Classifications

• • 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
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
  • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
  • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
  • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
  • C12N9/6424—Serine endopeptidases (3.4.21)
  • C12N9/6456—Plasminogen activators
  • C12N9/6459—Plasminogen activators t-plasminogen activator (3.4.21.68), i.e. tPA
• • 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
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
  • C12Y304/21—Serine endopeptidases (3.4.21)
  • C12Y304/21069—Protein C activated (3.4.21.69)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• This invention relates to a human tissue plasminogen activator composition made using recombinant DNA technology in myeloma cells. More particularly, it relates to a method of producing a tissue plasminogen activator composition of improved homogeneity relative to other activator preparations made in animal cells.
• tPA Human tissue plasminogen activator
• EPO publication number 41766 discloses a human melanoma cell line which secretes a tPA said to be indistinguishable both immunologically and in amino acid composition from human tissue plasminogen activator isolated from normal tissues.
• the cell line known as Bowe's melanoma, has provided those skilled in the art with a culturable source of mRNA encoding human tPA and a source of the protein itself.
• the methods involve the construction of radiolabelled oligonucleotide probes which are used in hybridization experiments against a cDNA library constructed with reverse transcriptase from size fractionated mRNA derived from the melanoma cell line. See, e.g., Pennica et al, Infra. Companies engaged in the exploitation of recombinant DNA technology have achieved expression of a protein having the amino acid sequence of human tPA in cells which do not normally produce it. See, for example, GB 2 119 804A, published November 23, 1983, which discloses expression of the tPA gene in Chinese Hamster Ovary (CHO) cells.
• tPA product produced in CHO cells, is currently proceeding through clinical testing. Generally, this tPA product has been found to induce sometimes significant peripheral bleeding, suggesting that its affinity for fibrin is less than optimal, and to exhibit a disappointing level of activity when used in vivo as an intravascular fibrinolytic agent. These observations suggest that the currently available human recombinant tPA products may be different from the natural protein at the tertiary structural level. Accordingly, while these commercial products may correspond in amino acid sequence to naturally produced human tPA, in this important aspect they may not be the same substance as endogeneous human tPA.
• myeloma cells constitute an ideal cell type for the expression and post-translational modification of human tissue plasminogen activator.
• Myeloma cells transformed with vectors designed to confer the ability to express the tPA gene can produce a mixture of structurally related proteins having tPA activity and having improved homogeneity as compared with tPA products made in other known types of transformants or in Bowe's melanoma cells.
• myeloma cells, as a class generally are relatively easily cultured and can be induced to secrete tPA into the extracellular medium in the absence of serum supplements. This very significantly simplifies purification of the product.
• EACA epsilon amino caproic acid
• the invention comprises a process for producing a human tissue plasminogen activator composition.
• the process comprises the steps of expressing a DNA comprising the coding sequence of human tPA (see figure 2) or allelic variants thereof, a different DNA encoding the same amino acid sequence or allelic variant, or a DNA sequence encoding an analog sequence of amino acids, in a culturable myeloma cell line, and then harvesting the tPA product.
• Transformants expressing tPA made by transfecting J558L cells, (deposited with the American Type Culture Collection, Rockville, Maryland, under accession number ATCC CRL 9132) are preferred.
• the J558 cell line is a murine myeloma which secretes lambda light chain of immunoglobulin A, but does not secrete intact immunoglobulin.
• the myeloma cell line secreting the tPA is maintained in medium free of added serum prior to harvest of the tPA, and the cells are cultured in porous microcapsules.
• EACA may be used in the tissue culture medium to inhibit proteolytic degradation of the single chain tPA protein. Purification may be accomplished using a protocol involving the steps of fractionating proteins in the medium on high surface area glass and on an affinity matrix comprising bound antibody, preferably monoclonal antibody, against an epitope of the activator.
• the activator is eluted from the immobilized antibody matrix using a citrate ion solution containing EACA. As the pH of the protein solution is raised, the EACA maintains solubility. This procedure permits one to produce a tPA product having high activity dissolved in physiologically compatible buffer comprising citrate and EACA.
• the resulting purified product is characterized as follows: a. Ninety percent or more of the total active protein contained in the preparation is a single-chain, high activity human tPA. b.
• the tPA composition when subjected to polyacrylamide gel electrophoresis as disclosed herein, is separated into two bands. differentiated by their respective levels of glycosylation, with the major, lower molecular weight band representing at least about 70% of the total active protein, and typically greater than about 90%.
• the composition also comprises at least about 70%, and preferably more than about 90%, of a protein consisting of the known amino acid sequence of full length human tPA protein minus its first 35 amino terminal residues ("mature form"). Typically, essentially all of the remaining active protein has the same sequence plus a Gly Ala Arg tripeptide on its amino end.
• This composition can be dissolved at high concentration in physiologically compatible solution containing citrate and EACA to provide a therapeutically effective fibrinolytically active injectable composition.
• Animal tests indicate that the tPA manufactured as disclosed herein is at least as active as the prior art material produced in CHO and Bowe's melanoma.
• In vitro tests using the chromogenic substrate S-2288 test system indicate that the tPA disclosed herein exhibits an increase in Vmax (and no decrease in Km), when activated by monomeric fibrin, and thus, at least at higher concentrations of substrate, it may be a catalytically more efficient preparation relative to prior art tPAs. Tests on primates designed to elucidate further the new tPA's pharmacokinetic properties should determine whether this difference has practical therapeutic consequences.
• Figure 1 is a two-dimensional representation of the full length human tPA protein showing what are believed to be its disulfide linkages, its glycosylation sites, and its amino acid sequence (amino acids identified in standard code). Residues -1 to -35 constitute a signal peptide and variable pro sequence which is cleaved from the mature protein;
• Figure 2 is the DNA sequence of the human tPA gene, including its signal peptide region and its start and stop signals, but excluding its 3' untranslated region;
• Figures 3A - 3D are the full DNA sequence of plasmid pEMpl - tPA, the preferred expression vector for use in the manufacture of tPA in accordance with the invention
• Figure 4 is a diagram of plasmid pEMpl - tPA
• Figure 5 is a graph of optical density vs. fraction number illustrating the affinity chromatography elution pattern of the high activity human tPA composition of the invention
• Figure 6 is a photographic reproduction of an exemplary polyacrylamide gel, electrophoresis separation of the tPA composition of the invention.
• Figure 7 is a graph of the reciprocal of the rate of plasminogen activation (optical density units per minute) versus the reciprocal of plasminogen concentration (as measured by the concentration of S-2288) in the presence and absence (open circles) of fibrin monomer. Descri tion
• the invention provides a novel human tissue plasminogen activator composition comprising an amino acid sequence which is the same as or analogous to the known, published sequence, but which differs from currently available tPA products in its pattern of glycosylation and protein subspecies distribution.
• the invention is based on the discovery that myeloma cells provide an intracellular environment capable of modifying the tPA protein after translation and before secretion to result in an improved, relatively homogeneous product. Animal and in.
• the tPA composition of the invention is similar to Bowe's melanoma tPA, excepting that the new tPA exhibits an increase in Vmax in the presence of fibrin, whereas Bowe's or CHO produced tPAs do not.
• Fig. 1 depicts a two-dimensional model of the single chain tPA protein.
• the standard one letter code for each of the amino acids is given in the open circles.
• the bars indicate the deduced location of disulfide bridges.
• the triangle indicates the cleavage site between the heavy and light chains.
• the labels Ser, Asp, His together define what is believed to be the protease active site (see Ny et al, Proc. Natl. Acad. Sci., USA, V. 81, 1984, p. 5358).
• four asparagine residues 10, 12, 14, and 16 are illustrated as being glycosylated.
• the first 35 residues are cleaved from the protein during maturation, and by convention are numbered -35 to -1, with the -1 residue adjacent the first residue on the mature protein (Ser + 1) .
• tPA made by expression in myeloma cells is structurally different from tPA expressed in melanoma or CHO cells. While the relationship between the structure of the novel tPA and prior art materials has not as yet been elucidated fully, it has been established that tPA produced from Bowe's melanoma comprises two closely related species: one (approx. 70% by weight) wherein sugar residues are attached to glycosylation sites 10 and 16; and one (approx. 30% by weight) wherein sugar residues are attached to sites 10, 12, and 16. No sugar residues appear at glycosylation site 14 in the Bowe's melanoma tPA.
• the glycosylation pattern of tPA made in myeloma cells is not yet known. However, two species are produced at a ratio that typically exceeds the 70:30 ratio of tPA species from Bowe's melanoma cells. In production runs using the process of the invention, greater than a 90:10 ratio is often achieved, with the major fraction being lightly glycosylated and having a lower molecular weight than the minor fraction. Polyacrylamide gel electrophoresis separations of the composition often results in an apparent single band of the lower molecular weight form.
• the glutamine - glutamic acid linkage labelled B in the drawing of Fig. 1 is believed to delimit the signal peptide from the reminder of the uncleaved tPA molecule. Further cleavage must occur as tPAs isolated from Bowe's cell culture and other natural sources have several additional residues cleaved off as pro sequences. Enzymes in the myeloma cells differentially cleave the N-terminus of the protein, resulting in production of a preponderance of a protein subspecies beginning with serine (amino acid 36 of Figure 1, + 1 of the mature protein). Small amounts of a longer protein, beginning with glycine at amino acid 33 of the drawing (-3), often are also produced. Typically, about 70% and preferably more than 90% of the total active protein is the shorter subspecies.
• Arg-Ile amino acids 275-276
• a cleavage site open to attack by endoprotease which results in a disulfide linked two chain form of the protein.
• protease inhibitors such as epsilon amino caproic acid in the culture media and the harvested product hinders cleavage of the single chain such that typically more than 90% of active protein in the compositions of the invention is in the single chain form.
• the recombinant DNA techniques for manufacturing expression vectors and myeloma transformants useful in this invention are well-known and developed. They involve the use of various restriction enzymes which make sequence-specific cuts in DNA to produce blunt ends or cohesive ends, DNA ligases, techniques enabling enzymatic addition of sticky ends to blunt-ended DNA molecules, cDNA synthesis techniques, synthetic probes for isolating genes having a particular function, conventional transfection techniques, and equally conventional techniques for cloning and subcloning DNA.
• Various types of vectors may be used such as plasmids and viruses including animal viruses and phages.
• the vectors will normally exploit various marker genes which impart to a successfully transfected cell a detectable phenotypic property which can be used to identify which individuals of a population of cells have successfully incorporated the recombinant DNA of the vector.
• Preferred markers for use in myeloma cell lines comprise DNAs encoding an enzyme normally not expressed (or expressed only at low levels) by the cells which enables the cells to survive in a medium containing a toxin.
• TK thymidine kinase
• APRT adenosine phosphoribosyltransferase
• HPRT hypoxanthine phosphoriboxyl transferase
• TK thymidine kinase
• APRT adenosine phosphoribosyltransferase
• HPRT hypoxanthine phosphoriboxyl transferase
• DHFR dihydrofolate reductase
• XGPRT xanthine-guanosine phosphoribosyl transferase
• Fig. 2 discloses what is believed to be the full coding sequence of natural human tPA, taken from the published literature, starting with the ATG initiation codon and ending with the TGA translation stop signal.
• the gene includes the bases coding for the signal peptide region but omits a 3' untranslated region and the polyadenylation site normally present beyond the stop signal.
• the base designations are numbered from 20 - 1710.
• This coding sequence is what has been used to construct expression vectors which have been transfected into myeloma cell lines to produce the tPA claimed herein.
• a different DNA from that set forth in Fig. 2 can be constructed which encodes the same protein sequence.
• Natural allelic variations of the gene may exist from individual to individual. Also, it is possible that various insertions or deletions, base substitutions, or additions in the coding sequence would produce a human tPA amino acid sequence which is modified after translation in myeloma cells like the protein encoded by the natural gene. All such proteins made from genes specifying a different but functionally operating amino acid sequence are herein referred to as an analog sequence. All such analog sequences are considered within the scope of this invention as equivalent materials.
• Figures 3a - 3d and 4 disclose, respectively, the nucleotide sequence of the preferred plasmid vector used in the tPA production process of this invention, and a restriction map of the plasmid.
• This preferred vector is designated pEMpl-tPA, and comprises 7533 base pairs. It includes an enhancer element which enhances transcription of mRNA from the cDNA encoding tPA (see Gillies et al, Cell, 1983, infra and U.S. 4,663,281), and exploits vector construction principles which regulate selectively the enhancer function in accordance with the disclosure of copending U.S. application Serial No. 837,595, filed March 7, 1986. The natural 3' untranslated region of the full length tPA DNA was truncated to promote mRNA stability.
• the vector disclosed in Figs. 3 and 4, and other suitable expression vectors, are used in accordance with the invention to transfeet myeloma cells, preferably of murine origin, such as cell line J558 (ATCC-TIB 6), Sp2/0-Agl4 (ATCC CRL 1581), and P3X63-Ag8.653 (ATCC CRL 1580).
• the preferred myeloma cell line is J558L (ATCC CRL 9132, See Oi et al, Proc. Natl. Acad. Sci., USA, V. 80, p. 825 1983).
• These cells comprise malignant transformants of B cells from the circulatory system of the Balb/C mouse, and are derived from myeloid tissue.
• myeloma cells of murine or rat origin is preferred over human myeloma cells as the possibility of contamination of the recombinant tPA by human viruses is greatly reduced.
• myeloma cells of the type set forth above may be cultured in suspension or preferably within microcapsules in accordance with the procedure disclosed in US Patent No. 4,409,331 to F. Lim, the disclosure of which is incorporated herein by reference.
• the cells be cultured in serum-free medium and in the presence of epsilon amino caproic acid.
• the extracellular protein may be harvested daily as the medium is replaced. This approach has the advantage of producing the product substantially free of the effect of contaminating proteolytic enzymes, tPA complexing agents, and other inactivating factors often present in bovine or equine sera.
• the preferred J558L cell secretes lambda light chain of immunoglobulin A in significant quantities. This and other contaminating proteins can be readily separated from the tPA product using the simple purification procedure disclosed below. The tPA product purified in accordance with this procedure retains the activity of the molecule as secreted.
• TPA binds to the glass under suitable conditions of pH whereas, for example, immunoglobulins have only low non-specific affinity and will pass through or may be selectively eluted from a controlled pore glass column. Fractions rich in the tPA may be eluted from the glass by treatment with a suitable elution reagent such as Tris buffered saline.
• a suitable elution reagent such as Tris buffered saline.
• the controlled pore glass fractionation can result in a 40 - 50 fold increase in concentration and a 30 - 40 fold purification of the tPA product from the harvested media.
• An additional step in the purification involves conventional affinity chromotography using immobilized antibodies, preferably a monoclonal antibody, against an epitope of the activator.
• the tPA may be eluted from the antibody matrix at pH 3 using citrate ion solution. Subsequently, the tPA containing fractions may be neutralized to physiologic pH (EACA prevents tPA precipitation at this pH.
• This solution often has a protein content comprising greater than 90% single chain, active, human tPA, typically containing only small amounts of two chain materials. It may be suitable for therapeutic use as is, as citrate buffer and EACA have been approved for intravenous injection.
• An expression vector, pEMpl was constructed from the following fragments: (a) a 2.25 PvuII-BamHI fragment from pSV2- ⁇ pt (Mulligan and Berg, Science; V. 209, 1422-1427, 1980) containing the SV40 enhancer and early region promoter, the E. coli qpt gene, the SV40 small tumor-antigen intervening sequence, and the SV40 transcription termination and polyadenylation signals; (b) a 2.3 kb PvuII-EcoRI fragment from pBR322 containing the ampicillinase gene and the bacterial origin of replication (Sutcliffe, Proc. Natl. Acad. Sci, USA, V.
• cDNA for tPA was obtained by standard techniques (Maniatis, supra) .
• PolyA+ RNA isolated from cultured Bowe's cells was primed with oligo (dT) for first strand synthesis with reverse transcriptase, nick-translated with RNase H and DNA polymerase I for second strand synthesis (Gubler & Hoffman, Gene, V. 25, 263, 1983), methylated with EcoRI methylase, ligated to EcoRI linkers, and inserted into the EcoRI site of the lambda phage vector gtlO.
• Phage plaques were screened with radioactively labelled oligonucleotide tPA-specific probes, the sequences of which were determined from the published DNA sequence of the tPA cDNA (Pennica et al. Nature, V. 301, pp. 214-231, January, 1983). TPA clones containing the entire coding region, and approximately 800 base pairs of the 3' UT region were identified and confirmed by nucleotide sequencing. The extremely long 3' UT of the tPA cDNA was discovered to cause messenger RNA instability. The bulk of the 3* UT region was therefore eliminated by cleaving at the Sau3A site located 34 nucleotides downstream of the translation stop site. A 1.7 kb fragment containing the tPA cDNA with the truncated 3' UT was ligated to Xhol linkers.
• the tPA cDNA fragment was then inserted into the unique Xhol site present in pEMpl vector to produce recombinant plasmid pEMpl-tPA.
• a diagram of vector pEMpl-tPA is given in Figure 4; the complete nucleotide sequence of the vector is disclosed in Figures 3A-3D.
• the tPA-containing plasmids were transfected into J558L myeloma cells by the protoplast fusion method (Gillies et al, supra) .
• Cells containing the plasmid, and therefore the apt gene, were selected by culturing in media containing mycophenolic acid. Resistant colonies containing pEMpl-tPA were subcloned and screened for tPA expression.
• the synthesis and secretion of tPA into the medium was determined by means of an assay in which tPA converts plasminogen to plasmin, which then cleaves the chromogenic tripeptide S2251 (Pennica et al, supra) .
• transformants were cultured for 48 hours in serum-free medium before assay. Activity was measured in international units (IU) based on a standard supplied by the World Health Organization and confirmed by a tPA standard obtained from American Diagnostics, Inc. Sixteen of the transformants obtained with pEMpl-tPA, were assayed for tPA activity in the medium. Seven of the 16 were positive, with activities ranging between 1000 and 6000 IU/ml.
• a clone of the transformed J558L cells designated was selected for further experiments involving the laboratory scale production of tPA.
• a seed culture of the clone was grown in Dulbecco's modified Eagle Medium (Gibco, Catalogue #320-1965) supplemented with 10% fetal calf serum (Hazelton) , penicillin, streptomycin and 2 mM glutamine.
• cells medium were encapsulated in accordance with the method of Lim (See U.S., 4,409,331), and cultured in medium containing double the normal amount of amino acids and reduced CaCl, concentration (EMA) .
• Lim See U.S., 4,409,331
• EMA reduced CaCl, concentration
• the cell culture was mixed with 1.6% sodium alginate which was formed into spheres and added to a CaCl, solution to produce gel balls containing cells. These were suspended in a sodium chloride solution containing 25 TI units/ml aprotinin.
• the calcium alginate gel balls were washed in saline three times, suspended six minutes in an aqueous solution of poly L lysine (750 mg/1 in a 0.9% sodium chloride) to deposit a hydrogel membrane about the gel balls.
• the capsules were allowed to settle, the supernatant was aspirated off, and the capsules were washed in 60 ml 2% CHES buffer, pH 8.4, in an aqueous solution comprising 0.6% NaCl and 0.2% CaCl,. After three minutes, the capsules were washed in 0.3% CaCl- for 3 minutes, physiological saline for three minutes, and then coated a second time with the poly L lysine (approximately 62,000 dalton average molecular weight), using a 400 mg/1 solution of PLL in 0.9% NaCl wash for 5 minutes.
• the capsules were then settled, aspirated, washed in saline, and treated for 4 minutes in a 0.15% sodium alginate (low viscosity) solution in saline.
• the capsules were then washed once more with saline and twice, (10 minutes, 6 minutes) in 55 mM sodium citrate (in 0.9%
• the capsules contained approximately
• the capsules were washed in serum-free. sterile media and then suspended in a serum-free medium comprising the EMA medium supplemented with 1 mg/ml bovine serum albumin, 20 micromolar ferric fructose, 1 microgram per ml of each of linoleic and oleic acids, 5 mM EACA and 1.2 micrograms per ml ethanolamine.
• the encapsulated culture is maintained in this medium for an additional 12 to 30 days. About two-thirds of the media volume may be changed daily and saved.
• the cell Before changing to serum free medium, the cell
• Samples of the medium were taken starting on day 15 through day 25 and assayed using an activity assay and an ELISA to demonstrate tPA production.
• the tPA in the media was purified, first by subjecting it to a high surface area glass matrix (controlled pore glass, Sigma PG-1,000-200) packed into a column.
• the column was washed with 4 liters of 100 mM NaOH, followed by 4 liters of 100 mM HCl and 4 liters deionized water.
• the column was then loaded with the serum-free medium from the culture. Serum-containing medium may be used, but it greatly reduces the capacity of the column.
• the medium was filtered through a 6 micrometer Sealclean filter, and loaded onto the CPG column at a flow rate of 255 cm/hour.
• the column was washed with 4 liters of buffer containing 200 mM Tris HCl, pH 8.3, 5 mM EDTA, 0.01% surfactant (Tween 80), 5 mM EACA and 500 mM NaCl.
• This step elutes weakly bound protein, for example, any aprotinin, and the IgA light chain secreted by the tPA-producing clones.
• the tPA is eluted with 4 liters of buffer containing 500 mM Tris HCl, pH 8.3, 5 mM EDTA, 0.01% Tween 80, 5 mM EACA, and 1.5 M sodium chloride. This step elutes the bound tPA, producing a fraction containing approximately 50% tPA mixed with other proteins.
• the column may then be washed with 4 liters 100 mM NaOH followed by 4 liters 100 mM HCl to elute any remaining protein for recycle.
• This crude material was then further purified by affinity chromatography.
• affinity chromatography For this step, the procedure of Kohler and Milstein was used to produce a hybridoma which secretes a monoclonal antibody specific to an epitope of the human tPA product.
• a Balb/C mouse was immunized with human tPA obtained from American Diagnostics Inc. On day zero, 10 micrograms of material in complete Freunds adjuvant were injected intraperitoneally (IP). Fourteen to fifteen days later, an additional 10 micrograms of the tPA in incomplete Freunds adjuvant was injected IP as a booster; and at day 21, the mouse serum was screened for anti-tPA activity by ELISA. A 50% titer measured at a dilution greater than 1/1,000 was taken as a positive reading.
• the positive animals were boosted with 10 micrograms tPA in phosphate buffered saline introduced IP and sacrificed three days later. Their spleens were forced through a screen to form a single cell suspension.
• the B cells and the SP2/0 myeloma were suspended together at a ratio of 4:1 B cells to myeloma. Fusion was conducted with 45% polyethylene glycol (1000 daltons). Selection of clones was conducted in HAT medium for 7 days, and surviving clones were grown in HT Dulbecco's Minimum Essential Medium supplemented with 10% fetal calf serum. The fusion frequency was 1-3x10 .
• the tPA antibody secreting clones were then further screened for a hybridoma which secreted an antibody which reacted with both the double and single chain tPA species, but did not react with urokinase or with tPA complexed with serum tPA inhibitor.
• the selected hybridoma accordingly produced monoclonal antibody against an epitope of the tPA not involved with its enzymatic activity.
• An antibody gel column was produced by immobilizing the tPA monoclonal antibody on cyanogen bromide activated Sepharose (Pharmacia) according to the method of Kohn and Wilchek (Biochem. Biophys. Res. Comm., V. 107, pp. 878-884, (1982).
• the gel is activated in 60% acetone at a -15 to -20° C. with cyanogen bromide using triethylamine as a "cyano transfer" reagent.
• the activated gel is then transferred to water (4 ⁇ C) and the antibody is added suspended in sodium carbonate buffer, pH 8.3, containing sodium chloride. After mixing for two hours at room temperature, the unsubstituted groups are blocked with ethanolamine in the carbonate buffer.
• the anti-tPA Sepharose is packed into a 2.6 cm diameter column, 4 cms high, and equilibrated with 0.1M sodium citrate, pH 7.0, IM NaCl, 20mM epsilon amino caproic acid, and 0.01% Tween 80.
• the caproic acid is required as the antibody is contaminated with protease which might reduce the yield of tPA in the absence of a protease inhibitor.
• the partly purified tPA product from the controlled pore glass step contains 500 ml Tris, 1.5 M NaCl, EDTA and surfactant. Its pH is adjusted to 7, and it is applied to the column.
• the column is washed in the citrate buffer for two column volumes, and then the tPA is eluted with 0.15 M citric acid, pH 3.0, supplemented with 20 mM epsilon amino caproic acid, and 0.01% Tween 80.
• An exemplary elution profile is set forth in Fig. 5. TPA is collected and the pH of the solution is adjusted to 7. Of the total protein content of these elution volumes, greater than 95% is human tPA.
• the purified tPA composition comprises single chain tPA species, as shown by separation on reducing gels.
• the purified product was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis.
• the results of the experiment are shown in Figure 6.
• the procedure was conducted on 10% gel under reducing conditions according to the method of Laemmli, NATURE, 227. 680-685 (1970). Lanes one, two, and nine were loaded with low molecular weight standards from pharmacia. Each of lanes 3-8 were loaded with 5.0 micrograms of various tPA compositions. Lanes 3 and 8 contained the tPA composition of the invention. Lanes 4 and 7 contained tPA from separate sources made by expression in CHO cells. Lanes 5 and 6 contained duplicate samples of a tPA composition from Bowe's melanoma.
• CHO tPA migrates slower than either melanoma or myeloma tPA. Both CHO and Bowe's material migrate as two distinct bands, indicating a molecular weight heterogeneity due to differences in glycosylation. The upper band is thought to be due to the form of tPA with 3 N-linked glycosylation sites, and the lower band thought to be due to the form with 2 N-linked glycosylation sites.
• the myeloma tPA of this invention has essentially the same mobility as Bowe's melanoma tPA, indicating that its glycosylation probably closely resembles that of the "natural" tPA, unlike the CHO-derived material.
• N-terminal sequencing of myeloma tPA on Applied Biosystems 470 and 477A gas phase sequencers indicate the presence of two species of tPA, one which starts at Ser + 1 and the other having the sequence GLY ALA ARG before Ser + 1 form as determined from the ratio of primary to secondary sequence obtained during N terminal analysis. In Bowe's melanoma tPA the ratio between the two forms is 1:1 (Wallen et al Eur J. Biochem 132 p. 681-686 1983).
• TPA compositions produced as set forth above have been tested extensively in in vitro assays and in animal models using established protocols to assess its efficacy as an intravascular fibrinolytic agent versus other tPA compositions and other fibrinolytic preparations.
• DPA recombinant, predominantly single chain tPA composition of this invention
• tPA produced by Bowe's melanoma from several different sources: single and double chain species from American Diagnostic, a double chain species from Bioscot (BSdc), and a single chain species.
• the fibrinolytic activity of the different tPA species was measured by determining their ability to activate elements in human plasma (including plasminogen) to lyse an 125I-fibrinogen-labelled clot. The extent of thrombolysis was calculated from the amount of radioactivity released from the clot into the plasma.
• Fibrinogenolytic activity was determined by measuring the amount of 125I remaining in plasma after exposure to tPA and therefore available for clotting. These in vitro studies demonstrated dose-related effects within the range tested. However, no significant differences in the lytic activities of the various tPA's were observed.
• Direct and Indirect in vitro activity assays of DPA versus a standard tPA have also been conducted.
• the indirect assay involves the chromogenic substrate S-2251; the direct assay involves the substrate S-2288.
• the test procedures are well known (See, e.g., Current Status of Activity
• the activity of the test sample is determined by measuring color development of S-2251 as it is cleaved by plasmin.
• the plasmin is produced as plasminogen is cleaved by the tPA in the presence of fibrinogen.
• the direct assay the tPA is cleaved with plasmin to produce the double-chain subspecies, the plasmin is inactivated, and S-2288 is added to the reaction mixture.
• the two-chain tPA acts directly on the substrate * Activity is proportional to color intensity. In both procedures, substrate concentrations in excess of Km were used.
• DPA in the presence of fibrinogen displayed an activity approximately twice that of the standard tPA (about 10 units/mg); in the direct assay, the activity of both DPA and the standard were essentially identical.
• the activity of the DPA was assayed using a modified direct test involving S-2288.
• Matrex Pd 102 beads (Amicon) were coated with fibrin monomer. The beads are 1-2 microns in diameter, and comprise polyacrylonitrile including N hydroxysuccini ide carboxylate ester groups and free carboxyl groups for covalent coupling with free amino groups on protein. Fibrinogen was coupled to the beads, and then converted to fibrin monomer with thrombin. This reagent is then used in the direct activity assay.
• DPA DPA in the presence of fibrin
• fibrinogen in the indirect test with excess substrate DPA activity is increased relative to natural material
• DPA is characterized by distinct pharmacokinetic behavior, with an increase in Vmax in the presence of fibrin.
• One group of in vivo experiments compared the thrombolytic effects and half-lives of DPA with those of BSdc and urokinase (UK) in rabbits. Plasma samples obtained from rabbits infused with the different activator solutions were examined for their fibrinolytic activity by dilute blood clot lysis time assay.
• thrombolytic agents The effects of these thrombolytic agents on the coagulation system was determined by measuring prothrombin time (PT) which detects abnormalities in clotting factors VII, X, II, V, and I of the extrinsic pathway, and partial thromboplastin time (PTT) which assesses deficiencies of clotting factors VIII, IX, X, XII, prekallikrien, and kininogen of the intrinsic pathway (as well as of II, V, X, and I) of plasma. No significant differences were observed between DPA, or BSdc treated plasma and control. However, a prolongation of PT was evident in the UK treated plasma.
• PT prothrombin time
• PTT partial thromboplastin time
• Fibrinogenolytic parameters of blood were examined during and after infusion with these thrombolytic agents. No significant differences were observed in the concentrations of fibrinogen or plasminogen in the tPA treated groups as compared with controls; UK treatment resulted in decreases of both factors. Antiplasmin levels in DPA treated rabbits decreased to 62% of -control, and that of BSdc to 46% of control during infusion, but these levels were restored to control levels immediately after infusion; levels in UK treated rabbits dropped to 1% of control during and after infusion.
• DPA's thrombolytic activity at any concentration tested was greater than that of STR as determined by measurement of the thrombus mass, and this activity behaved in a dose-dependent manner.
• PT, PTT, and fibrinogen analysis were performed essentially as described above to determine what effects intravenous DPA had on circulating coagulation factors.
• Measurable fibrin/fibrinogen degradation or split products (FSP) were an indication of the proteolytic digestion of fibrin/fibrinogen.
• PT, PTT, FSP, and fibrinogen levels were significantly abnormal during and after infusion of high doses of the DPA drug.
• a reduction in the plasma concentration of fibrinogen concomitant with an increase in fibrin/fibrinogen degradation products (FSP) was observed.
• FSP fibrin/fibrinogen degradation products
• these measurements were abnormal during infusion, but returned to near normal following infusion.
• PT and PTT were comparable to control both during and after infusion, and there was only a slight reduction in the plasma fibrinogen concentration together with a moderate increase in FSP.
• a thrombus was formed in the jugular vein from 125I-fibrinogen, blood, and thrombin. Thrombolysis was determined by measuring isotope released into the blood. The ability of increasing doses of DPA to lyse the clot was compared with that of tPA derived from Bowe's melanoma and determined in other studies using the identical model. At every concentration tested DPA had greater lytic activity in this model than tPA from Bowe's melanoma. These results suggest that DPA may have greater clinical value in some in. vivo models than in others.
• DPA tissue plasminogen activators from mouse myeloma cells
• CHO tPA Chinese hamster ovary cells
• Bowe's tPA Bowe's melanoma cells
• the CHO tPA was approximately 75% single chain, and the Bowe's tPA was approximately 90% single chain.
• the jugular vein clot was prepared as described by Collen, and allowed to age for 30 minutes before the clamps were removed.
• the tissue plasminogen activator was delivered by a constant rate pump over a period of four hours into the contralateral vein.
• the dose was diluted into 20 ml of carrier buffer, (0.3 M NaCl with 0.01% Tween-80) . Two milliliters were given as a bolus injection and the remaining 18 ml were given over the 4 hour period. Blood plasma and blood serum samples were taken at 0, 1.0, 3.0 and 4.5 hours.
• the serum samples were collected into an aprotinin solution and were used for fibrinogen degradation product determinations. At 4.5 hours the clot was removed and the extent of thrombolysis calculated as a difference between the radioactivity originally incorporated into the clot and that remaining in the vein segment. The results were expressed in percent of the original radioactivity.
• the plasma samples were analyzed for the standard thrombin time and for plasminogen levels after activation with urokinase.
• the serum samples were also used for fibrinogen degradation product determinations.
• Fibrinogen degradation products were measured using the latex particle method purchased from Wellcome Diagnostics. The test was run only on the samples from the 0.5 mg/kg dose. These samples exhibited no measurable fibrinogen degradation products when compared with the standard positive controls provided with the diagnostic kit. Since no fibrinogen degradation products were seen with the highest dosage, it was concluded that the lower doses would also be negative.
• the invention may be embodied in other specific forms.

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• 1987
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