DE $i(STREPTOMYCES SP) - Google Patents

ENDOSTATINE RECOMBINEE, SOLUBLE, ET PROCEDE DE PREPARATION A PARTIR DE $i(STREPTOMYCES SP) Download PDF

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Publication number
WO2000060945A1
WO2000060945A1 PCT/US2000/009747 US0009747W WO0060945A1 WO 2000060945 A1 WO2000060945 A1 WO 2000060945A1 US 0009747 W US0009747 W US 0009747W WO 0060945 A1 WO0060945 A1 WO 0060945A1
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endostatin
soluble
human
streptomyces
recombinant
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PCT/US2000/009747
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English (en)
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Charles L. Desanti
William R. Strohl
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Merck & Co., Inc.
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Priority to AU43420/00A priority Critical patent/AU4342000A/en
Publication of WO2000060945A1 publication Critical patent/WO2000060945A1/fr

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    • 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
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/76—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Actinomyces; for Streptomyces
    • 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
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • 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
    • A61K38/39—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence

Definitions

  • This invention relates to compositions of soluble recombinant endostatin and methods of producing soluble recombinant endostatin from a prokaryotic host cell of the genus Streptomyces.
  • Endostatin is a 183 amino acid C-terminal fragment of collagen XVIII with a molecular mass of approximately 20 kDa. Endostatin has been reported to have anti-angiogenic activity sufficient to induce the regression of a variety of implanted, primary tumors in mice (O'Reilly et al., 1997; WO 97/15666). However, endostatin does not directly inhibit the proliferation of tumor cells nor does it induce apoptosis in endothelial cells in pre-existing vessel systems.
  • endostatin The anti-angiogenic activity of endostatin is reported to prevent the formation of new blood vessels which are recruited by fast growing tumors (O'Reilly et al., 1997; Hanahan and Folkman, 1996). Therefore, it is believed that endostatin may exert anti-tumor effects through starvation of the tumor mass. The mechanism by which endostatin induces not only tumor starvation but also tumor regression is not known. Repeated cycles of endostatin treatment did not cause the development of endostatin-resistant tumors. That result indicated that endostatin might be a promising cure for cancer (Boehm et al., 1997; Kerbel, 1997). Several attempts to produce recombinant endostatin in an active, soluble form have been reported in the literature.
  • Hohenester et al. reported that the X-ray structure of this form of murine endostatin was determined to 1.5 A resolution and revealed a structural similarity to the C-type lectin family but lacking the characteristic Ca 2 + binding site.
  • Boehm et al. also produced murine endostatin from a mammalian tumor cell line, B16F10 melanoma cells. While the murine endostatin so produced was reported to have a full length N-terminus, no data was presented to indicate that the murine endostatin was active or that it was soluble. Dhanabal et al., 1999, also report on the production of murine endostatin in the yeast P. pastoris. This report does not specifically note the cleavage of the N-terminus described by Boehm et al., 1998, as related to expression in the P.
  • Dhanabal et al. did classify their yeast produced endostatin at about 20 kDa and characterized that size estimate as being in agreement with the 22- 24 kDa size reported by others.
  • Dhanabal et al. 's P. pastoris derived murine endostatin may be similar in size to the inactive, N-terminal truncated endostatin also prepared in P. pastoris by Boehm et al., 1998, Dhanabal et al., report that their 20 kDa murine endostatin produced anti-angiogenic effects in vivo and in vitro.
  • Dhanabal et al. also expressed murine endostatin in bacteria according to the method of O'Reilly et al., 1997. It is noteworthy that Dhanabal et al. observed a size of 22-24 kDa for the murine endostatin they produced in bacteria and also observed dimer-sized complexes in the 44-46 kDa range. Like O'Reilly et al., Dhanabal et al., were unable to produce soluble endostatin from bacteria.
  • the Ding et al. publication reported a crystal structure of human endostatin to 2.9 A resolution and indicated that the molecule contains a Zn2+ binding site formed by three Histidinel > 3, 11 and one Aspartic acid76 residues.
  • the Zn2+ was reported to be ligated at HislN , His N ⁇ and Hisl iN ⁇ .
  • Ding et al. also reported that human endostatin is expected to form zinc-dependent dimers. This statement is in agreement with the 44-46 kDa dimer-sized endostatin material observed by Dhanabal et al., 1998.
  • the Gram-positive streptomycetes When compared to eukaryotic host cells and the enteric Gram-negative host Escherichia coli, the Gram-positive streptomycetes have a combination of qualities which make them attractive over these counterparts.
  • the members of the genus produce a great variety of extracellular proteins (Chang, 1987) and thus many potential signal sequences are available for one to try to adapt for secretion-expression systems (Gilbert, et al., 1995). Further, secretion of heterologous proteins can be simplified because streptomycetes do not contain an outer membrane. Finally, streptomycetes do not produce endotoxin and there is no evidence that proteins expressed in Streptomyces are modified by acylation or glycosylation.
  • mTNF mouse tumor necrosis factor
  • S. lividans When expressed using the promoter and leader peptide of alpha-amylase, approximately 1000-fold less recombinant mTNF was observed as compared to the production of alpha-amylase from that host (Lammertyn, et al, 1996).
  • soluble secreted human CD4 was produced by recombinant S. lividans at levels of ca. 300 mg/L (Fornwald, et al, 1993). Therefore, while production of proteins from streptomycetes shows promise, the literature teaches that it is often a trial and error process.
  • the present invention solves an existing problem in the field of angiogenesis by providing soluble recombinant endostatin and a method of producing said endostatin from Streptomyces.
  • the endostatin can be from any mammal but human endostatin is preferred.
  • Compositions containing soluble recombinant endostatin made from Streptomyces are also provided by this invention.
  • this invention provides assays conducted using soluble recombinant endostatin produced from Streptomyces. These assays are advantageous over those known in the art because the endostatin used is easily made, plentiful, inexpensive and a soluble recombinant product.
  • FIG. 1 The leader peptides of SnpA and alpha-amylase, compared with other examples of streptomycete leader peptides.
  • the sequences are SEQ ID NOs: 7, 8, 8, 20, 21, 22, 23 and 24, respectively.
  • FIG. 2. A map ofpIJ303.
  • FIG. 3. A map of pANT826.
  • FIG. 4. A map ofpANT1201.
  • FIG. 5 An outline showing of the construction of the plasmids which confer secretion of soluble endostatin on S. lividans.
  • FIG. 6. The sequence of the human endostatin gene cloned in pMALcH#15. The sequences are SEQ ID NOs: 25 (DNA) and 26 (amino acid).
  • FIG. 7. Plasmids pANT3042, pANT3045 and pANT3052.
  • FIG. 8A-8B Expression and secretion of endostatin.
  • FIG. 8A shows a growth curve of S. lividans TK24 carrying plasmid pANT3052.
  • FIG. 8B shows immunologically reactive endostatin protein detected in cell- free culture broth.
  • FIG. 9A-9B This figure shows the sequence of the region in pANT1201 spanning from the transcriptional terminator to the end of the snpR gene. All of the sequence between the two Kpn ⁇ sites shown is included in the final set of secretion vectors. Both the mmr transcriptional terminator and the multiple cloning site were constructed using synthetic oligonucleotides. The sequences are SEQ ID NOs: 27 (DNA) and 28 (amino acid).
  • FIG. 10A-10B Annotated sequence of the vaa-human endostatin gene fusion and snpR/snpA promoter region in plasmid pANT3052. The sequences are SEQ ID NOs: 29 (DNA) and 30 (amino acid).
  • FIG. 11 A map of the plasmid pANT3052.
  • the present invention provides soluble recombinant endostatin from Streptomyces, a method of producing the endostatin, compositions including the endostatin and assays and methods employing the endostatin.
  • the soluble recombinant endostatin of this invention is useful in the treatment of cancer, the inhibition of tumor growth, the inhibition of angiogenesis, the isolation of receptors for endostatin and in assays for the identification of anti- angiogenic compounds.
  • An advantage of the present invention is that the endostatin protein is produced as a secreted, soluble protein which needs no refolding. Additionally, the protein as produced is stable in the fermentation broth and is produced in large enough quantities that it presents a potentially commercially-viable process. Finally, because Streptomycetes are amenable to cultivation in large fermentations (Binnie, et al, 1997), the process described herein also is scaleable, allowing for large quantities of soluble endostatin to be produced by fermentation.
  • the soluble recombinant endostatin can be made using genetic material from any animal.
  • soluble recombinant endostatin is produced using genetic material from primates including human, chimpanzee and ape, or from other animals including murine, pig, goat, dog, bovine, avian, whale, porpoise, etc. Soluble recombinant endostatin made from primate genetic material is preferred and that made from human genetic material is most preferred.
  • leader peptides direct the secretion of polypeptides out of the streptomycete cell and can also be referred to as secretion signal sequences or simply signal sequences.
  • secretion signal sequences or simply signal sequences.
  • two different leader peptides were chosen for testing in a secretion-based system. The first was the leader peptide of the small neutral (metallo-) protease, SnpA.
  • This protein is a secreted protease of Streptomyces sp. strain C5, encoded by the snpA gene (Lampel, et al, 1992). The second was the leader peptide of the secreted alpha- amylase of Streptomyces venezuelae. The amylase is encoded by the vaa gene (Lammertyn, et al, 1996). These SnpA and alpha-amylase leader peptide sequences are shown in FIG. 1, together with the leader peptide sequences of six other streptomycete secreted proteins.
  • each of the leader sequences include the N-terminal positively-charged region, the core hydrophobic region, and the leader peptidase recognition sequence (from approximately -4 to +4 around the cleavage site.
  • the leader peptidase cleavage site can be referred to as a cleavable linker.
  • the typical leader peptidase recognition sequence includes the amino acids Ala-Xaa-Ala, followed by the cleavage site and two relatively small-sized amino acid residues. A proline residue often can be found to occupy the -4 position, giving Pro- Ala-Xaa-Ala - ⁇ -Xaa-Xaa (SEQ ID NO:l) as the conserved sequence around the cleavage site.
  • Cleavable linkers and leader peptides that function in streptomyces are appropriate for use in the method of this invention.
  • the basic strategy for accomplishing the secretion of human endostatin in cultures of recombinant S. lividans includes four steps: (i) Utilization of a strong promoter to drive expression of the fusion constructs. We prefer the SnpR (LysR-like activator gene)-activated snpA -promoter (Lampel, et al, 1992). (ii) The generation of polynucleotides encoding in-frame fusions of various leader peptide sequences with human endostatin. (iii) Insertion of these polynucleotides into high copy number streptomycete vectors. And (iv), growth of the recombinant S. lividans cultures in a liquid medium. The level of secretion human endostatin in the culture broths can be assessed by various protocols including Western blots using an anti-endostatin antibody.
  • the genomic DNA of Streptomyces is typified by high guanine plus cytosine mol%, usually around 65-70%. This fact correlates with the observance of a considerable degree of codon bias in the open reading frames of streptomycete genes (Wright and Bibb, 1992). Codons with a guanine or cytosine residue in the third position are relatively abundant, and naturally this imbalance extends to the tRNA pools utilized during translation of mRNA transcripts. Conversely, streptomycete tRNAs recognizing rare codons, such as TTA and CTA (both leucine) are expressed at very low levels. Evaluation of the cDNA encoding human endostatin revealed the gene contained 60% guanine and cytosine, and was devoid of TTA and CTA codons, making it an acceptable candidate for expression in Streptomyces.
  • the preferred streptomycete host is Streptomyces lividans.
  • Many strains of S. lividans are known and used in the art. Any of those strains commonly used for the expression of recombinant proteins can be used in the method of this invention.
  • the most preferred host is S. lividans strain TK24. It is preferred that the streptomycete host cells are grown in a soluble, sucrose rich medium so that the growth characteristics and recombinant gene expression are optimal, and the secreted product is stable in the extracellular milieu. Soluble media including Tryptic Soy Broth, Dextrose, Yeast Extract, and Sucrose are appropriate.
  • a streptomycete expression vector with a strong promoter is preferred.
  • Many streptomycete vectors and several strong promoter systems are known in the art and can be used interchangeably.
  • the melCl (Leu, et al, 1989) and ermE* (Bibb and Janssen, 1986; also called "ermE-up") promoters are widely known to be strong promoters used for the expression of genes in S. lividans (Schmidt- John and ⁇ ngels, 1992). In one example study, these promoters gave 27-fold and 47-fold better expression, respectively, of tendamistat than did the ⁇ /?A7-promoter.
  • promoters gave 7-fold and 12.5-fold better expression, respectively, of tendamistat in S. lividans than did the thiostrepton-induced tipA -promoter (Schmitt- John and ⁇ ngels, 1992). Either of these two strong promoters, or promoters of comparable strength, can be used in the present invention.
  • Streptomyces sp. strain C5 SnpR-activated swp -promoter is more than 50-fold stronger than melCl -promoter, as measured by the production of the reporter product AphH (assayed using ELISA technology as practiced by those familiar with the art), suggesting that the SnpR-activated snpA- promoter is one of the strongest streptomycete promoters known.
  • the use of the SnpR-activated sM/? -promoter-polylinker cassette is the preferred embodiment of this approach.
  • the strong promoter is at least about 40 times as active as the melCl promoter, more preferably at least about 50 to 75 times as active as the melCl promoter, and also preferably at least about 100 times as active as the melCl promoter.
  • the strength of any given promoter can be compared to the melCl promoter by generating a pair of plasmid constructs.
  • the expression of a reporter gene is driven by the melCl promoter and in the other, the expression of the reporter gene is driven by the promoter of interest.
  • a reporter gene can be an enzyme, immunogen, green fluorescence protein (GFP), etc.
  • the strength of a candidate promoter is assessed at the translational level, i.e., by assessing the amount of protein produced.
  • An immunologic or other assay can be used.
  • high levels of transcription does not always lead to high levels of translation.
  • operably linked it is meant that nucleotide sequences are aligned in a vector such that they operate together to produce a protein of a particular sequence or regulate the expression of one nucleic acid sequence due to its proximity or alignment with another nucleic acid sequence.
  • operably linked means that amino acid sequences are aligned in a polypeptide in a fashion appropriate to the operation of the amino acid sequence.
  • a leader sequence is operably linked to a polypeptide if the leader sequence acts to direct secretion of the polypeptide from the host cell.
  • an isolation tag is operably linked to an amino acid sequence if it appears in the polypeptide sequence in a position that allows one to isolate the polypeptide through some aspect of the isolation tag.
  • an isolation tag appears at a terminus of a polypeptide and is operably linked thereto by a cleavable linker, that is, by an amino acid sequence that contains an endopeptidase cleavage site.
  • endostatin is isolated from the culture medium by methods known and used in the art. For example, one can perform size exclusion separations such as gel chromatography or centrifugal ultrafiltration, in addition to ion-exchange and heparin-affinity chromatography.
  • cleavable linker sequences are known in the art. These linker sequences are useful for removing leader peptide sequences and/or isolation tags from recombinantly expressed protein. For example, factor Xa, caspases, enterokinase and thrombin cleavage sites are used in this manner.
  • a cleavable linker native to streptomycetes and more preferably, use a cleavable linker naturally associated with the leader peptide employed for secretion of the endostatin.
  • the endostatin was made using a linker associated with the leader peptide and cleavable by an endopeptidase endogenous to the streptomycete host. That linker leaves five additional amino acids at the N-terminus of the endostatin, Val-Pro-Pro-Gly-Ser (SEQ ID NO:2). It is preferred that after cleavage no more than five amino acids are added to the N-terminus. If one employs a linker that is not native to the host cell, i.e., that is not cleaved by a peptidase native to the host cell, then the linker can be cleaved at any appropriate time.
  • endostatin For consistency herein, the numbering of the amino acids of endostatin is +1 for the native N-terminal amino acid and +183 for the native C-terminus. Where additional amino acids are present at the N-terminus, the additional amino acid adjacent to the native N-terminal amino acid is at position -1, the next additional amino acid is -2, an so on.
  • endostatin having a native N-terminus however, endostatin with additional amino acids are also useful.
  • endostatin having the N-terminal sequence Val-5Pro-4Pro- Gly-2Ser-l His+lSer + 2His + 3 (SEQ ID NO:2) is a most preferred endostatin of this invention.
  • Soluble, secreted endostatin can be isolated by routine biochemical methods used in the art including chromatographic separations, immunologic separations, or the use of isolation tags.
  • isolation tags are known in the art and can be used to assist in the purification of recombinant endostatin from the cellular material, including thioredoxin and hexa-histidine.
  • Endostatin synthesized with a hexa-histidine tag can be isolated on a Ni2+ resin as practiced routinely in the art.
  • affinity columns including Ni-Ta resin from QIAGEN or Talon resin from CLONTECH, can be employed.
  • cleavable linker sequence between the tag and the N-terminus of endostatin.
  • the cleavable linker site is not cleaved by a peptidase native to the host cell.
  • Assays of this invention use soluble recombinant endostatin produced from Streptomyces. These assays are advantageous over those known in the art because one employs a plentiful, readily available, inexpensive, soluble recombinant endostatin produced from Streptomyces. Therefore, the assays of the present invention can be performed at lower cost and higher throughput than present assays.
  • the soluble endostatin is used as a standard in the ex vivo rat aortic ring assay.
  • endothelial cell tube outgrowth is regulated by autocrine, paracrine, and junxtacrine interactions among endothelial cells, pericytes and fibroblasts.
  • This ex vivo model comprises the necessary sequence of events leading to the formation of new vessels including proliferation, migration and canalization.
  • the soluble endostatin is used as a standard in a high throughput screening (HTPS) assay based on the endothelial cell proliferation assays.
  • HTPS high throughput screening
  • endothelial and non-endothelial control cells are seeded into 96-well plates and the rate of their proliferation is measured either by 3H-thymidine incorporation or conversion of (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4- sulfophenyl)-2H-tetrazolium, inner salt (MTS reagent) into a soluble formazan dye by metabolically active cells.
  • MTS reagent inner salt
  • the MTS assay can be used not only to measure relative proliferation but also compound toxicity by comparing treated cells with untreated controls.
  • the soluble endostatin is used as a standard in the semi-HTPS endothelial cell migration assay.
  • endothelial and non-endothelial control cells are evaluated for their ability to migrate from the top layer of a 8 ⁇ m membrane to the bottom layer in response to a chemoattractant such as a growth factor or tumor cell conditioned medium.
  • Escherichia coli DH5 D was obtained from LIFE TECHNOLOGIES,
  • Streptomyces lividans TK24 and S. lividans TK24(pU303) were obtained from the NPDD Culture Collection, MERCK & CO, INC.
  • Plasmids pANT826 source of 5tt-minus plasmid sequences; FIG. 3) and pANT1201 (source o ⁇ snpR, .swp ⁇ -promoter, and a synthetic multiple cloning site; FIG. 4, FIG. 9A-9B) were constructed essentially from the snp locus (divergently transcribed snpR and snp A genes and their promoter regions (see WO9744439, Published Nov.
  • pJJlOl a pJJlOl derivative was described by Katz et al, 1983.
  • the plasmid pLT303 is also a derivative of pLJlOl the construction of which is described by Kieser et al, 1982.
  • the Genbank accession number for pIJlOl is M21778.
  • the nucleotide sequence encoding human endostatin (FIG. 6) is known in the art, was isolated from a human cDNA library and cloned into plasmid pMALcH#15. Anti-endostatin polyclonal antibodies were produced by standard methods.
  • Luria-Burtani (LB) medium as either liquid or solid (containing 15 g agar 1 " ) was used to cultivate E. coli (Ausubel, et al, 1994-98). When necessary for plasmid maintenance, ampicillin or neomycin was added at 50 mg-ml " or 15 mg-ml "
  • TSBP-S contains (in g*l " ): BBL tryptic soy broth, 30; Difco yeast extract, 2; dextrose, 6; and sucrose, 150. Where required, thiostrepton or neomycin was added to the growth medium to 50 mg-ml " or 15 mg-ml " , respectively.
  • DNA oligonucleotides encoding the leader peptide sequences of Streptomyces sp. strain C5 SnpA and of S. venezuelae alpha-amylase (Lammertyn, et al, 1996) shown in FIG. 1 for each strand were synthesized. The appropriate oligonucleotides were annealed to generate a double-stranded DNA with overhanging ends.
  • synthetic oligonucleotide linkers with Clal and BamHl cohesive ends suitable for ligation into Clal-BamRI digested pANT1201, were prepared in the following manner.
  • the two complementary single stranded oligonucleotides comprising the desired linker were combined in 1:1 stoichiometry, 30 mM each oligonucleotide, in annealing buffer containing lOmM Tris-Cl, pH 8.0 and 20mM MgCl2- The mixture was then incubated at 98°C for 10 minutes, and then allowed to cool to room temperature over two to three hours.
  • the annealed linker preparation was then diluted for use in later ligations with Clal-BamHI digested pANT1201.
  • the pertinent single stranded oligonucleotides used were: SnpA leader sequence linker:
  • SNP1 5'CGATGCGCATGCCCCTGTCCGTTCTCACCGCCGCCGGACTGAGCCTGGC GACCCTCGGTCTCGGCACCGCCGGTCCGGCCTCGGCGACCCCCACCG3' (SEQ ID NO:3)
  • SNP2 5'GATCCGGTGGGGGTCGCCGAGGCCGGACCGGCGGTGCCGAGACCGAGG GTCGCCAGGCTCAGTCCGGCGGCGGTGAGAACGGACAGGGGCATGCGCA T3' (SEQ ID NO:4)
  • VAA alpha-amylase
  • the human endostatin gene (FIG. 6) was cloned from pMALcH#15 as a BamHI- HinaSH (5' and 3' ends, respectively) fragment, compatible with the leader sequence derivatives of pANT1201.
  • the sti locus is a dispensable ca. 200 bp sequence that is the site of second strand initiation for the plasmid replication. Inclusion of the sti locus eliminates the accumulation of plasmid ssDNA and significantly increases plasmid stability and plasmid copy number (Deng et al, 1988; Zaman, et al, 1993).
  • Protoplasts of Streptomyces lividans TK24 were transformed with ⁇ ANT3045, pANT3042, or ⁇ ANT3052. After regeneration, the respective recombinant strains were streaked on R2YE medium for short term storage. The spore material from several plates was resuspended in 10% glycerol and frozen at - 20°C for long term storage. A typical fermentation involved using single R2YE grown colony of a recombinant S. lividans strain as the inoculum for 5 ml of TSBP-S in a 16x125 mm glass culture tube containing 5-7 4 mm glass beads.
  • the denatured samples were applied to a precast 12% Tris- HC1 acrylamide gel (BIORAD, Hercules, CA) and electrophoretically separated in SDS-PAGE buffer, running at 6 V-c ⁇ T . Upon completion of the electrophoresis, the gel was removed, equilibrated in electroblotting buffer, and transferred to a polyvinyl- difluoride membrane at 100V for 1 hour at 4°C.
  • TBS tris-buffered saline
  • NFDM non-fat dry milk
  • Polyclonal rabbit anti-human endostatin antibody was diluted 1:20,000 in TBS-0.1% Tween-20 (TTBS) containing 0.2% NFDM for use as the primary reagent.
  • the blocked membrane was incubated with the primary reagent overnight at 4°C. After several rinses with TTBS, the membrane was incubated with the secondary reagent, alkaline phosphatase-conjugated goat anti-rabbit IgG at a 1 :3,000 dilution in TTBS, for 1 hour at room temperature.
  • the membrane was soaked in CDP-STAR chemiluminescent phosphatase substrate (BIORAD) for 5 minutes at room temperature, and sealed in a plastic heat-sealable pouch.
  • KODAK X-OMAT AR film was exposed to the sealed membrane for 10 seconds to 5 minutes, and developed with an automatic developer.
  • Table 3 summarizes the results obtained using the plasmids described above.
  • the SnpA leader sequence-human endostatin fusion resulted in the accumulation of higher molecular weight, Western-blot reactive proteins internal to the S. lividans mycelia, but very little properly-sized human endostatin was secreted.
  • the S. venezuelae alpha-amylase leader sequence-human endostatin fusion in the sti- minus plasmid resulted in the production of a small amount of extracellular, properly- sized antibody-reactive endostatin protein (FIG. 8B, final lane, denoted with arrow).
  • the expected N-terminal sequence of the human endostatin produced by this process is: (NH2-VPPGSHSH... (SEQ ID NO:2)), where the sequence up to and including the first serine residue is derived from the vector as a remnant of the leader peptidase recognition sequence.
  • mice endostatin derived either from recombinant E. coli (O'Reilly et al, 1997) or from recombinant Pichia pastoris (Dhanabal et al, 1999), was reported to be biologically active in vivo.
  • endostatin activity it is thought that as long as the critical HSH (human endostatin) or HTH (mouse endostatin) domains, which are reported to be required for binding the Zn 2+ ligand
  • EGM media CLONETICS EGM bullet kit - Cat # CC-3125.
  • Reduced growth factor matrigel BECTON DICKINSON/COLLABORATIVE BIOMEDICAL PRODUCTS cat# 40230C.
  • Vascular endothelial cell growth factor (VegF) R&D SYSTEMS, Cat # 293-VE. Aortas are isolated from rats with a weight range of 120 - 160 g.
  • the aorta After isolation, the aorta is immediately placed in EGM media without hydrocortisone.
  • the connective tissue is peeled back from the aorta under a dissecting microscope and each end is cut and discarded to avoid using any tissue injured with the hemostat during isolation.
  • the aortas and rings are kept on ice until being embedded.
  • the aortas are sliced into rings of approximately 1 mm width while bathed in media in a petri dish. Great care should be taken to discard the rings that contain small branching vessels - this eliminates an additional cut edge as a variable. Rings from each aorta are rinsed 5 times in 5 ml each with EGM (CLONETICS) medium without hydrocortisone added.
  • the rings are placed in individual wells of a 48 well plate that has been pre-coated with 125 ⁇ L of reduced growth factor (RDF) matrigel.
  • the rings are randomized by mixing all aortic rings in one tube prior to placement in the 48 well plate. This helps to diminish the possibility of a single dose group being affected by rat to rat variation as well as possible distal and proximal differences. Alternatively, to diminish the possibility that a single dose group is affected by rat to rat variation, the rings from individual aortas are kept separated. A control group is included within each aorta and the dosed rings within that aorta are compared to the corresponding control group.
  • RDF reduced growth factor
  • RGF matrigel is thawed on ice and is kept on ice during the coating.
  • the matrigel is added to each well with a pipette tip and the tip is drawn in a circular motion around the well to insure complete coating.
  • the matrigel will solidify at room temperature after approximately 10 min. This time can be shortened by incubating the plates at 37°C.
  • the matrigel becomes solid EGM media without hydrocortisone containing 100 ng/ml VegF and any other inducer or inhibitors is added at a total volume of 500 ⁇ L/ well. Only the internal 24 wells of the plate are used and PBS is added to the outer wells to avoid evaporation problems in the sample wells. The plates are incubated at 37°C in a 5% CO2 incubator and checked daily for growth.
  • Tube outgrowth typically starts after 3-4 days.
  • Photographs are taken of each well using EKTACHROME 64T slide film with pseudo dark field microscopy using a 4x objective. Outgrowth is quantitated visually on a 4 point grading scale according to the following: massive growth — less than massive growth — little growth — no growth.
  • images of the growth from the rings are captured with a
  • EGM media CLONETICS EGM bullet kit - Cat #CC- 3125.
  • Endothelial cell (EC)trypsin/EDTA CLONETICS, Cat# CC-5012.
  • Vascular endothelial cell growth factor (VegF) R&D SYSTEMS, Cat # 293-VE.
  • Basic fibroblast growth factor (bFGF) R&D SYSTEMS, Cat # 233-FB.
  • Fetal Bovine serum (FBS) Life Technologies, Cat# 10082147.
  • Human umbilical vein endothelial cells (HUVEC) CLONETICS, Cat# CC-2519.
  • HUVECs are grown and maintained in EGM medium. Sub-confluent cells at no greater than the 7th passage are removed from their flasks with EC trypsin/EDTA, resuspended in DMEM, 10% FBS and centrifuged at 1000 rpm for 5 minutes at 4°C . The supernatant fluid is discarded and the cell pellet is resuspended in an appropriate volume of DMEM, 10% FBS. The cell number is determined with a hemacytometer using trypan blue vital staining solution. After counting the appropriate dilution is made with DMEM, 10% FBS to achieve a cell concentration of 40,000 cells/ml.
  • 100 ⁇ l of this suspension is added to each well of a 96 well plate to achieve a final concentration of 4,000 cells/ well.
  • Cells are growth-arrested for 24 hours at 37°C in a humidified atmosphere containing 5% CO2- The medium is replaced with 100 ⁇ l fresh DMEM, 10% FBS containing either 1 ng/ml bFGF or 50 ng/ml VegF +/- endostatin or compounds to be evaluated.
  • Control wells are dosed with the appropriate vehicle (e.g. 0.25% [v/v] DMSO).
  • each well is dosed with 10 ⁇ l of an 80 ⁇ Ci/ ml solution of 3H-thymidine to yield a final concentration of 8 ⁇ Ci/ well.
  • 5% C02 the medium is removed and each well is washed twice with 350 ⁇ l of PBS containing 1 mg/ml bovine serum albumin.
  • 100 ⁇ l of 1.5 N NaOH is added to each well and incubated for 30 min at 37°C.
  • the cell lysates are transferred to 7 ml glass scintillation vials containing 150 ⁇ l of water. Scintillation cocktail (5 ml) is added and the cell associated radioactivity is determined by liquid scintillation spectroscopy.
  • Human endothelial cell line ECV304 ATCC Cat# CRL-1998.
  • Dulbecco's minimal essential media (DMEM) LIFE TECHNOLOGIES, Cat# 11965- 092.
  • Fetal Bovine serum (FBS) LIFE TECHNOLOGIES, Cat# 10082147.
  • Vascular endothelial cell growth factor (VegF) R&D SYSTEMS, Cat # 293-VE.
  • ECV304 cells are grown and maintained in Medium 199 supplemented with 10% FBS and 2 mM L-glutamine. Sub-confluent cells are removed from their flasks with EC trypsin/EDTA, resuspended in Medium 199, 10% FBS and centrifuged at 1000 rpm for 5 minutes at 4°C . The supernatant fluid is discarded and the cell pellet is resuspended in an appropriate volume of Medium 199, 10% FBS. The cell number is determined with a hemacytometer using trypan blue vital staining solution. After counting the cells are diluted to 150,000 cells/ml with Medium 199 containing 0.5% FBS.
  • the lower chamber is filled with Medium 199 contaimng 25 ng/ml bFGF.
  • the upper chamber is seeded with 7,500 cells/ well +/- endostatin or compounds to be evaluated. Control wells are dosed with the appropriate vehicle (e.g. 0.25% [v/v] DMSO). Cells are allowed to migrate for 4 hours at 37°C. After this incubation the media is removed from the upper chamber and the cells on the upper chamber side of the membrane removed with a cotton swab and the cells on the under side of the membrane (migrated) are fixed and stained with the LEUKOSAT stain kit according to the manufacturers' directions. The number of stained nuclei on the under side of the membrane are quantitated using IMAGE PRO PLUS software after capturing the image with a digital camera and saving it as black and white TIF file.
  • the soluble endostatin can be used to isolate the putative receptor of endostatin.
  • a first approach utilizes endostatin protein covalently linked to a chromatography resin and, as an alternative, endostatin protein bound to a endostatin antibody-resin (an antibody raised to endostatin). These resins are used to capture the receptor from solubilized endothelial cell membranes.
  • Various solubilization methods can be employed. Proteins that are found to be bound to endostatin columns may be eluted with salt gradients in the case of the covalently linked endostatin columns and low pH solution in the case of the antibody-linked endostatin column. Any proteins that are eluted are evaluated by mass spectrometry and N-terminal sequencing as well as peptide mapping after proteolytic cleavage.
  • the soluble endostatin can be immobilized on plastic plates and used to "pan" for transiently transfected cells expressing the putative receptor.
  • the source of the DNA for these transfections is an endothelial cell cDNA library. This method allows for the identification and cloning of the putative endostatin receptor
  • the endostatin produced by the method of this invention is mixed with a pharmaceutically acceptable carrier to make a pharmaceutical composition.
  • the composition is administered to a patient in need of treatment for a condition that can be ameliorated by prophylactic or therapeutic treatment with an inhibitor of angiogenesis.
  • injectable preparations of soluble endostatin are preferred.
  • injectable diluents known and used in the art can be used to make appropriate injectable compositions.
  • Streptomyces sp. C5 s/ipA MRMP SVLTAAG SLATLGLGTAGPASA ⁇ ⁇ TAEG pANT3045 A small quantity of antibody-reactive (SEQ ID NO: 7) protein was produced. Most of the recombinant protein remained intracellular, and by size, appeared to still contain signal sequence.
  • INS h into fermentation a
  • the sti locus confers higher copy number and stability on the plasmids.
  • the results of these experimentals were obtained by SDS-PAGE followed by Western blot of the proteins onto PVDF membrane and reaction with anti- human endostatin polyclonal antibody, c
  • the -1 and +1 refer to the relative position of amino acids with respect to the predicted signal sequence cleavage site, d The S.
  • MOUSE (Picl ⁇ ia-C) EFHTHQDFQPVLHLVALNT . 12 Dhanabal et al., 1999 O MOUSE (B 16F10 Melanoma cells) HTHQDFQPVLHLVALN . 13 Boehm et al., 1998

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Abstract

L'invention concerne un procédé de préparation d'une endostatine recombinée, soluble, dans Streptomyces. Elle concerne également des préparations contenant cette endostatine recombinée, soluble, destinée à inhiber l'angiogenèse ou destinée au traitement du cancer. L'invention concerne également des dosages dans lesquels on utilise cette endostatine recombinée, soluble.
PCT/US2000/009747 1999-04-13 2000-04-12 ENDOSTATINE RECOMBINEE, SOLUBLE, ET PROCEDE DE PREPARATION A PARTIR DE $i(STREPTOMYCES SP) WO2000060945A1 (fr)

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AU43420/00A AU4342000A (en) 1999-04-13 2000-04-12 Soluble recombinant endostatin and method of making same from (streptomyces sp)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001060839A3 (fr) * 2000-02-18 2002-02-21 Us Health Procede de renaturation d'endostatine de recombinaison
WO2005021756A1 (fr) * 2003-08-29 2005-03-10 Children's Medical Center Corporation Peptides anti-angiogeniques comprenant une extremite n-terminale d'endostatine
US7524811B2 (en) 2003-08-29 2009-04-28 Children's Medical Center Corporation Anti-angiogenic peptides from the N-terminus of endostatin

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997015666A1 (fr) * 1995-10-23 1997-05-01 The Children's Medical Center Corporation Compositions therapeutiques anti-angiogenese et procedes associes
US5854205A (en) * 1995-10-23 1998-12-29 The Children's Medical Center Corporation Therapeutic antiangiogenic compositions and methods

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997015666A1 (fr) * 1995-10-23 1997-05-01 The Children's Medical Center Corporation Compositions therapeutiques anti-angiogenese et procedes associes
US5854205A (en) * 1995-10-23 1998-12-29 The Children's Medical Center Corporation Therapeutic antiangiogenic compositions and methods

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001060839A3 (fr) * 2000-02-18 2002-02-21 Us Health Procede de renaturation d'endostatine de recombinaison
WO2005021756A1 (fr) * 2003-08-29 2005-03-10 Children's Medical Center Corporation Peptides anti-angiogeniques comprenant une extremite n-terminale d'endostatine
WO2005042566A3 (fr) * 2003-08-29 2005-10-06 Childrens Medical Center Peptides pour traitement et prevention de l'endometriose
JP2007525972A (ja) * 2003-08-29 2007-09-13 チルドレンズ メディカル センター コーポレーション エンドスタチンのｎ末端からの抗血管新生性ペプチド
US7524811B2 (en) 2003-08-29 2009-04-28 Children's Medical Center Corporation Anti-angiogenic peptides from the N-terminus of endostatin
US7645735B2 (en) 2003-08-29 2010-01-12 Children's Medical Center Corporation Anti-angiogenic peptides for treating or preventing endometriosis

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