RU2261276C2 - Isolated dna molecule encoding human erythropoietin (variants), expressing plasmid or viral dna-vector, glycoprotein erythropoietin (variants) and method for its preparing, pharmaceutical composition (variants), mammalian cell line (variants) - Google Patents

Abstract

FIELD: biology, genetic engineering, biotechnology, medicine.

SUBSTANCE: invention relates to preparing glycosylated polypeptide (glycoprotein) as a component of human erythropoietin by using the technology of recombinant DNAs. This polypeptide shows ability to increase production of reticulocytes and erythrocytes, to enhance the level of hemoglobin synthesis and consumption of iron by marrow cells and characterized by the higher molecular mass as compared erythropoietin isolated from human urine. Invention describes variants DNA sequences encoding this polypeptide that comprise vector constructions with these sequences, a method for preparing transformed mammalian cell lines producing the recombinant human erythropoietin, and a method for its preparing and purification. Also, invention proposes pharmaceutical compositions comprising glycosylated polypeptide (glycoprotein) of erythropoietin as an active component. Applying this invention provides scaling the process for preparing active human erythropoietin useful for its using in medicine.

EFFECT: improved preparing method, valuable properties of polypeptide.

10 cl, 4 dwg, 21 tbl, 12 ex

Description

This is a partial continuation of the pending application for US patent No. 561024, filed December 13, 1983; 582185, filed February 21, 1984, and 655841, filed September 28, 1984.

BACKGROUND OF THE INVENTION

The present invention relates to the manipulation of genetic materials and, in particular, to recombinant techniques for producing polypeptides having partially or fully primary structural conformation and / or one or more of the biological properties of naturally occurring erythropoietin.

A. Manipulation of genetic materials

Genetic materials can be broadly defined as those chemicals that program and control the production of cell and virus components and direct cell and virus responses. A long-chain polymer substance known as deoxyribonucleic acid (DNA) contains the genetic material of all living cells and viruses, with the exception of some viruses programmed with ribonucleic acid (RNA). The structural units in DNA polymers are four different nucleotides, each of which consists of purine (adenine or guanine) or pyrimidine (thymine or cytosine) linked to a deoxyribose sugar to which a phosphate group is attached. The addition of nucleotides in the form of a linear polymer occurs by fusion of the 5'-phosphate of one nucleotide with the 3'-hydroxyl group of another nucleotide. Functional DNA occurs in the form of stable double-stranded communities of single nucleotide strands (known as deoxy oligonucleotides), whose communities are formed by hydrogen bonding between purine and pyrimidine bases [ie "complementary" communities existing either between adenine (A) and thymine (T), or between guanine (G) and cytosine (C)]. According to the convention, it is customary to call nucleotides according to the names of their purine or pyrimidine bases, and to call complementary nucleotide communities in double-stranded DNA (ie, AT and G-C) as “base pairs”. Ribonucleic acid is a polynucleotide containing adenine, guanine, cytosine and uracil (U), rather than thymine associated with a ribose and a phosphate group.

In short, the programming function of DNA is accomplished primarily through a process in which specific DNA nucleotide sequences (genes) are "transcribed" into relatively unstable polymers of messenger RNA (mRNA). In turn, mRNA serves as a matrix for the formation of structural, regulatory, and catalytic proteins from amino acids. This mRNA “translation” process involves the functioning of small RNA strands (tRNAs), which transport and align individual amino acids along mRNA strands, allowing nucleotides to form in the proper amino acid sequences. “Information” mRNA from DNA and provides the basis for the delivery of tRNA and orientation of any of twenty amino acids for the "expression" of the polypeptide, is in the form of triplet "codons" - consecutive groups of three nucleotide bases. In a sense, protein formation is the ultimate form of "expression" of programmed genetic information provided by the nucleotide sequence of a gene.

"Promoter" DNA sequences typically "precede" a gene in a DNA polymer and provide a site for initiation of transcription in RNA. “Regulatory” DNA sequences are usually also “upstream” (ie, preceding) a gene in a given DNA polymer, linking proteins that determine the frequency (or speed) of transcriptional initiation. Collectively referred to as the “promoter / regulatory” or “control” DNA sequence, these are sequences that precede a selected gene (or series of genes) in a functional DNA polymer that help determine whether transcription will occur and, ultimately, gene expression. DNA sequences that "accompany" a gene in a DNA polymer and provide a signal for terminating transcription in mRNA are referred to as "terminator" transcription sequences.

The focus of microbiological technology of the last decade is an attempt at industrial production and pharmaceutically important substances using microorganisms that either initially do not have genetically encoded information related to the desired product included in their DNA, or (in the case of mammalian cells in culture) not express the usual by the chromosomal gene at a noticeable level. Simply put, a gene that establishes the structure of a desired polypeptide product is either isolated from a "donor" microorganism, or chemically synthesized and then stably introduced into another microorganism, preferably a self-replicating single-celled microorganism, such as bacteria, yeast, or mammalian cells in culture. Existing machinery for gene expression in “transformed” or “transfected” microbial host cells works to create the desired product using exogenous DNA as a template for transcription of mRNA, which is then translated into a continuous sequence of amino acid residues.

This technical field is widely represented in patent and literary publications related to “recombinant RNA” methodologies for isolation, synthesis, purification and amplification of genetic materials used in the transformation of selected host microorganisms. US Patent No. 4,237,224 to Cohen, et al., For example, relates to the transformation of unicellular host microorganisms with a “hybrid” viral or circular plasmid DNA comprising selected exogenous DNA sequences. Patent Techniques Cohen, et al. for the first time include obtaining a transformation vector by enzymatically cleaving viral or circular plasmid DNA to form linear DNA strands. Selected foreign (“exogenous” or “heterologous”) DNA strands, usually including sequences encoding the desired product, are obtained in a linear form using similar enzymes. Linear viral or plasmid DNA is incubated with foreign DNA in the presence of cross-linking enzymes capable of carrying out the recovery process, and "hybrid" vectors are formed containing a selected segment of exogenous DNA, "woven" into the plasmid of viral or circular DNA.

The transformation of compatible single-cell host microorganisms with a hybrid vector results in the formation of multiple copies of exogenous DNA in the host cell population. In some cases, the desired result is simply the amplification of foreign DNA, and the collected culture-grown “product” is DNA. More often, the goal of transformation is expression by exogenous DNA host cells in the form of a large-scale synthesis of secreted amounts of industrially important fragments of a protein or polypeptide encoded by a foreign DNA. See also, for example, US Pat. Nos. 4,264,731 (issued to Shine), 4,273,875 (issued to Manis), 4,293,652 (issued to Cohen) and European Patent Application No. 093619, published November 9, 1983.

The development of specific DNA sequences for plexus into DNA vectors is achieved through the use of a number of technical methods, depending to a large extent on the degree of "foreignness" of the "donor" in relation to the projected host, as well as on the size of the polypeptide expressed in the host. Fearing excessive simplification, it can be argued that there are three main alternative methods: (1) "isolation" of a double-stranded DNA sequence from the genomic DNA of the donor; (2) chemically obtaining the DNA sequence generating the code for the polypeptide of interest, and (3) in vitro synthesis of the double-stranded DNA sequence by enzymatic reverse transcription of mRNA isolated from donor cells. The above methods, which include the formation of DNA “complement” mRNA, are commonly referred to as “cDNA” methods.

Obtaining DNA sequences is often the method of choice when the complete sequence of amino acid residues of the desired polypeptide is known. DNA production procedures described in US Patent Application Serial No. 483451, Alton, et al. (filed April 15, 1983, and the corresponding PCT application US 83/00605, published November 24, 1983 under the number WO 83/04053), for example, create means to achieve such extremely desirable results, such as: the provision of alternating codons that are often found in a host microorganism selected for expression (for example, the provision of “preferred” codons of yeast or E. coli); avoid the presence of untranslated "intron" sequences (usually found in mammalian genomic DNA sequences and their mRNA matrices) that are not easily processed by prokaryotic host cells; Avoiding undesired “leading” polypeptide sequences, usually encoded by genomic DNA and cDNA sequences, however, often not easily cleaved from the polypeptide of interest using bacterial or yeast host cells; providing for easy insertion of DNA into suitable expressive vectors in communities with the desired promoter / regulatory and terminator sequences; and providing for the easy construction of genes encoding polypeptide fragments and analogues of the desired polypeptides.

When the complete sequence of amino acid residues of the desired polypeptide is not known, the direct preparation of DNA sequences is not possible and the isolation of DNA sequences encoding the polypeptides using the cDNA method becomes a selection method, despite possible shortcomings in the ease of assembly of expression vectors capable of providing high levels of microbial expressivity indicated higher. Among standard techniques for the isolation of cDNA sequences is the preparation of plasmid-bearing cDNA "libraries" derived from reverse transcription of mRNA, which is often found in donor cells selected as responsible for the expression of high level genes (for example, cDNA libraries derived from pituitary cells that express relatively large amounts of growth hormone products). Where significant portions of the amino acid sequence of the polypeptides are known, the labeled, probe sequences of single-stranded DNA duplicating the sequence, presumably in the “planned” cDNA, can be used in DNA / DNA hybridization techniques performed on clonal copies of cDNA that has been denatured into a single-stranded form. [See, mainly, the disclosure and discussion of this technical field presented in US patent 4394443, issued in the name of Weissman, et al. and recent demonstrations of the use of long oligonucleotide hybridization probes reported by Wallace, et al., Nuc. Acids Res., 6, pp. 3543-3557 (1979), Reyes, et al., P.N.A.S. (U.S.A.), 79, pp. 3270-3274 (1982) and Jaye, et al., Nuc. Acids Res., 11, pp. 2325-2335 (1983). See also US patent No. 4358535, issued in the name of Falkow, et al., Concerning methods of DNA / DNA hybridization in the diagnosis; published European patent applications No. 0070685 and No. 0070687 relating to light emitting labels on single-stranded polynucleotide probes; Davis, et al., “A Manual for Genetic Engineering, Advanced Bacterial Genetics,” Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1980) at pp. 55-58 and 174-176 regarding colony hybridization and epidemic disease techniques; and New England Nuclear (Boston, Mass.) Brochures on the "Gene Screen" Hybridization Transfer Membrane materials, providing guidelines for the transfer and hybridization of DNA and RNA, Catalog No. NEF-972].

Among the most significant recent advances in hybridization techniques for screening recombinant clones is the use of labeled mixed oligonucleotide probes, each of which is potentially a complete complement of a specific DNA sequence in a hybridization sample that includes a heterogeneous mixture of single-stranded DNA and RNA. These techniques are recognized as particularly useful in identifying cDNA clones derived from sources that produce extremely small amounts of mRNA sequences for the polypeptide of interest. In short, the use of strict hybridization conditions aimed at avoiding nonspecific binding may allow, for example, autoradiographic visualization of a specific cDNA clone at the end of the planned DNA hybridization to the only probe in the mixture that is its complete complement. See mainly Wallace, et al., Nuc. Acids Res., 9, pp. 879-897 (1981); Suggs, et al., P.N.A.S. (U.S.A.), 78, pp. 6613-6617 (1981); Choo, et al., Nature, 299, pp. 178-180 (1982); Kurachi, et al., P.N.A.S. (U.S.A.), 79, pp. 6461-6464 (1982); Ohkubo, et al., P.N.A.S. (U.S.A.), 80, pp. 2196-2200 (1983); and Kornblihtt, et al., P.N.A.S. (U.S.A.), 80, pp. 3218-3222 m (1983). In general, mixed probe techniques in Wallace, et al. (1981) above, were expanded by various developers to the point of obtaining reliable results in the isolation of cDNA clones using a 32-membered mixed pool of oligonucleotide probes 16 bases in length (16-mer) of uniformly changing DNA sequences together with a single 11-mer for performing on-off "positive" confirmation of the presence of cDNA of interest. See Singer-Sam, et al., P.N.A.S. (U.S.A.), 80, pp. 802-806 (1983).

The use of genomic DNA isolates is the least common of the three methods described above for the development of specific sequences for use in recombinant techniques. This is especially true in the field of recombinant techniques aimed at ensuring the microbial expression of mammalian polypeptides, and is mainly due to the complexity of the mammalian genomic DNA. Thus, although reliable methods exist for the development of phage-bearing libraries of the genomic DNA of humans and other mammalian species [see, for example, Lawn, et al., Cell, 15, pp. 1157-1174 (1978), relating to the methods to generate a human genomic library, commonly referred to as the "Maniatis Library"; Karn, et al., P.N.A.S. (U.S.A.), 77, pp.5172-5176 (1980), concerning a human genomic library based on an alternative method for limiting endonuclease fragmentation; Blattner, et al., Science, 196. pp. 161-169 (1977), describing the construction of a bovine genomic library], several relatively successful attempts have been made to use hybridization techniques to isolate genomic DNA in the absence of far-reaching predictions of amino acid or DNA sequences. One example is the work of Fiddes, et al., J. Mol. and App. Genetics, 1, pp. 3-18 (1981), which reports the successful isolation of a gene encoding the alpha subunit of the pituitary glycoprotein hormones of the person from the Maniatis Library by using an “extremely long” probe, including the complete fragment of 621 base pairs of the pre-isolated cDNA sequences for the alpha subunit. As another example, in the work of Das, et al., P.N.A.S. (U.S.A.), 80, pp. 1531-1535 (1983), reports the isolation of human genomic clones for human HLA-DR using a synthetic oligonucleotide with 175 base pairs. And finally, in Anderson, et al., P.N.A.S. (U.S.A.), 80, pp. 688-6842 (1983), reports the isolation of a genomic clone for bovine pancreatic trypsin inhibitor (BPTI) using a single probe having 86 base pairs in length and constructed in accordance with the known BPTI amino acid sequence. The authors note the determination of modest prospects for the isolation of mRNA suitable for the synthesis of a cDNA library, due to the obvious low levels of mRNA in the initially targeted sources of the parotid gland and lung tissue. Expressing hopes for success in probing the genomic library using a mixture of labeled probes, they state: "For the most part, mixed sequence oligodeoxynucleotide probes were used to isolate protein genes of an unknown sequence from cDNA libraries. Such probes are usually a mixture of 8-32 oligonucleotides and are long 14-17 nucleotides, being typical representatives of each possible combination of codons for a slight stretching (5-6 residues) of the amino acid sequence. In severe hybridization conditions, which put poorly paired nitrogen bases into probes, these mixtures can localize specific gene sequences in low-complexity clonal libraries, however, due to their small length and heterogeneity, mixed probes are often devoid of the specificity needed to probe such complex sequences, such as the mammalian genome, which makes this method impractical for the isolation of mammalian protein genes when the corresponding mRN not available. " (Links omitted).

Thus, in the art there remains a need to improve methods for performing fast and efficient isolation of cDNA clones in cases where little is known about the amino acid sequence and when the "enriched" sources of mRNA tissue are not very accessible so that they can be used in building cDNA libraries. Such improved methods would be especially useful if they were used to isolate mammalian genomic clones where loose information is available regarding the amino acid sequences of the polypeptide encoded by the gene of interest.

B. Erythropoietin as a polypeptide of interest

Erythropoiesis, the formation of red blood cells, is carried out continuously throughout a person's life to compensate for destroyed cells. Erythropoiesis is an extremely precisely regulated physiological mechanism that allows enough red blood cells to be produced in the blood for proper oxygenation of the tissue, but not so large that the cells obstruct blood circulation. The formation of red blood cells occurs in the bone marrow under the control of the hormone erythropoietin.

Erythropoietin, an acidic glycoprotein with a molecular weight of approximately 34,000 Daltons, can occur in three forms: α, β and asialo. Forms α and β differ slightly in carbohydrate components, however, they have the same potency, biological activity and molecular weight. The asialo form is an α or β form with a cleaved terminal carbohydrate (sialic acid). Erythropoietin is present in very low plasma concentrations when the body is in a healthy state in which the tissues receive sufficient oxygenation from the existing number of red blood cells. This normal low concentration is sufficient to stimulate the replacement of red blood cells, which are usually lost during aging.

The amount of erythropoietin in the blood circulation increases in conditions of hypoxia, when oxygen transfer by blood cells during blood circulation decreases. Hypoxia can be caused by the loss of a large amount of blood during bleeding, the destruction of red blood cells due to radiation, a decrease in oxygen consumption due to high altitudes or a prolonged unconscious state, as well as various forms of anemia. Under the influence of tissues subject to hypoxic stress, erythropoietin begins to increase the formation of red blood cells by stimulating the conversion of primary anterior cells in the bone marrow into proerythroblasts, which are subsequently brought to maturity, synthesize hemoglobin and are realized as red blood cells in the blood circulation. When the number of red blood cells in the blood circulation is greater than is necessary for normal tissue oxygen demand, erythropoietin in the blood circulation decreases.

See mainly the work of Testa, et al., Exp. Hematol., 8 (Supp. 8), 144-152 (1980); Tong, et al., J. Biol. Chem., 256 (24), 12666-12672 (1981); Goldwasser, J. Cell. Physiol., 110 (Supp. 1), 133-135 (1982); Finch, Blood, 60 (6), 1241-1246 (1982); Sytowski, et al., Expt. Hematol., 8 (Supp. 8), 52-64 (1980); Naughton, Ann. Clin. Lab. Sci., 13 (5), 432-438 (1983); Weiss, et al., Am. J. Vet. Res., 44 (10), 1832-1835 (1983); Lappin, et al., Exp. Hematol., 11 (7), 661-666 (1983); Baciu, et al., Ann. N.Y. Acad. Sci., 414, 66-72 (1983); Murphy, et al., Acta. Haematologica Japonica, 46 (7), 1380-1396 (1983); Dessypris, et al., Brit. J. Haematol., 56, 295-306 (1984); and Emmanouel, et al., Am. J. Physiol., 247 (1 Pt 2), F 168-76 (1984).

Since erythropoietin is important in the formation of red blood cells, the hormone has potential useful applications both in the diagnosis and in the treatment of blood disorders characterized by low or imperfect formation of red blood cells. See mainly the work of Pennathur-Das, et al., Blood, 63 (5), 1168-71 (1984) and Haddy, Am. Jour. Ped. Hematol./Oncol., 4, 191-196, (1982) regarding erythropoietin in possible therapies for sickle cell diseases, and also Eschbach, et al., J. Clin. Invest., 74 (2), pp.434-441, (1984), which describes a therapeutic regimen for a uremic sheep based on an in vivo response to an infusion of erythropoietin-rich plasma and proposes a dosage of 10 U EPO / kg per day for 15-40 days as a correction of anemia of the type associated with chronic renal failure. See also Krane, Henry Ford Hosp. Med. J., 31 (3), 177-181 (1983).

It has recently been estimated that the presence of large quantities of erythropoietin will treat 1,600,000 people in the United States each year of anemia. See, for example, Morrison, "Bioprocessing in Space - an Overview", pp. 577-571 in The World Biotech Report 1984, Volume 2: USA, (Online Publications, New York, N.Y. 1984). Recent studies have created the basis for predicting the effectiveness of erythropoietin therapy in a variety of disease states, disorders and hematologic disorders: Vedovato, et al., Acta. Haematol., 71, 211-213 (1984) (beta-thalassemia); Vichinsky, et al., J. Pediatr., 105 (1), 15-21 (1984) (cystic fibrosis); Cotes, et al., Brit. J. Obstet. Gyneacol., 90 (4), 304-311. (1983) (pregnancy, menstruation disorders); Haga, et al., Acta. Pediatr. Scand., 72, 827-831 (1983) (early premature anemia); Claus-Walker, et al., Arch. Phys. Med. Rehabil., 65, -370-374 (1984) (spinal cord injury); Dunn, et al., Eur. J. Appl. Physiol., 52, 178-182 (1984) (space flight); Miller, et al., Brit. J. Haematol., 52, 545-590 (1982) (acute blood loss); Udupa, et al., J. Lab. Clin. Med. 103 (4), 574-580 and 581-588 (1984); and Lipschitz, et al., Blood, 63 (3), 502-509 (1983) (aging); and Dainiak, et al., Cancer, 51 (6), 1101-1106 (1983) and Schwartz, et al., Otolaryngol., 109, 269-272 (1983) (various neoplastic disease conditions accompanied by abnormal erythropoiesis).

Previous attempts to obtain erythropoietin with a good output from plasma or urine have been proven to be relatively unsuccessful. A sophisticated and refined laboratory technique inevitably and, as a rule, leads to the collection of very small amounts of contaminated and unstable extracts containing erythropoietin.

US Pat. No. 3,033,753 describes a method for partial purification of erythropoietin from sheep blood plasma, which provides small yields of a total pure extract containing erythropoietin.

Initial attempts to isolate erythropoietin from urine led to unstable, biologically inactive hormone preparations. US Pat. No. 3,865,801 describes a method for stabilizing the biological activity of a general substance containing erythropoietin reconstituted from urine. The resulting preparation containing erythropoietin inhibits 90% of the activity of erythropoietin and is stable.

Another method for purifying human erythropoietin from the urine of patients suffering from aplastic anemia is described in Miyake, et al., J. Biol. Chem., Vol. 252; No. 15 (August 10, 1977), pp. 558-5564. This seven-step technique involves ion exchange chromatography, ethanol precipitation, gel filtration, adsorption chromatography and produces pure erythropoietin with a potency of 70,400 units / mg protein in 21% yield.

US Pat. No. 4,397,840 to Takezawa, et al. Describes methods for producing an “erythropoietin product” from healthy human urine samples with weakly basic ionic changes and claims that the resulting low molecular weight products “have no inhibitory effects against erythropoietin.”

UK patent application No. 2085887, Sugimoto, et al., Published May 6, 1982, describes a method for producing human hybrid lymphoblastoid cells and reports that production levels range from 3 to 420 units of erythropoietin per ml cell suspension (distributed in cultures after propagation by the host mammal with a content of up to 10 ⁷ cells per ml). At the highest production levels that are claimed to be achieved, the erythropoietin production rate can be calculated to be from 40 to 4000 units / 10 ⁶ cells / 48 hours in an in vitro culture, followed by transfer of cells from in vivo propagation systems ( See also equivalent US patent No. 4377513). Numerous suggestions have been made regarding the isolation of erythropoietin from tissue sources, including neoplastic cells, but the production yields were completely low. See, for example, Jelkman, et al., Expt. Hematol., 11 (7), 581-588 (1983); Tambourin, et al., PNAS (USA), 80, 6269-6273 (1983); Katsuoka, et al., Gann, 74, 534-541 (1983); Hagiwara, et al., Blood, 63 (4), 828-835 (1984); and Choppin, et al., Blood, 64 (2), 341-347 (1984).

Other isolation techniques used to produce purified erythropoietin included immunological techniques. A polyclonal serum derivative antibody directed against erythropoietin is developed by injecting an animal, preferably a rat or rabbit, with human erythropoietin. Injected human erythropoietin is recognized as a foreign antigenic substance by the animal’s immune system and causes the production of antibodies against the antigen. Various cells that respond to stimulation by antigenic substances produce and release antibodies in the bloodstream that differ slightly from those produced by other reacting cells. Antibody activity remains in the serum of the animal when its blood is extracted. Although crude serum or antibody preparations purified as a serum fraction of immunoglobulin G can then be used in samples to detect complexation with human erythropoietin, the substances experience major inconvenience. This serum antibody, consisting of all the various antibodies produced by individual cells, is polyclonal in nature and forms complexes with components in common extracts differently than single erythropoietin.

Of interest to the premises of the present invention are recent advances in the development of complete cell cultures capable of producing a single antibody species that is specifically immunologically active with a single antigenic determinant of the selected antigen. See mainly Chisholm, High Technology, Vol. 3, No. 1, 57-63 (1983). Attempts have been made to use cell fusion and hybridization techniques to develop "monoclonal" antibodies to erythropoietin and to use these antibodies in the isolation and quantification of human erythropoietin. One example is the message that appeared in annotated form in Lee-Huang, Abstract No. 1463 of Fed. Proc., 41, 520 (1982), which refers to the successful development of mouse-mouse hybridoma cell lines that secrete monoclonal antibodies to human erythropoietin. As another example, a detailed description of the preparation and use of a monoclonal, anti-erythropoietin antibody appeared in Weiss, et al., P.N.A.S. (U.S.A.), 79, 5465-5469 (1982). See also works by Sasaki, Biomed. Biochim. Acta., 42 (11/12), 5202-5206 (1983); Yanagawa, et al., Blood, 64 (2), 357-364 (1984); Yanagawa, et al., J. Biol. Chem., 259 (5), 2707-2710 (1984); and US patent No. 4465624.

For the background of the invention, reports of the immunological activity of synthetic peptides are also of interest, which essentially duplicate the amino acid sequence preserved in naturally occurring proteins, glycoproteins and nucleoproteins. More specifically, relatively low molecular weight polypeptides are shown to be involved in immune responses that are similar in duration and length to immune responses of physiologically important proteins such as viral antigens, polypeptide hormones and the like. Among the immune responses of such polypeptides, provoking the formation of specific antibodies in immunologically active animals is also included. See, for example, Lerner, et al., Cell, 23, 309-310 (1981); Ross, et al., Nature, 294, 654-656 (1981); Walter, et al., P.N.A.S. (U.S.A.), 77, 5197-5200 (1980); Lerner, et al., P.N.A.S. (U.S.A.), 78, 3403-3407 (1981); Walter, et al., P.N.A.S. (U.S.A.), 78, 4882-4886 (1981); Wong, et al., P.N.A.S. (U.S.A.), 78, 7412-7416, (1981); Green, et al., Cell, 28, 477-487 (1982); Nigg, et al., P.N.A.S. (U.S.A.), 79, 5322-5326 (1982); Baron, et al., Cell, 28, 395-404 (1982); Dreesman, et al., Nature, 295, 158-160 (1982); and Lerner, Scientific American, 248, No. 2, 66-74 (1983). See also Kaiser, et al., Science, 223, pp. 249-255 (1984), regarding the biological and immunological activity of synthetic peptides, which approximately possess secondary structures of peptide hormones, however, may not have their primary structural conformation. The above studies relate, of course, to amino acid sequences of proteins other than erythropoietin, a substance for which no essential information about the amino acid sequence has been published. In a joint application for US patent No. 463724, filed February 4, 1983 J. Egrie, published August 22, 1984 as European patent application No. 0116446, describes a cell line of a hybridoma mouse-mouse (A.T.S.C. No. HB8209) , which produces a highly specific monoclonal, anti-erythropoietin antibody, which is also specifically immunoactive with a polypeptide containing the following amino acid sequence:

NH ₂ -Ala-Pro-Pro-Arg-Leu-Ile-Cys-Asp-Ser-Arg-Val-Leu-Glu-Arg-Tyr-Leu-Leu-Glu-Ala-Lys-COOH.

The polypeptide sequence is that sequence that is assigned to the first twenty amino acid residues of mature human erythropoietin isolated in accordance with the method described by Miyake, et al., J. Biol. Chem., 252, 5558-5564 (1977), and over which an amino acid analysis was performed using a gas phase sequencer (Applied Biosystems, Inc.) in accordance with the methodology of Hewick, M., et al., J. Biol. Chem., 256, 7990-7997 (1981). See also Sue, et al., Proc. Nat. Acad. Sci. (USA), 80, pp. 3651-3655 (1983), concerning the development of polyclonal antibodies against synthetic 26-mer based on a different amino acid sequence, and the work of Sytowski, et al., J. Immunol. Methods, 69, pp. 181-186 (1984).

Although polyclonal or monoclonal antibodies, as described above, have very useful materials for use in immunoassays to detect and quantify erythropoietin and may be useful in the related purification of erythropoietin, it seems unlikely that these materials for large-scale isolation can provide sufficient amounts of erythropoietin from mammalian sources for further analysis, clinical examination and possible broadly targeted therapeutic Using the treatment agent, for example, chronic kidney disease, in which the affected tissue is not able to maintain the production of erythropoietin. Therefore, it is assumed that the best prospects for the complete characterization of mammalian erythropoietin and the creation of large quantities of it for possible diagnosis and clinical use include the use of recombinant techniques for the implementation of large-scale microbial synthesis of the compound.

Despite the fact that considerable efforts have been made in an attempt to isolate DNA sequences encoding erythropoietin of humans or other mammalian species, none of them have been successful. This is mainly due to the deficiency of tissue sources, especially human tissue sources, enriched in mRNA so as to allow the construction of a cDNA library, from which, using known methods, it is possible to isolate a DNA sequence encoding erythropoietin. In addition, little is known about the continuous sequence of amino acid residues of erythropoietin, and therefore it is impossible to build, for example, long polynucleotide probes suitable for reliable use of cDNA and especially genomic DNA libraries in screening for DNA / DNA hybridization. Illustratively, a sequence of twenty amino acids used to generate the above monoclonal antibody produced by A.T.S.C. No. HB8209, does not allow the construction of a unique 60-basic oligonucleotide probe in the manner described by Andersen, et al., Above. It was found that the human gene for erythropoietin can manifest itself as a “single copy gene” in the human genome, and anyway, the genetic material encoding human erythropoietin probably accounts for less than 0.00005% of the total human genomic DNA that should be present in the genomic library .

To date, the most successful of the known attempts in recombinant methods for creating DNA sequences suitable for use in the microbial expression of isolated amounts of mammalian erythropoietin have not reached the goal. An example is Farber, et al., Exp. Hematol., 11, Supp. 14, Abstract 101 (1983), which reports the extraction of mRNA from the renal tissues of baboons treated with phenylhydrazine and the injection of mRNA into Xenopus laevis oocytes with a rather temporary result of an in vitro mixture of “translation products” that showed the biological properties of erythropoietin. More recently, Farber, et al., Blood, 62, No. 5, Supp. No. 1, Abstract, 392, at page 122a (1983) reported on in vitro translation of human kidney mRNA by frog oocytes. The resulting translation product mixture was evaluated as containing about 220 mU translation product having erythropoietin activity per microgram of mRNA introduced. Although such levels of in vitro translation of exogenous mRNA encoding erythropoietin were found to be completely low (compared to even previously reported levels of translation of baboon mRNA into the desired product), it was estimated that the results confirm the existence of a human kidney as a erythropoietin expression site that allows the construction of cDNA - libraries of enriched human kidneys from which the desired gene can be isolated. [Cm. also Farber, Clin. Res., 31 (4), 769A (1983)].

Since US patent applications No. 561024 and 582185 have been filed, there has been a single report on the cloning and expression of what is recognized as human erythropoietin cDNA in E. coli. In short, a number of cDNA clones were introduced into E. coli plasmids, and β-lactamase fusion products were noted to be immunoreactive with a monoclonal antibody to the unidentified “human erythropoietin epitope”. See Lee-Huang, Proc. Nat. Acad. Sci. (USA) 81, pp. 2708-2712 (1984).

Summary

Newly purified and isolated polypeptide products are proposed for the first time, having partially or completely primary structural conformation (i.e., a continuous sequence of amino acid residues) and one or more biological properties (e.g., immunological properties and biological activity in vivo and in vitro) of naturally occurring erythropoietin including its allelic variants. These polypeptides also clearly differ in that they are the product of expression of a prokaryotic or eukaryotic host (e.g., bacterial, yeast, and mammalian cells in culture) of exogenous DNA sequences obtained by genomic or cDNA cloning, or by gene synthesis. The products of microbial expression in vertebrate cells (e.g., mammals and birds) may also differ in that they are free from community with human proteins or other contaminants that may be associated with erythropoietin in its natural environment for mammalian cells or in extracellular fluids such as blood plasma or urine. The products of typical yeast (e.g., Saccaromyces cerevisiae) or prokaryotic (e.g., E. coli) host cells are free from community with any mammalian protein. Depending on the host used, the proposed polypeptides may be glycosylated by mammals or other eukaryotic carbohydrates, or may be non-glycosylated. The proposed polypeptides may also include the amino acid residue of the initial methionine (at position - 1).

The proposed new glycoprotein products include those that have a primary structural conformation, sufficiently duplicating the conformation of naturally occurring (e.g. human) erythropoietin to enable one or more of the biological properties of the latter, and those that have an average carbohydrate composition that differs from a composition of naturally occurring (e.g. human) erythropoietin.

Vertebrate cells (e.g., COS-1 and CHO) of the present invention contain the first cells that are always available, which can multiply continuously in vitro and which, during growth in culture, are capable of producing more than 100U in the growth medium (preferably more than 500U and most preferably, more than 1000U-5000U) of erythropoietin per 10 ⁶ cells in 48 hours, as determined by radioimmunoassay.

Synthetic polypeptides are also proposed that fully or partially duplicate continuous sequences of amino acid residues of erythropoietin, which are first explained here. These sequences, by separating primary, secondary, or tertiary structural and conformational characteristics with naturally occurring erythropoietin, may possess biological activity and / or immunological properties together with a naturally occurring product so that they can be used as biologically active or immunological erythropoietin substituents in therapeutic and immunological processes. Accordingly, monoclonal and polyclonal antibodies are provided that are produced by standard methods that are immunoreactive with such polypeptides and, preferably, also immunoreactive with naturally occurring erythropoietin.

The present invention illustrates the cloned DNA sequences of the monkey and human species principles, as well as the polypeptide sequences conveniently derived from there and representing, respectively, the primary structural conformation of the monkey and human species principles.

New, biologically functional vectors of viral and circular plasmid DNA are also encompassed, encompassing the DNA sequences of the invention, and microbial (e.g., bacterial, yeast, and mammalian cells) host organisms that are stably transformed or transfected with such vectors. Accordingly, new methods are proposed for producing useful polypeptides, including cultivating the cultivation of such transformable or transfectable microbial hosts under conditions that facilitate the large-scale expression of exogenous, vector-bearing DNA sequences and the isolation of the desired polypeptides from culture media, cell lysates or fractions of cell membranes.

Isolation and purification of the microbially expressed polypeptides of the present invention can be carried out using conventional methods, including, for example, separation by preparative chromatography and immunological separation of preparations containing monoclonal and / or polyclonal antibodies.

Having clarified the sequence of the amino acid residues of erythropoietin, the present invention provides the complete and / or partial construction of DNA sequences encoding erythropoietin and containing such favorable characteristics as the introduction of codons “preferred” for expression by selected non-mammalian hosts, providing cleavage sites by restrictive endonuclease enzymes and providing additional initial, final or intermediate DNA sequences that contribute to construction is readily expressed vectors. Accordingly, it is proposed to construct (and develop through specific mutagenesis of cDNA and genomic DNA) DNA sequences encoding the microbial expression of polypeptide analogues or derivatives of erythropoietin that differ from naturally occurring forms in terms of the identity and location of one or more amino acid residues (i.e. Deletion analogues containing a smaller amount of all residues defined for EPO and / or substitution analogues in which one or more established residues are replaced E residues, and / or other analogues in which one or more amino acid residues are added to the end or middle portion of the polypeptide); and which possess some or all of the properties of naturally occurring forms.

The proposed new DNA sequences include all sequences suitable for providing expression in prokaryotic or eukaryotic host cells of polypeptide products having at least a portion of the primary structural conformation and one or more of the biological properties of erythropoietin, which are encompassed by: (a) DNA- the sequences shown in Tables V and VI, or their complementary threads; (b) DNA sequences that hybridize (under the hybridization conditions illustrated here, or more stringent conditions) to DNA sequences as defined in (a), or fragments thereof; and (c) DNA sequences which, due to degeneration of the genetic code, must hybridize to the DNA sequences defined in (a) and (b) above. Particularly covered in part (b) are genomic DNA sequences encoding the forms of allelic variants of monkey and human erythropoietin and / or encoding erythropoietin of other mammalian species. Particularly covered in part (c) are the constructed DNA sequences encoding EPO, EPO fragments and EPO analogues, whose DNA sequences may include codons that facilitate the translation of messenger RNA in invertebrate hosts.

This class of polypeptides is encoded by the invention, which is encoded by parts of the DNA complement to the top of the genomic DNA sequence shown in Table VI, i.e. “complementary inverted proteins” as described by Tramontano, et al., Nucleic Acids Research, 12, pp. 5048-5059 (1984).

Also encompassed by the invention are pharmaceutical compositions containing effective amounts of the polypeptide products of the present invention together with suitable diluents, stimulants and / or carriers that allow the creation of erythropoietin therapy, especially in the treatment of anemic disease states and, above all, such anemic conditions that lead to chronic renal failure .

The polypeptide products of this invention can be “co-labeled” with a detectable marker (eg, radiolabeled ¹²⁵ I) to provide reagents suitable for the detection and quantification of erythropoietin in solid tissue and liquid samples such as blood or urine. DNA products can also be labeled with detectable markers (such as radiolabels and non-isotope labels such as biotin) and used in DNA hybridization processes to localize the position of the erythropoietin gene and / or the position of any related gene family in the chromosome map of humans, monkeys, and others species of mammals. They can also be used to identify erythropoietin gene abnormalities at the DNA level and used as gene markers to identify neighboring genes and their abnormalities.

As described in detail below, the present invention also provides significant improvements in methods for detecting specific single-stranded polynucleotides of an unknown sequence in a heterogeneous cell or viral sample, including multiple single-stranded polynucleotides, in which:

(a) a mixture of labeled probes of single-stranded polynucleotides with uniformly varying base sequences is obtained, each of these probes being potentially specifically complementary to a base sequence that is supposedly unique to the detected polynucleotide,

(b) fixing the sample on a dense substrate,

(c) treats a substrate having a sample fixed thereon to weaken further binding of polynucleotides to it, with the exception of the hybridization pathway to polynucleotides in said sample,

(d) a treated substrate having a sample fixed on it is temporarily brought into contact with said mixture of labeled probes under conditions conducive to hybridization only between fully complementary polynucleotides, and

(e) a specific polynucleotide is detected by monitoring for a hybridization reaction between it and a fully complementary probe within the indicated mixture of labeled probes, as proved by the presence of the highest density of labeled material on the substrate at the locus of a specific polynucleotide compared to the background density of labeled material resulting from non-specific binding of labeled probes with the substrate.

These methods are especially effective in situations that require the use of 64, 128, 256, 512, 1024 or more mixed polynucleotide probes having a length of 17 to 20 bases in DNA / DNA, or RNA / RNA, or DNA / RNA hybridization.

As described below, the aforementioned improved methods have visually identified cDNA clones encoding erythropoietin of monkeys within a library derived from mRNA from renal cells of anemic monkeys. More specifically, a mixture of 128 uniformly varying 20-mer probes based on amino acid sequence information derived from alternating fractions of human erythropoietin was used in colony hybridization techniques to identify seven "positive" erythropoietin cDNA clones within a total of 200,000 colonies. Even more than that, the practice of using the improved methods of the present invention allowed us to quickly isolate three positive clones from the shielding limits of the 1,500,000 sterile spots that make up the human genomic library. This was accomplished by using the above mixture of 128 20-mer probes together with a second set of 128 17-mer probes based on an amino acid analysis of a different continuous sequence of human erythropoietin.

The above illustrative methods constitute the first known example of the use of multiple mixed oligonucleotide probes in DNA / DNA hybridization processes aimed at isolation of mammalian genomic clones, and the first known example of the use of a mixture of more than 32 oligonucleotide probes in the isolation of cDNA clones.

Numerous aspects and advantages of the invention will become apparent to those skilled in the art upon consideration of the following detailed description of the invention, providing an example of the practical application of the invention in preferred embodiments.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, DNA sequences have been isolated and characterized that encode a partially or fully polypeptide sequence of a human and ape erythropoietin species (hereinafter “EPO”). In addition, the DNA of monkeys and humans has become the subject of eukaryotic and prokaryotic expression, creating secreted amounts of polypeptides that exhibit biological (e.g. immunological) properties of naturally occurring EPO, as well as biological activities in vivo and in vitro EPO.

Monkey species DNA was first isolated from a cDNA library constructed by mRNA, which was derived from monkey kidney tissue in a chemically induced anemic state and whose serum was determined immunologically to contain higher levels of EPO compared to normal monkey serum. The required cDNA clones containing EPO-encoding DNA were isolated by hybridization of DNA / DNA colonies using a pool of 128 mixed, radiolabeled, 20-mer oligonucleotide probes, and included rapid screening of 200,000 colonies. The calculation of oligonucleotide probes was based on amino acid sequence information represented by enzymatic fragmentation and sequence determination of a small sample of human EPO.

Human DNA was isolated from the human genomic DNA library. Clones containing EPO-encoding DNA were isolated by hybridization of DNA / DNA plaques using the above 128 mixed 20-mer oligonucleotide probes, as well as a second pool of 128 radiolabeled 17-mer probes, whose sequences were based on amino acid sequence information obtained from different enzymatic fragment of human EPO.

Positive colonies and plaques were verified by dideoxy programming of the clonal DNA sequence using a set of 16 sequences within a pool of 20-mer probes, and selected clones were analyzed for the nucleotide sequence with the final deduction of the primary structural conformation of the EPO polypeptides encoded by it. The deduced polypeptide sequences showed a high degree of homology with respect to each other and to the partial sequence generated by amino acid analysis of human EPO fragments.

The selected monkey positive cDNA clone and the selected human human genomic clone were introduced into the shuttle DNA vector, which was amplified in E. coli and used to transfect mammalian cells in culture. Cultural growth of transfected host cells was expressed in preparations floating on the surface of the culture medium containing, as estimated, up to 3000 mU EPO per ml of culture fluid.

The following examples are presented to illustrate the invention and are particularly directed to methods previously carried out to identify EPO encoding monkey cDNA clones and human genomic clones, to methods resulting from this identification, as well as to sequences, development of expression systems and immunological confirmation of EPO expression in such systems.

More specific. Example 1 aims to determine the amino acid sequence of human EPO fragments and construct mixtures of radiolabeled probes based on the results of this sequence determination. Example 2 is mainly aimed at methods included in the identification of positive cDNA clones of monkeys, and thus provides information regarding the treatment of animals and preliminary analysis of radioimmunoassay (RIA) of animal sera. Example 3 is aimed at obtaining a cDNA library, screening colony hybridization and checking for positive clones, determining the DNA sequence of a positive cDNA clone, as well as generating information on the primary structural conformation (amino acid sequence) of monkey EPO polypeptides. Example 4 is directed to methods included in the identification of positive genomic human clones, and thus provides information related to the source of the genomic library, plaque hybridization methods, and verification of positive clones. Example 5 aims to determine the DNA sequence of a positive genomic clone and generate amino acid sequence information of human EPO polypeptides, including comparison with monkey EPO sequence information. Example 6 is directed to methods for constructing a vector, including EPO-coding DNA, derived from a positive monkey cDNA clone, the use of the vector for transfection of COS-1 cells and cultural growth of transfected cells. Example 7 is directed to methods for constructing a vector comprising EPO-encoding DNA derived from a positive human genomic clone, using the vector for transfection of COS-1 cells and cultural growth of transfected cells. Example 8 is directed to methods of immunoassays conducted on supernatant fluids of media obtained from cultured transfected cells in accordance with Examples 6 and 7. Example 9 is directed to the biological activity of in vitro and in vivo microbial expressed EPO of Examples 6 and 7.

Example 10 is directed to the development of mammalian host expression systems for EPO cDNA of monkey species and human genomic DNA, including Chinese hamster ovary cells ("CHO"), and to the immunological and biological activity of the products of such expression systems, as well as the characterization of such products. Example 11 aims to obtain genes encoding human EPO and EPO analogues, the genes of which include a number of preferred codons for expression in E. coli and yeast host cells, as well as systems for such expression. Example 12 relates to profiles of the immunological and biological activities of the expressed products of the systems described in Example 11.

Example 1

A. Determination of the amino acid sequence of a fragment of human EPO

Human EPO is isolated from urine and subjected to tryptic cleavage, leading to the development and isolation of 17 separate fragments in amounts approximately expressed in 100-150 picomoles.

The numbers are randomly assigned to the fragments and analyzed for amino acid sequence by microanalysis using a gas phase sequencer (Applied Biosystems) to obtain sequence information shown in Table I below, where letter codes are used and “X” denotes a residue that is not clearly defined.

Table I Fragment No. Sequence Analysis Result T4a A-P-P-R T4b G-K-L-K T9 A-L-G-A-Q-K T13 V-L-E-R T16 A-V-S-G-L-R T18 L-F-R T21 K-L-F-R T25 Y-L-L-E-A-K T26a L-I-C-D-S-R T26b L-Y-T-G-E-A-C-R T27 T-I-T-A-D-T-F-R T28 E-A-I-S-P-P-D-A-A-M-A-A-P-L-R T30 E-A-E-X-I-T-T-G-X-A-E-H-X-S-L- N-E-X-I-T-V-P T31 V-Y-S-N-F-L-R T33 S-L-T-T-L-L-R T35 V-N-F-Y-A-W-K T38 G-Q-A-L-L-V-X-S-S-Q-P-W- E-P-L-Q-L-H-V-D-K

B. Calculation and construction of mixtures of oligonucleotide probes

The amino acid sequences shown in Table 1 are considered in the sense of genetic code degeneration in order to determine whether mixed probe techniques can be applied to DNA / DNA hybridization techniques on cDNA and / or genomic DNA libraries. This analysis reveals that within fragment T35 there is a series of 7 amino acid residues (Val-Asn-Phe-Tyr-Ala-Trp-Lys), which can be unambiguously characterized as encoded by one of 128 possible DNA sequences spanning 20 base pairs . Therefore, the first set of 128 20-mer oligonucleotides is synthesized using standard phosphoamidite methods, see, for example, the work of Beaucage, et al., Tetrahedron Letters, 22, pp. 1859-1862 (1981) on a solid support, in accordance with the sequence given below in Table II.

Additional analysis reveals that within the T38 fragment, there is a series of 6 amino acid residues (Gln-Pro-Trp-Glu-Pro-Leu), based on which a pool of 128 mixed 17-mer oligonucleotide probes can be obtained, as shown in Table III below.

Oligonucleotide probes are labeled at the 5'-end of gamma ³² P-ATP, 7500-8000 Ci / mmol (ICN) using T ₄ polynucleotide kinase (NEN).

Example 2

A. Monkey Processing Techniques and RIA Analysis

Cynomolgus female Masas fasicularias monkeys (2.5-3 kg, 1.5-2 years) are treated by subcutaneous administration of a phenylhydrazine hydrochloride solution (pH 7.0) at a dose of 12.5 mg / kg on days 1, 3 and 5. The hematocrit is monitored before each administration. On day 7, or as soon as the hematocrit level drops below 25% of the initial level, serum and kidneys are collected after administration of ketamine hydrochloride at a dosage of 25 mg / kg. The collected materials are immediately frozen in liquid nitrogen and stored at -70 ° C.

B. RIA for EPO

A radioimmunological study is used to quantify EPO in samples in accordance with the following methods:

An erythropoietin standard or an unknown sample is incubated with antiserum for two hours at 37 ° C. After two hours of incubation, the sample tubes are cooled on ice, erythropoietin labeled with ¹²⁵ I is added, and the tubes are incubated at 0 ° C for at least another 15 hours. Each test tube contains 500 μl of the incubation mixture containing 50 μl of diluted immune serum, 10,000 cpm ¹²⁵ I-erythropoietin, 5 μl of trasilol and 0-250 μl of either EPO standard or an unknown sample, and PBS containing 0.1% BSA for replenishment of the remaining volume. The serum used is the blood of a rabbit deflated in a second sample, immunized with a 1% pure human urinary erythropoietin preparation. The final dilution of the antiserum is adjusted so that the content of ¹²⁵ I-erythropoietin bound to the antibody does not exceed 10-20% of the total input amounts. In general, this corresponds to a final dilution of antiserum from 1: 50,000 to 1: 100,000.

¹²⁵ I-erythropoietin bound to the antibody is precipitated by the addition of 150 μl of Staph A. After a 40 minute incubation, the samples are centrifuged and the pellet washed twice with 0.75 ml of a solution of 10 mM Tris-HCl having a pH of 8.2 containing 0 15 M NaCl, 2 mM EDTA and 0.05% Triton X-100. The amount of washed residues was determined using a gamma-ray counter to determine the percentage of ¹²⁵ I-erythropoietin bond. Amounts associated with pre-immune sera are subtracted from all final values to correct for non-specific precipitation. The erythropoietin content of unknown samples is determined by comparison with a standard curve.

The above procedure is used on the monkey serum obtained above, in part A, and also not on the serum of the treated monkeys. Normal serum levels are determined to be approximately 36 mU / ml, while for serum on treated monkeys, they are between 1000 and 1700 mU / ml.

Example 3

A. Construction of the monkey cDNA library

Informational DNA was isolated from normal and anemic monkey kidneys using the guanidinium thiocyanate technique described by Chirgwin, et al., Biochemistry, 18, p. 5294 (1979), and the poly (A) ⁺ mRNA was purified by passing twice through a chromatographic column oligo (dT) cellulose, as described on pages 197-198 by Maniatis, et al., "Molecular Cloning, A Laboratory Manual" (Cold Springs Harbor Laboratory, Cold Springs, Harbor, NY, 1982). A cDNA library is constructed in accordance with a modification of the basic techniques set forth in Okayama, et al., Mol. and Cell. Biol., 2, pp. 161-170 (1982). The key points of the now preferred methods are as follows: (1) pUC8 is used as a plantar vector, cut with PstI and then provided with a tail of 60-80 bases in length using oligo dT; (2) HincII cleavage is used to cleave the oligo dT tail from one end of the vector; (3) the synthesis of the first strand and the attachment of the tail using oligo dG is carried out in accordance with the published methodology; (4) BamHI cleavage is used to cleave the oligo dG tail from one end of the vector; and (5) the replacement of the RNA strand with DNA is carried out in the presence of two connecting units (GATCTAAAGACCGTCCCCCCCCCC and ACGGTCTTTA) with a triple molar excess with respect to the vector with oligo dG tail.

B. Colony hybridization techniques for screening monkey cDNA libraries

Transformed E. coli are scattered with a density of 9000 colonies on 10 × 10 cm nutrient plates containing 50 μg / ml ampicillin. GeneScreen filters (New England Nuclear Catalog No. NEF-972) are pre-moistened on a BHI-CAM plate (37 g / L brain / heart bactoextract, 2 g / L Casamino acids and 15 g / L agar containing 500 μg / ml chloramphenicol) and used to raise colonies from a cup. Colonies are grown in the same medium for 12 hours or more to amplify the number of plasmid copies. Amplified colonies (colony sides) are processed serially by placing filters over two pieces of 3 MM Whatman paper saturated with the following solutions:

(1) 50 mM glucose - 25 mM Tris-HCl (pH 8.0) - 10 mM EDTA (pH 8.0) for 5 minutes,

(2) 0.5 M NaOH for 10 minutes, and

(3) 1.0 M Tris-HCl (pH 7.5) for three minutes.

The filters are then dried in air in vacuo for two hours at a temperature of 80 ° C.

The filters are then digested with Proteinase K through treatment with a solution containing 50 μg / ml of the protease enzyme in Buffer K [0.1 M Tris-HCl (pH 8.0) - 0.15 M NaCl - 10 mm EDTA (pH 8.2) 0.2% SDS]. Specifically, 5 ml of solution is added to each filter, and cleavage is continued at 55 ° C. for 30 minutes, after which the solution is removed.

The filters are then treated with 4 ml of pre-hybridization buffer (5 × SSPE - 0.5% SDS - 100 μg / ml SS E. coli DNA - 5 × BFP). Preliminary hybridization treatment is carried out at a temperature of 55 ° C, usually for 4 hours or more, after which the solution is removed.

The hybridization process is carried out as follows. To each filter, add 3 ml of hybridization buffer (5 × SSPE - 0.5% SDS - 100 μg / ml yeast tRNA) containing 0.025 picomoles of each of the 128 probe sequences shown in Table II (the complete mixture is designated as EPV mixture), and the filters are kept at a temperature of 48 ° C for 20 hours. This temperature is 2 ° C lower than the lower of the values of dissociation temperature (Td) determined for any of the probes.

After hybridization is complete, the filters are washed three times for 10 minutes on a rocking chair with a solution of 6 × SSC - 0.1% SDS at room temperature and washed two to three times with a solution of 6 × SSC - 1% SDS at a hybridization temperature (48 ° C).

Autoradiography filters identify seven positive clones among the screened 200,000 colonies.

The initial sequence analysis of one of the imaginary monkey cDNA clones (designated clone 83) is carried out to verify the results by modifying the methodology described by Wallace, et al., Gene, 16, pp.21-26 (1981). In short, a monkey plasmid from cDNA clone 83 of a monkey is linearized by digestion with EcoRI and denatured by heating in a boiling water bath. The nucleotide sequence is determined by the dideoxy method described by Sanger, et al., P.N.A.S. (U.S.A.), 74, pp. 5463-5467 (1977). A set of EPV mixture of probes consisting of 16 sequences is used as seed for sequence reactions.

C. Determination of the cDNA sequence of monkey EPO

The analysis of the nucleotide sequences of clone 83 is carried out by the methods described by Messing, Methods in Enzymology, 101, pp.20-78 (1983). Table IV presents the results of the analysis of the preliminary restriction map for the EcoRI / HindIII cloned fragment of clone 83 with approximately 1600 base pairs. The approximate locations of the recognition sites of restrictive endonuclease enzymes are expressed by the number of bases 3 ′ and the E. coli site at the 5 ′ end of the fragment. The determination of the nucleotide sequence is carried out by finding the sequence of individual restrictive fragments, with the aim of pairing overlapping fragments. For example, the overlap of sequence information represented by nucleotide analysis in the restriction fragment is denoted by C113 (Sau3A at ~ 111 / SmaI at ~ 324), and the sequence definition with the reverse order is denoted by C73 (AluI at ~ 424 / BstEII at ~ 203).

Table IV Limit enzyme recognition site Estimated Location (s) EcoRI 1 Sau3a 111 Smai 180 BstEII 203 Smai 324 KpnI 371 Rsai 372 Alui 424 PstI 426 Alui 430 Hpai 466 Alui 546 PstI 601 PvuII 604 Alui 605 Alui 782 Alui 788 Rsai 792 PstI 807 Alui 841 Alui 927 Ncoi 946 Sau3a 1014 Alui 1072 Alui 1115 Alui 1223 PstI 1301 Rsai 1343 Alui 1384 HindIII 1449 Alui 1450 HindIII 1585

Sequencing approximately 1342 base pairs (within the region from the 3 ′ Sau3A site to the EcoRI site and the HindIII site) and analysis of all possible reading systems contribute to the development of DNA and amino acid sequence information shown in Table V. In the table, the imaginary initial amino acid residue of the amino terminus of mature EPO (as verified by correlation of the previously indicated sequence analysis of twenty amino terminal residues) is indicated by the number +1. The presence of a methionine-determining ATC codon (designated -27), an “upstream” final alanine residue of the original amine, as the first residue designated for the amino acid sequence of the matured protein, is an indication of the likelihood that EPO is initially expressed in the cytoplasm in the form of a precursor, including The "leading" region of 27 amino acid residues, which is cut out before the ripened EPO enters the blood circulation. Potential glycosylation sites within the polypeptide are indicated by asterisks. The calculated molecular weight of the translation region is 21117 daltons, and the molecular weight of 165 residues of the polypeptide that make up the monkey’s mature EPO is 18236 daltons.

The polypeptide sequence in Table V can be easily analyzed for the presence of extremely hydrophilic regions and / or secondary conformational characteristics that are indicative of possibly very immunogenic regions, for example, using the methods proposed by Norr, et al., P.N.A.S. (U.S.A.), 78, pp. 3824-3828 (1981), and Kyte, et al., J. Mol. Biol., 157, pp. 105-132 (1982), and / or Chou, et al., Biochem., 13, pp. 222-245 (1974) and Advances in Enzymology, 47, pp. 45-47 (1978) ) Computerization of the analysis in accordance with the method of Norr, et al., Is possible thanks to the program designated PEP Reference Section 6.7, presented by Intelligenetics, Inc., 124 University Avenue, Palo Alto, California.

Example 4

A. Human Genomic Library

The human fetal liver genomic library carrying the Ch4A phage, obtained in accordance with the methods of Lawn, et al., Cell, 18, pp.533-543 (1979), is stored for use in plaque hybridization studies.

B. Plaque hybridization techniques for screening the human genomic library

Phage particles are lysed and DNA is fixed on filters (50,000 plaques per filter) according to the methods described by Woo, Methods In Enzymology, 68, pp. 389-395 (1979), except that GeneScreen Plus filters (New England were used) Nuclear Catalog No. NEF-976) and NZYAM cups (NaCl, 5 g; MgCl ₂ -6H ₂ O, 2 g; NZ-Amine A, 10 g; yeast extract, 5 g; casamino acid, 2 g; maltose, 2 g ; and agar, 15 g per liter).

Air-dried filters are fired at 80 ° C for 1 hour and then digested with proteinase K, as described in Example 3, part B. Pre-hybridization is carried out using 1 M NaCl - 1% SDS buffer at 55 ° C for 4 hours or more, after which the buffer is removed. Hybridization and posthybridization washes are carried out as described in Example 3, part B. A mixture of 128 20-mer probes designated by EPV and a mixture of 128 17-mer probes of Table III (designated as EPQ mixture) are used. Hybridization is carried out at a temperature of 48 ° C using a mixture of EPV probes. Hybridization using a mixture of EPQ probes is carried out at a temperature of 46 ° C, i.e. 4 degrees below the lowest calculated Td for mixture members. Removal of hybridized probes for re-hybridization is performed by boiling in 1 × SSC - 0.1% SDS for two minutes. Autoradiography of the filters reveals three positive clones (active with both mixtures of probes) among the scanned 1,500,000 phage plaques. Verification of the positive clones for EPO coding is achieved by determining the DNA sequence and electron micrographic imaging of the heteroduplex formation with cDNA of monkey of Example 3. This technique also provides evidence of the presence of multiple introns in the genomic DNA sequence.

Example 5

The analysis of the nucleotide sequences of the positive clones (designated λhEl), and the results obtained to date, are shown in Table VI.

In Table VI, the initial continuous DNA sequence denotes the upper thread of 620 bases in what appears to be the untranslated sequence immediately preceding the translated portion of the human EPO gene. More specifically, the sequence contains the 5'-end of the gene, which leads to a translated region of DNA encoding the first four amino acids (-27 to -24) of the leader sequence ("pre-sequence"). Four base pairs in the sequence preceding the last one encode the leader’s beginning, however, have not yet been unambiguously identified and are therefore indicated by “X”. This is followed by an intron of approximately 639 base pairs (of which 439 base pairs have a sequence identified and the remaining 200 base pairs are designated “I.S.”), immediately preceding the codon for glutamine, which is designated as the remainder of the -23 translated polypeptide. The immediately following exon sequence encodes amino acid residues through the alanine residue (designated as the +1 residue of the amino acid sequence of the matured human EPO) to the codon that determines threonine at position +26, followed by the second intron, consisting of 256 bases, as specifically indicated. The exon sequence following this intron for amino acid residues 27–55 continues with the third intron containing 612 base pairs. The subsequent exon encodes residues 56-115 of human EPO, followed by the fourth nitron of 134 base pairs, which continues to exon encoding residues 116-166 and the stop codon (TGA). Finally, in Table VI, a sequence of 568 base pairs is identified in what constitutes the untranslated region 3 ′ of the human EPO gene, the two base pairs of which (“X”) have not yet been uniquely identified for the sequence.

Table VI is used to identify the primary structural conformation (amino acid sequence) of the matured human EPO, including 166 established amino acid residues (with calculated molecular weight = 18399). The Table also shows a DNA sequence encoding a leader sequence of 27 residues together with a 5 'and 3' DNA sequence, which may be important for promoter / operator functions performed by the gene operon. Plots for potential glycosylation of a mature EPO human polypeptide are indicated by asterisks in the Table. It should be noted that the specific amino acid sequence in Table VI probably constitutes the sequence of the naturally occurring allelic form of human erythropoietin. The reason for this situation was found in the results of continuous attempts to determine the sequences of urinary isolates of human erythropoietin, which made it possible to have information that a significant number of erythropoietin molecules have methionine at residue 126 in contrast to serine, as shown in the Table.

Table VII below illustrates the degree of homology of the polypeptide sequence between human EPO and monkey. In the upper continuous line of the Table, letters are used to represent the deduced translated sequences of human EPO, starting at residue -27, and the lower continuous line shows the deduced polypeptide sequence of monkey EPO, starting from -27, representing the remainder. Asterisks are used to highlight sequence homology. It should be noted that the deduced sequences of human and monkey EPO reveal an “additional” lysine (K) residue at position 116 (for humans). The reference to Table VI means that this residue is located at the edge of the imaginary mRNA compound in the genomic sequence. The presence of a lysine residue in a human polypeptide sequence is verified by determining the sequence of a human cDNA clone derived from mRNA isolated from COS-1 cells that are transformed with the human genomic DNA in Example 7 below.

Example 6

In initial attempts at microbiological synthesis of isolated amounts of erythropoietin of a polypeptide material encoded by monkey cDNA obtained by the procedure of Example 3, the selected expression system is a system containing mammalian host cells (e.g., COS-1 cells, A.T.C.C., No.CRL-1650). These cells are transfected with a shuttle vector capable of spontaneous replication in the E. coli host (due to the presence of DNA derivatives from pBR322 plasmids) and to mammalian hosts (due to the presence of DNA derivatives from SV40 viruses).

More specifically, the expression vector is constructed in accordance with the following methods. Clone plasmid 83 obtained in Example 3 was amplified in E. coli bacteria and approximately 1.4 kb were isolated by digestion with EcoRI and HindIII. Monkey DNA encoding erythropoietin. 4.0 kbp were isolated from plasmid pBR322 separately. HindIII / SalI fragment. About 30 base pairs of the EcoRI / SalI linker fragment are obtained from DNA containing M13mp10 RF DNA repeats (P and L laboratories). Said “linker” includes sequentially an EcoRI-sticky end, followed by SstI, SmaI, BamHI and XbaI recognition sites and a sticky SalI end. The above three fragments are crosslinked to give about 5.4 kb. intermediate plasmid ("pERS"), EPO-DNA which borders on one side when using the "bank" of useful sites restriction recognition of endonuclease. After that, the pERS plasmid was digested with HindIII and SalI to obtain erythropoietin DNA and the transformation of EcoRI into a SalI "linker" (M13mp10). The above 1.4 kb DNA fragment stitch with a BamHI / SalI site of approximately 4.0 kbp. plasmids pBR322 and another "linker" of the M13mp10 HindIII / BamHI RF fragment, which also has about 30 base pairs. The M13 linker fragment is the HindIII-sticky end, followed by recognition sites of PstI, SalI, XbaI and the sticky end of BamHI. The cross-linking product again becomes a useful intermediate plasmid (“pBR-EPO”) containing EPO DNA, which is bounded on both sides by “accumulated rows” of the restriction region.

The vector selected for the expression of EPO DNA in COS-1 cells ("pDSVL1") is pre-constructed to allow selection and self-duplication in E. coli. These characteristics are achieved due to the beginning of replication and the sequence of DNA genes resistant to the action of ampicillin, which are present in the region that carries out the contraction of nucleotides 2448 to 4362 of the plasmid pBR322. The specified sequence is structurally modified by introducing a "linker", which provides recognition of HindIII immediately upon adjoining nucleotide 2448 before inclusion in the specified vector. Along with the selection of vectors for expression of EPO DNA, other useful properties include the ability to autoduplicate in COS-1 cells and the presence of a sequence that acts as a viral promoter in mammalian cells. These characteristics are achieved by starting the sequence of DNA replication and the sequence of the “late” DNA gene, which is a viral enhancer that is present in a chain containing 342 base pairs, which pulls together the number of nucleotides from 5171 to 270 of the SV40 genome. A unique restriction region (BamHI) is formed on a vector and immediately adjoins a number of viral promoters through the use of a commercially available “linker” sequence (Collaborative Research). Additionally, a sequence of 237 base pairs (a derivative as a nucleotide number from 2553 to 2770 of the SV40 virus) is added to the vector, containing the late gene polyadenylation signal of the viral mRNA (commonly known as a transcription terminator). The specified fragment is placed in the vector in the appropriate orientation relative to the viral promoter of the "late gene" through a unique plot of BamHI. In this vector, another mammalian gene is also presented, which is in a non-material position relative to the possible transcription of a gene that is located on the indicated unique region between the sequences of the viral promoter and the terminator. [This mammalian gene contained approximately 2500 base pairs of mouse dihydrofolate reductase (DHFR) minigen from plasmid pMG-1, Gasser, et al., P.N.A.S. (U.S.A.), 79, pp. 6522-6526, (1982)]. Again, most of the active components of plasmid pDSVL1 consist of 2448-4362 nucleotides of plasmid pBR322 along with nucleotides 5171-270 (342 base pairs) and 2553-2770 (237 base pairs) of viral DNA containing the SV40 virus.

In accordance with the methods described previously, for example, Maniatis, et al., Supra, erythropoietin-encoding DNA was isolated from the pBR-EPO plasmid as a BamHI fragment and sutured into the pDSVL1 plasmid excised with BamHI. To confirm the introduction of the EPO gene in the correct orientation in two of the obtained cloned vectors (duplicated vectors H and L), an analysis was performed using the restriction enzyme (see FIG. 2 illustrating the plasmid pDSVL1-MkE). Vectors with an incorrect orientation of EPO genes are left as regulators of negative values in transfection experiments designed to determine the expression levels of EPO in hosts that are transformed by vectors with the correct orientation of EPO DNA.

Vectors H, L, F, X, and G are combined with a DNA carrier (DNA of the liver and mouse spleen). For transfection, doublet 60 mm plates are used, using calcium phosphate microprecipitation methods. Duplicate 60 mm plates are also transfected with a DNA carrier as an “imitation” of negative transformation transformation. After 5 days, the entire culture medium is tested for the presence of polypeptides that have the immunological properties of naturally occurring erythropoietin.

Example 7

A. The expression system of the original EPO using COS-1 cells

The system selected at the initial stage of the microbiological synthesis of the amounts of human erythropoietin polypeptide material encoded by the EPO clone of human genomic DNA is selected, the system also includes expression in mammalian host cells (for example, COS-1, A.T.C. No. CRL-1650). The human EPO gene is initially subcloned into a "shuttle" vector, which has the ability to autonomously replicate in both hosts of the E. coli bacterium (thanks to the DNA derivative of plasmid pBR322) and in the cell line of the mammalian COS-1 (due to the presence of the SV-40 virus derivatives of DNA). The shuttle vector containing the EPO gene is then transfected into COS-1 cells. EPO polypeptide material is formed in transfected cells and secreted into the cell culture medium.

More specifically, a trait expression vector is constructed in accordance with the following methods. DNA isolated from the lamb λhE1 clone containing the genomic EPO gene was digested with BamHI and HindIII restrictive endonucleases and 5.6 kb were isolated. A DNA fragment was found to contain the entire EPO gene. The indicated DNA fragment is mixed and crosslinked with the bacterial plasmid pUC8 (Bethesda Research Laboratories, Inc.), which is similarly cleaved, creating an intermediate plasmid "pUCS-HuE", thus forming a suitable source of the limiting fragment.

Selected vectors for the expression of EPO DNA in COS-1 cells are built in advance. Plasmid pSV4SEt, which contains DNA sequences, allows selection and auto-duplication in E. coli. The indicated characteristics are achieved by the onset of replication and the sequence of the ampicillin resistant DNA gene, which is present in the region contracting nucleotides from 2448 to 4362 of the bacterial plasmid pBR322. Change the structure of the indicated sequence by introducing a linker, providing the HindIII recognition site immediately upon contact with nucleotide 2448. The plasmid pSV4SEt is also capable of self-duplication in COS-1 cells. The specified characteristics are achieved by using a fragment of 342 base pairs containing the viral origin of replication using the SV40 virus (nucleotides from 5171 to 270). The indicated fragment is modified by introducing a linker that provides an EcoRI recognition site adjacent to nucleotide 270 and a linker that provides a SalI recognition site adjacent to nucleotide 5171. This vector also contains fragment 1061 of the SV40 virus base pair (nucleotides from 1711 to 2772 s an additional linker that provides a SalI recognition site adjacent to the next nucleotide 2772). Within the indicated fragment, there is a unique BamHI recognition sequence. Summarizing the above, the plasmid pSV4SEt contains unique recognition sites BamHI and HindIII, allowing the introduction of the human EPO gene. These sequences enable replication and selective selection in E. coli and replication in COS-1 cells.

To introduce the EPO gene into plasmid pSV4SEt, plasmid pUC8-HuE was digested with BamHI and HindIII to a limiting endonuclease and 5.6 kb were isolated. EPO encoding a DNA fragment. Plasmid pSV4SEt was also digested with BamHI and HindIII, and the main part of the fragment containing 2513 base pairs was isolated (while retaining all the necessary functions). These fragments are mixed and crosslinked, forming the final vector "pSVgHuEPO" (see figure 3). This vector is diluted in E. coli, and the DNA vector is isolated. To confirm the input of the EPO gene, a restriction enzyme is analyzed.

The plasmid pSVgHuEPO DNA is used to express the characteristics of EPO polypeptide material in COS-1 cells. In detail, the plasmid pSVgHuEPO DNA was combined with a carrier DNA and transfected into 60 mm triplicate platelets of COS-1 cells. As a control, DNA alone is transfected in COS-1 cells. The cell culture medium is selected after 5 and 7 days and tested for the presence of polypeptides that have immunological properties characteristic of naturally occurring human EPO.

B. Second expression system of EPO signs in COS-1 cells.

Another expression system is used to produce improved products of human erythropoietin-polypeptide material encoded using a clone of human genomic DNA that forms EPO in COS-1 cells (A.T.C.S. No. CRL-1650).

In the previous system, EPO signs are expressed in COS-1 cells using its own promoter, which is present within the BamHI restriction fragment relative to HindIII, having a length of 5.6 kb. In the following construction, the EPO gene is alternated to obtain expression of the signs using the late promoter of the SV40 virus.

More specifically, a restrictive fragment of a human genomic EPO representing a cloned 5.6 kb BamHI relative to HindIII, modify in accordance with the following procedure. As described above, the plasmid pUC8-HuE was digested with BamHI and restrictive endonuclease BstEII. BstEII restrictive endonuclease cleaves within 5.6 kb. The EPO gene at a position that represents 44 base pairs 5 ′ to the initiating ATG, encoding a peptide precursor and about 680 base pairs 3 ′ to the HindIII restriction site. Then a fragment comprising approximately 4900 base pairs is isolated. A synthetic linker DNA fragment containing Sall and BstEII sticky ends and an internal BamHI recognition site is synthesized and purified. These two fragments are mixed and crosslinked with plasmid pBR322, which is cut using Sall and BamHI to obtain the intermediate plasmid pBRgHE. The human EPO genomic gene can be isolated from it in the form of a BamHI cleavage fragment with 4900 base pairs, carrying the complete gene structure of a single ATG 44 base pair 3 'relative to the BamHI site adjacent to the amino acid coding termination region.

The resulting fragment was isolated and introduced as a BamHI fragment into the plasmid pDSVL1 of the cleaved BamHI expression vector (see Example 6). The resulting plasmid pDSVLgHuEPO, as shown in FIG. 4, is used to express the characteristics of EPO polypeptide material from COS-1 cells, as described in Examples 6 and 7A.

Example 8

The culture medium grown from 6 transfected cell cultures of COS-1 of Example 6 was analyzed by radioimmunoassay according to the procedure described in Example 2, Part B. Each sample was analyzed with aliquots of 250, 125, 50 and 25 μl. Supernatants from cell growth "imitated" transfected or transfected with vectors with the wrong orientation of EPO genes had negative values for immunoreactivity. For each sample of two supernatant fluid layers obtained from the growth of COS-1 cells transfected with vectors (H and L) with the correct orientation of the EPO DNA, the% inhibition of ¹²⁵ I-EPO associated with the antibody ranged from 72 to 88%, all whose values are placed in the upper position of the standard curve. Therefore, it is impossible to accurately calculate the true concentration of erythropoietin in the floating layers of the culture. In preliminary calculations, it was 300 mU / ml, however, performed with the largest aliquot (250 μl).

A sample of the culture fluid obtained according to Example 6, and the 5- and 7-day culture fluids described in Example 7A were subjected to radioimmunoassay to compare the activity of recombinant monkey and human EPO materials with respect to naturally occurring human EPO meeting the standard. The results are presented in graphical form in figure 1. In short, the results presented unexpectedly showed that the recombinant monkey EPO was substantially competitive for the anti-human EPO antibody, although it was not possible to completely inhibit the binder under the test conditions. The maximum% inhibition values for recombinant human erythropoietin, however, almost approaches standard values. The parallel nature of the dose-response curve implies the immunological identity of the sequences (epitopes) in general. Before calculating the concentration of monkey EPO in the culture fluids, repeated evaluations were carried out at the indicated high dilution values and it was found that they ranged from 2.91 to 3.12 U / ml. The calculated levels of human EPO production were established, respectively, at 392 mU / ml for a sample with 5-day growth and 567 mU / ml for a sample with seven-day growth. The calculated levels of monkey EPO production in Example 7B expression systems were similar or better.

Example 9

The culture fluids obtained according to examples 6 and 7 were subjected to in vitro analysis to determine the activity of EPO in accordance with the method of Goldwasser, et al., Endocrinology, 97, 2, pp. 315-323 (1975). The calculated values of the EPO monkey for the test culture fluids ranged from 3.2 to 4.3 U / ml Human EPO culture liquids were also active in the obtained calculations in an in vitro assay and, moreover, this activity can be neutralized by the action of an anti-EPO antibody. The culture fluids of the recombinant monkey EPO of Example 6 were also quantified in vivo biological activity according to the general method of Cotes, et al., Nature, 191, pp. 1065-1067 (1961), and Hammond, et al., Ann. N.Y. Acad. Sci., 149, pp. 516-527 (1968). The indicated activity levels ranged from 0.94 to 1.24 U / ml.

Example 10

In the previous examples, recombinant human or monkey EPO material is obtained from vectors that are used to transfect COS-1 cells. These vectors replicate in COS-1 cells due to the presence of the SV40 virus T antigen inside the cell that initiates the occurrence of SV40 replication on these vectors. Although the obtained vectors reproduce the useful qualities of EPO in COS-1 cells, their severity is only temporary (within 7-14 days) due to possible loss of the vector. In addition, only a negligible% of COS-1 reproductions becomes productively transfected with these vectors. The present example describes trait expression systems using DHFR cells ^- Chinese hamster ovary (CHO) and a selectable marker, DHFR. [For a discussion of related expression systems, see US Pat. No. 4,399,216 and European Patent Application 117058, 117059 and 117060, published August 29, 1984].

In CHO cells, DHFR ^- (Dux-Bll) CHO K1, Urlaub, et al., Proc. Nat. Acad. Sci. (USA), Vol. 77, 4461 (1980), the dihydrofolate reductase enzyme (DHFR) is lacking due to mutations occurring in structural genes, and therefore, glycine, hypoxanthine, and thymidine must be present in the culture medium. Plasmid pDSVL-MkE (Example 6) or pDSVL-gHuEPO (Example 7) were transfected with carrier DNA in DHFR ^- CHO cells are grown in a medium containing hypoxanthine, glycine and thymidine in the culture plates 60 mm. Plasmid pSVgHuEPO (Example 7A) was mixed with the plasmid pMG2 containing the dihydrofolate reductase mouse gene cloned into the bacterial plasmid pBR322 vector (according to Gasser, et al., Supra). A mixture of plasmids and carrier DNA is transfected into DHFR ^- CHO cells. (Cells that capture one plasmid will usually also capture a second plasmid). After 3 days, the obtained cells are dispersed by trypsinization on several 100 mm culture plates in an environment in which hypoxanthine and thymidine are deficient. Only those cells that are transformed into a stable state through the DHFR gene show survival in the EPO gene in this medium. After 7-21 days, colonies of living cells become visible. These dispersion colonies after dispersion by trypsinization can multiply continuously in a medium with hypoxanthine and thymidine deficiency, creating new cell strains (for example, CHO pDSVL-MkEPO, CHO pSVgHuEPO, CHO pDSVL-gHuEPO).

The culture fluids obtained from the above cell strains are subjected to radioimmunoassay for the presence of a recombinant EPO of a monkey or human. The medium for CHO strains pDSVL-MkEPO contains EPO with the same immunological properties as EPO obtained from COS-1 cells transfected with plasmid pDSVL-MkEPO. A 65-hour sample of culture fluid contains 0.60 U / ml EPO monkey.

Culture fluids derived from CHO pSVgHuEPO and CHO pDSVL-gHuEPO contain recombinant human EPO having the same immunological properties as EPO obtained from COS-1 cells transfected with pSVgHuEPO or pDSVL-gHuEPO plasmid. A 3-day sample of culture fluid obtained from CHO pSVgHuEPO contains 2.99 U / ml of human EPO, and a 5.5-day sample of culture fluid obtained from CHO pDSVL-gHuEPO contains 18.2 U / ml of human EPO , as shown by the results of radioimmunological analysis.

The quality of EPO obtained through the above cell strains can be improved by enhancing genes, creating new cell strains with greater efficiency. The dihydrofolate reductase enzyme (DHFR), which is a product encoded by the DHFR gene, can be inhibited by methotrexate (MTX). In particular, cells propagated in an environment deficient in hypoxanthine and thymidine are inhibited or killed by methotrexate. Under appropriate conditions (for example, at a minimum concentration of MTX), cells that are stable and capable of growth in MTX can be obtained. It was found that these cells become resistant to MTX by enhancing a number of their DHFR genes, resulting in increased production of the DHFR enzyme. Living cells can, in turn, be treated with elevated concentrations of methotrexate, resulting in an increase in the number of DHFR genes in cell strains. “Passing genes” (for example, EPO), transferred to or transformed with the DHFR gene or transformed with it, are often found to increase their number of reproductions.

As an example of the practical implementation of the amplification system, the CHO cell strain pDSVL-MkE is treated with methotrexate by increasing its concentration (0 nM, 30 nM and 100 nM). Samples representing a 65-hour culture medium at each stage of amplification, according to radioimmunological analysis, contain 0.60, 2.45 and 6.10 U / ml erythropoietin, respectively. The CHO cell strain pDSVL-gHuEPO is subjected to treatment with methotrexate (MTX) with a serial increase in its concentration: 30 nM, 50 nM, 100 nM, 200 nM, 1 μM and 5 μM. A sample representing a 3-day supernatant, at the stage of its treatment with methotrexate (MTX) with a concentration of 100 nM, contains 3089 ± 129 U / ml of human erythropoietin according to radioimmunological analysis. Samples representing a 48-hour culture medium treated with methotrexate with a concentration of 100 nM and 1 μM contain, according to radioimmunological analysis, 466 and 1352 U / ml of human erythropoietin, respectively (average number obtained from a 3-time test of samples). According to the specified method in 60 mm Petri dishes containing 5 ml of medium, seeded in 1 × 10 ⁶ cells. After 24 hours, the medium is removed, replacing it with 5 ml of fresh medium in which there is no serum (high glucose DMEM, to which 0.1 nM non-basic amino acids and L-glutamine are additionally added). Erythropoietin is allowed to accumulate for 48 hours in a serum-free medium. Then the medium is collected for testing by radioimmunoassay, and the cells are trypsinized and then counted. The average values of the samples as a result of radioimmunological analysis are about 467 and 1352 U / ml erythropoietin for cells grown by treatment with methotrexate with a concentration of 100 nM and 1 μM, respectively, provide the actual yield of erythropoietin of the order of 2335 and 6750 U per cup. The average number of cells per dish is 1.94 × 10 ⁶ and 3.12 × 10 ⁶ cells, respectively. Thus, the rate of efficient production under the indicated culture conditions is 1264 and 2167 U / 10 ⁶ cells in 48 hours.

The cell cultures described above are a genetically heterogeneous population. In order to isolate genetically heterogeneous clones with the highest ability to produce, standard selection methods are used (see Section A, Part 2, “Points to Consider in the Characterization of Cell Lines Used to Produce Biologies”, June 1, 1984, Department of Scientific Biologics Testing, Center for Medicines and Biologics, United States Food and Drug Administration).

The above-described productivity of CHO cell lines producing erythropoietin can be improved by using appropriate cell culture techniques. Propagation of mammalian animal cells in the culture medium usually requires the presence of serum in the growth medium. The method of producing erythropoietin from CHO cells in serum-free medium greatly facilitates the rectification of erythropoietin from the culture medium. The method described below enables the economical production of erythropoietin in large quantities in a serum-free medium sufficient for production.

Cells of the CHO pDSVL-gHuEPO line grown under standard cell culture conditions use transplanted cell culture vials for plating. These cells are propagated as a suspension cell line in vials containing a transplantable cell culture in a 50:50 mixture of a high glucose DMEM and F12 Ham mixture supplemented with 5% fetal bovine serum (FCS), L- glutamine, penicillin and streptomycin, 0.05 nM non-essential amino acids and methotrexate with the appropriate concentration. Suspended cell cultures provide the ability to rapidly multiply to large volumes erythropoietin obtained from CHO cells. CHO cells grown in suspension are used for plating them in rotating vessels with an initial seeding density of 1.5 × 10 ⁷ viable cells per 850 cm ^{2 of the} indicated vessel containing 200 ml of medium. Over a 3-day period, these cells are allowed to grow to a relatively tightly adjacent cell line until a monolayer is formed. The medium used in this phase of cell growth is similar to the medium used to cultivate cells in suspension. By the end of the 3-day cell growth period, the serum-containing medium is separated and replaced with 100 ml of serum-free medium. The serum-free medium contains a 50:50 mixture of DMEM with a high content of glucose and F12 Ham, in which 0.05 mM non-basic amino acids and L-glutamine are additionally added. The rotating vessels with the prepared medium are placed for 2-3 hours in the incubator and then it is again removed, replacing 100 ml of fresh, serum-free medium. With 1-3-hour incubation in serum-free medium, a decrease in the concentration of protein contamination with serum occurs. Then, the rotating vessels are again placed for 7 days in an incubator, during which erythropoietin accumulates in a serum-free culture medium. At the end of the 7-day erythropoietin production phase, the old medium is removed, replacing it with fresh, serum-free medium necessary for the second production cycle. As an example of the practical implementation of this production system, a sample is presented that is in serum-free medium for 7 days, which according to the radioimmunological analysis contains 3892 ± 409 U / ml erythropoietin. Taking into account the data on the calculation of cell density of the order of 0.9-1.8 × 10 ⁵ cells per cm ² , each rotating vessel with a volume of 850 cm ² contains from 0.75 to 1.5 × 10 ⁸ cells, and based on this, thus, the erythropoietin production rate for 7 days in 100 ml of culture reaches values ranging from 750 to 1470 U / 10 ⁶ cells in 48 hours.

The supernatants from the CHO pDSVL-MkEPO cell line amplified in methotrexate at a concentration of 10 nM are subjected to radioimmunoassay to test the activity of erythropoietin in vitro and in vivo. A sample prepared in a concentrated medium during radioimmunological analysis contains 41.2 ± 1.4 U / ml; 41.2 ± 0.064 U / ml of Mk-erythropoietin, as determined according to an in vitro biological activity assay, and 42.5 ± 5 U / ml according to an in vivo biological activity assay. When determining the sequence of amino acid residues of polypeptide products, the presence of erythropoietin products is detected, and the dominant species includes 3 residues of the “leading” sequence adjacent to the proposed completion of the amino groups with alanine. At present, it is not clear whether this is the result of improper processing of the polypeptide nucleus in CHO cells, or reflects the difference in the structure of the terminal termination of the amino groups of monkey erythropoietin relative to human erythropoietin.

The supernatant prepared from the CHO cell line pDSVL-gHuEPO was subjected to three tests. According to radioimmunoassay, a 5.5-day sample contains about 18.2 U / ml recombinant human erythropoietin in the indicated medium; for in vitro analysis, 15.8 ± 4.6 U / ml; and for in vivo analysis, 16.8 ± 3.0 U / ml.

The supernatant prepared from CHO cell line pDSVL-gHuEPO with gradual amplification with methotrexate at a concentration of 100 nM was subjected to three tests. According to radioimmunological analysis, a 3-day sample contains 3089 ± 129 U / ml recombinant human erythropoietin; when testing the sample in vitro 2589 ± 71.5 U / ml; and when testing the sample in vivo 2040 ± 160 U / ml. The amino acid sequence shows the completion of the amino groups corresponding to those presented in table VI.

Cell-conditioned medium prepared from CHO cells, which was transfected with pDSVL-MkE plasmid in methotrexate at a concentration of 10 nM, was combined and methotrexate was dialyzed for several days, resulting in a culture with erythropoietin activity of the order of 221 ± 5.1 U / ml ( erythropoietin-cell conditioned medium) (EPO-CCM). To determine the effect of EPO-CCM on the hematocrit level in the Balb / C line of a normal mouse, the following experiment is carried out in vivo. Cell-conditioned medium prepared from non-transfected CHO cells (CCM) and EPO-CCM is regulated using a phosphorus buffered solution (PBS). Cell-conditioned medium is used in the control group of animals (3 mice), and for experimental groups (2 mice per group) 2 dosage levels of EPO-CCM are used: 4 units per injection and 44 units per injection. During a 5-week course, 7 mice are injected intraperitoneally 3 times a week. After the 8th injection, the average hematocrit in the control group was found to be 50.4%, for the group with the introduction of 4 units of EPO-CCM, they account for 55.1% and with the introduction of 44 units of EPO-CCM - 67.9%.

The expression products of mammalian animal cells can be removed without difficulty in a fairly purified form from the culture medium using HPLC (C ₄ ) with an ethanol gradient, preferably at pH = 7.

A preliminary attempt was made to determine the properties of recombinant glycoprotein products obtained from the conditioned medium for expression of COS-1 cells and CHO of the erythropoietin gene from human urine using both Western blot analysis (staining) and SDS-PAGE analysis. These studies show that the erythropoietin material produced by CHO cells has a slightly higher molecular weight than the expression products of COS-1 cells, which, in turn, is slightly larger than the human urinary extract of a bullet source. All products are somewhat heterogeneous. Treatment with a neuraminidase enzyme to remove sialic acid leads to the fact that the recombinant products of the COS-1 and CHO cells have approximately the same molecular weight, which, however, is one or the other more than the molecular weight of the resulting human urinary extract containing Asian groups. Treatment with the F-endoglucosidase enzyme (EC 3.2.1) of the recombinant CHO cell product and urinary extract product (to completely remove carbohydrates from both products) results in almost homogeneous products, which basically have the same molecular weight.

The purified erythropoietin obtained from human urine and the recombinant erythropoietin produced from CHO cells, erythropoietin according to the present invention, are analyzed for the determination of carbohydrate composition by the method described by Ledeen, et al., Methods in Enzymology, 83 (Part D, 139-191 (1982 ), which was modified using the hydrolysis method described by Nesser, et al., Anal. Biochem. 142, 58-67 (1984). Experimentally determined indicators of the carbohydrate composition for urinary isolate (expressed as molar ratios in these products) have following values: heck oza, 1.73; N-acetylglucosamine, 1; N-acetylneuraminic acid, 0.93; fucose, 0 and N-acetylgalactosamine, 0. Corresponding values for recombinant products (derived from CHO cell line pDSVL-gHuEPO 3-day culture medium enhanced by methotrexate at a concentration of 100 nM) has the following indicators: hexose, 15.09; N-acetylglucosamine, 1; N-acetylneuraminic acid, 0.998; fucose, 0 and N-acetylgalactosamine, 0.

The results obtained are consistent with the results of the Western blot and SDS-PAGE analyzes described above.

The glycoprotein products obtained according to the present invention are thus achievable products whose primary structural conformation sufficiently reproduces the conformation of naturally occurring erythropoietins to give them one or more biological properties, and whose quantitative composition of carbohydrates differs from the composition of naturally occurring erythropoietins.

Example 11

The proposed example relates to the entire cycle of erythropoietins production by assembling the nucleotide bases of two structural genes encoding the human erythropoietin sequence presented in Table VI and containing, respectively, “preferred” codons for expression of characters in E. coli and yeast cells (S. cerevisiae).

In this example, the construction of genes encoding erythropoietin analogues is further described. In short, the protocols used are generally presented in the above description of Alton, et al. (WO 83/04053). These genes are designed for the initial assembly of constituent oligonucleotides in complex duplex, which, in turn, are assembled in three discrete parts. These parts are constructed for amplification and, when removed from the amplification system, can be assembled sequentially or through the ligature of a fragment with a complex structure into a suitable expression vector.

Tables VIII-XIV show the layout and assembly of the resulting gene encoding the translation product of human erythropoietin, which lacks any leading or preceding sequence but contains the initial methionine residue at position -1. Moreover, the gene introduced into the main part of the E. coli cell prefers codons and, therefore, this construct is called the "ECEPO" gene.

In more detail, Table VIII illustrates the use of oligonucleotides to generate Part 1 of an ECEPO gene encoding the amino terminal residues of a human polypeptide. Oligonucleotides are collected in duplexes (1 and 2, 3 and 4, etc.), which are then crosslinked to obtain Part 1 ECEPO, as in Table IX. It should be noted that the assembled part includes the corresponding sticky ends of the final EcoRI and BamHI, with the XbaI restriction enzyme recognition site being directed “downstream” of the EcoRI, and the KpnI recognition site directed “upstream” of the sticky end of BamHI. Part 1 can be easily amplified using the phage vector M13 used to verify the sequence of this part. Some difficulties unexpectedly arise when a portion is isolated as an XbaI / KpnI fragment from RF DNA generated in E. coli, probably due to methylation of the bases of the KpnI recognition site within the host. Therefore, phage DNA is isolated and reproduced in double-stranded form in vitro by stretching with a primer, and then the desired double-stranded fragment is isolated.

Parts 2 and 3 of the ECEPO gene (Tables XI and XIII) are constructed in a similar manner from the oligonucleotides of Tables X and XII, respectively. Each part is amplified on an M13 vector, used for sequence checking, and isolated from phage DNA. As can be seen from Table XI, Part 2 of ECEPO is built with sticky ends EcoRI and BamHI and can be isolated as a fragment of KpnI / BglII. Similarly, Part 3 ECEPO is obtained with the sticky ends of BamHI and SalI and can be isolated from RF DNA as a BglII / SalI fragment. The three parts thus obtained can be easily assembled into a continuous DNA sequence (Table XIV) encoding the polypeptide of the complete human EPO with the amino-terminal methionine codon (ATG) for translational initiation of E. coli. It should be noted that a number of base pairs are directed upstream of the initial ATG, which duplicates the sequence of the ribosome binding site of the strongly expressed OMP-f E. coli gene.

Any suitable expression vector may be used for ECEPO transfer. A specific vector selected for the expression of the ECEPO gene as the "temperature-sensitive" plasmid pCFM536 is a derivative of the plasmid pCFM414 (A.T.S.C. 40076), as described in co-pending application for US patent No. 636727, filed August 6, 1984 g, Charles F. Morris. More specifically, pCFM536 cleave XbaI and HindIII; a large fragment is isolated and used in two-way crosslinking with the ECEPO gene. Parts 1 (XbaI / KpnI), 2 (KpnI / BgIII) and 3 (BglII / SalI) are pre-assembled in the correct order in M13 and the EPO gene is isolated from there as a single XbaI / HindIII fragment. This fragment includes a portion of the polylinker from M13 of the mp9 phage, contracting SalI and HindIII sections in it. Expression control of the resulting expression plasmid, p536, is performed using the _L Lambda promoter P _L , which itself can be controlled by the repressor C ₁₈₅₇ gene (such as that obtained in K12ΔHtrp of E. coli strand).

The constructed ECEPO gene can be modified in various ways to encode erythropoietin analogues, such as [Asn ² , des-Pro ² according to Ile ⁶ ] hEPO and [His ⁷ ] hEPO, as described below.

A. [Asn ² , des-Pro ² by Ile ⁶ ] hEPO

Plasmid 536, carrying the constructed ECEPO gene of Table XIV as an XbaI insert in HindIII, was digested with HindIII and XhoI. The ECEPO gene is cut with the endonuclease of the latter in a unique recognition site having 6 base pairs, tightening the last base of the codon encoding Asp ⁸ with the second base of the Arg ¹⁰ codon. An XbaI / XhoI linker sequence is constructed having the following sequence;

The XbaI / XhoI linker and a fragment of the ECEPO XhoI / HindIII gene sequence are inserted into a large fragment resulting from the cleavage of XbaI and HindIII of plasmid pCFM526, a derivative of plasmid pCFM414 (A.T.C. 40076), as described in the simultaneously pending patent application US No. 636727, filed August 6, 1984 by Charles F. Morris, to generate a plasmid-carrying DNA sequence encoding the expression of E. coli form Met ^{-1 of the} desired analogue.

B. [His ⁷ ] hEPO

Plasmid 536 was digested with HindIII and XhoI, as in Part A above. The XbaI / XhoI linker is built with the following sequence:

The linker and the ECEPO XhoI / HindIII sequence fragment are then inserted into pCFM526 to generate a plasmid-carrying DNA sequence encoding the expression of E. coli form Met ^{-1 of the} desired analogue.

The construction of the manufactured gene ("SCEPO"), including the yeast preferred codons, occurs as described in the following Tables XV-XXI. As in the case of the ECEPO gene, the complete construction contains the formation of three sets of oligonucleotides (Tables XV, XVII and XIX), which are formed into duplexes and assembled into parts (Tables XVI, XVIII and XX). It should be noted that the synthesis is partially facilitated by the use of some suboptimal codons in both SCEPO and ECEPO constructs, i.e. oligonucleotides 7-12 of part 1 of both genes were identical, as oligonucleotides 1-6 of part 2 were identical in each gene.

The assembled parts of SCEPO are aligned in M13 and Parts 1, 2 and 3 are isolated from phage as fragments of HindIII / KpnI, KpnI / BglII and BglII / SalI.

The currently preferred expression system for SCEPO gene products is a secretion system based on the secretion of S. cerevisiae α factor, as described in U.S. Patent Application No. 487,753, filed April 22, 1983, Grant A. Bitter, published October 31, 1984 of the year as European Patent Application 0123294. In short, the system includes constructs where the DNA encoding the leading sequence of the yeast α-factor gene is located just beyond 5 'to the coding region of the expressed exogenous gene. As a result, the translated gene product includes a leading or signal sequence that is “processed” by the endogenous yeast enzyme in the direction of secretion of the remainder of the product. Since the design makes it possible to use the α-factor translation initiation codon (ATG), it was not necessary to create such a codon at position -1 of the SCEPO gene. As can be concluded from Table XXI, the alanine (+1) encoding the sequence is behind the linker sequence, which allows direct insertion into a plasmid that includes DNA for the first 80 residues of the α-factor conductor following the α-factor promoter. A specific preferred construct for the expression of the SCEPO gene contains four-sided crosslinking, including the above fragments of the SCEPO portion and a large fragment of the HindIII / SalI cleavage of plasmid pαC3. The promoter and leader sequences of the α-factor, as well as the SCEPO gene, are isolated from the resulting plasmid pαC3 / SCEPO by cleaving the BamHI and stitching the cleaved BamHI into the pYE plasmid to form plasmid pYE / SCEPO expression.

Example 12

The present example relates to the expression of recombinant products constructed by ECEPO and SCEPO genes within the expression system of Example 11.

Using an expression system designed for use in E. coli host cells, the plasmid p536 of Example 11 is transformed into E. coli AM7 cells previously transformed with a suitable plasmid, pMW1, which has received the C ₁₈₅₇ gene. Cell cultures in Bouillon (ampicillin 50 μg / ml and kanamycin 5 μg / ml, preferably with 10 mM MgSO ₄ ) are maintained at a temperature of 28 ° C and during cell growth in culture to O.D. ₆₀₀ = 0.1, EPO expression is induced by raising the temperature to 42 ° C. Cells grow to approximately 40 OD, providing an EPO (gel estimated) of about 5 mg / OD l.

Cells are harvested, lysed, broken with a French press (10,000 psi) and treated with lysozyme and detergent NP-40. The precipitate obtained by centrifugation at 24000xg was dissolved in guanidine HCl and subjected to further single purification using C ₄ (Vydac) reverse phase HPLC (EtOH, 0-80%, 50 mM NH ₄ Ac, pH 4.5). The determination of the base sequence of the protein reveals that the product is more than 95% pure, and the resulting products reveal two different amino-ends, APPR ... and PPR ... with a relative quantitative ratio of 3: 1. This latest observation of hEPO and [des Ala ¹ ] hEPO products means that "processing" of the amino terminus within the host cells serves to remove the final methionine and, in some cases, the initial alanine. The activity determined by radioimmunoassay for isolates is at the level of 150,000-160,000 U / mg; activity detected in vitro is about 30,000-62,000 U / mg; and activity detected in vivo varies from 120 to 720 U / mg. (Compared with the standard human urinary isolate = 70,000 U / mg, in each test). The dose response curve for the recombinant product in the in vivo test is significantly different from the human urinary EPO standard curve.

The EPO analog plasmids formed in Parts A and B of Example 11 are each transformed into pMW1 transformed E. coli AM7 cells, and the cells are cultured as described above. The purified isolates are tested by both RIA and in vitro assays. The values obtained in the RIA and in vitro tests for the expression products of [Asn ² , des-Pro ² according to Ile ⁶ ] hEPO are approximately 11000 U / mg and 6000 U / mg of protein, respectively, whereas the analysis values for [His ⁷ ] hEPOs are about 41,000 U / mg and 14,000 U / mg protein, respectively. This means that analog products are 1 / 4-1 / 10 “active”, as well as the “parent” expression product in trials.

In an expression system intended for use in S. cerevisiae host cells, the pYE / SCEPO plasmid is transformed into two different strains, YSDP4 (genotype α re 4-3 trp1) and RK81 (genotype α α re 4-3 trp1). The transformed YSDP4 hosts are grown in 3D medium (Yeast Genetics Methods, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., p. 62 (1983)) replenished with casamino acids 0.5%, pH 6.5 at 30 ° C. The media collected when the cells grow to 36 O.D. contain EPO products at about 244 U / ml (97 μg / OD L, as detected by RIA). Transformed RK81 cells grown to 6.5 O.D. or 60 O.D., provide the creation of environments with EPO concentrations of about 80-90 U / ml (34 μg / OD l, as detected by the RIA). Preliminary analyzes reveal significant heterogeneity in products obtained through the use of an expression system, which is apparently due to changes in glycosylation of expressed proteins, as well as a relatively high mannose content of the attached carbohydrate.

Plasmids PαC3 and pYE in E. coli HB101 cells were submitted for registration in accordance with the U.S. Patent Office Rules of Procedure of September 27, 1984 with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland, under A.T.S. FROM. 39881 and A.T.S.S. 39882 respectively. Plasmids pCFM526 in AM7, pCFM536 cells in JM103 cells and pMW1 in JM103 cells were similarly registered on November 21, 1984 as A.T.S. 33932, 33934 and 33933, respectively. Strains YSPD4 and RK81 Saccharomyces cerevisiae were registered on November 21, 1984 as A.T.S. 20734 and 20733, respectively.

It should be apparent from the consideration of the above illustrative examples that the present invention in its many aspects offers extremely valuable products and methods.

The proposed polypeptides are noticeably useful materials, whether they are microbially expressed products or synthetic products, the primary, secondary or tertiary conformation of which became known for the first time thanks to the present invention.

As noted above, the recombinantly produced and synthetic products of the invention exhibit, to varying degrees, the biological activity of in vitro EPO isolates from natural sources and are therefore designed to be useful as substitutes for EPO isolates in culture media used for erythropoietin cell growth in culture. Also, since the polypeptide products of the present invention have the activity of in vivo natural EPO isolates, they are noticeably suitable for use in erythropoietin therapy techniques applied to mammals, including humans, to develop any or all of the inherent effects of EPO in vivo, for example, stimulating reticulocyte response , the development of ferrokinetic effects (such as the effects of the circulation of iron in plasma and the effects of the time of transition to the brain substance), red blood cell mass transfer, stimulation of the synthesis of emoglobin C (see Eschbach, et al., above) and, as noted in Example 10, an increase in hematocrit levels in mammals. The group of people treated with the proposed products included patients who usually need a blood transfusion, including victims of injuries, surgical patients, kidney patients, including dialysis patients, and patients with a variety of blood composition that causes disorders such as hemophilia, sickle-shaped cell disease, physiological anemiasis and the like. Minimizing the need for therapy with blood transfusion due to the use of erythropoietin therapy can be expected to lead to a reduced transfer of pathogens of the infectious disease. The proposed products due to their production using recombinant methods, it must be assumed that they do not contain pyrogens, natural inhibitory substances and the like, and therefore undoubtedly should provide increased overall returns in therapeutic methods in relation to natural products. Erythropoietin therapy using the proposed products should also be useful in increasing the oxygen permeability of individuals experiencing hypoxic environmental conditions and, possibly, in providing favorable cardiovascular effects.

Parenteral (e.g., intravenous, intramuscular, subcutaneous or intraperitoneal) routes are the preferred route of administration of the proposed polypeptide products, and the compositions administered should, as usual, contain therapeutically effective amounts of the product in combination with acceptable diluents, carriers and / or stimulants. Preliminary pharmacokinetic studies have indicated a longer in vivo half-life for monkey EPO products when administered intramuscularly, rather than intravenously. Effective dosages are expected to vary significantly depending on the conditions, however, therapeutic doses are now expected to range from 0.1 (~ 7U) to 100 (~ 7000U) μg / kg body weight of the active material. Standard diluents, such as human serum albumin, are contemplated for use in the pharmaceutical compositions of the present invention along with standard carriers such as saline.

Stimulating materials suitable for use in the proposed compositions include compounds indicated independently for erythropoietic stimulating effects, such as testosterones, stimulators of previous cells, growth factor insulin-like, prostaglandins, serotonin, cyclic AMP, prolactin and triidothyronine, as well as commonly used agents in the treatment of aplastic anemia such as methenolen, stanozole and nandrolone [(see, for example, Resegotti, et al., Panminerva Medica, 23, 243-248 (1981); McGonigle, et al., Kidney Int., 25 (2 ), 437-444 (1984); Pavlovic-Kantera, et al., Expt. Hematol., 8 (Supp. 8), 283-291 (1980); and Kurtz, FEES Letters, 14a (1), 105-108 (1982)]. It is also proposed to use substances that increase actions or synergistic erythropoietin or asialo-EPO, such as adrenergic agonists, thyroid hormones, androgens and BPA [see Dunn, "Current Concepts in Erythropoiesis", John Wiley and Sons (Chichester, England, 1983); Weiland, et al., Blut, 44 (3), 173-175 (1982); Kalmanti, Kidney Int., 22, 383-391 (1982); Shahidi, New. Eng. J. Med., 289, 72-80 (1973), Fisher, et al., Steroids, 30 (6), 833-845 (1977); Urabe, et al., J. Exp. Med., 149, 1314-1325 (1979); and Billat, et al., Expt. Hematol., 10 (1), 133-140 (1982)], as well as classes of compounds denoting "hepatic erythropoietic factors" [see Naughton, et al., Acta. Haemat., 69, 171-179 (1983)] and erythrotropins [as described by Congote, et al., In Abstract 364, Proceedings 7 ^th Intrenational Congress of Endocrinology (Quebec City, Quebec, July 1-7, 1984]; Congote, Biochem. Biophys. Res. Comm., 115 (2), 447-483 (1983) and Congote, Anal. Biochem. 140, 428-433 (1984)] and erythrogenins [as described by Rothman, et al., J. Surg. Oncol., 20, 105-108 (1982)]. Preliminary screenings designed to measure the erythropoietic responses of ex-hypoxic polycythemic mice pretreated with either 5-α-dihydrotestosterone or nandrolone, and then the proposed erythropoietin, showed dubious results.

The diagnostic uses of the proposed polypeptides are also extensive and include the use of labeled and unlabeled forms in a variety of immunoassay methods, including RIA, ELISA and the like, as well as in a variety of assays conducted to detect activity in vitro and in vivo. See, for example, Dunn, et al., Expt. Hematol., 11 (7), 590-600 (1983); Gibson, et al., Pathology, 16, 155-156 (1984); Krystal, Expt. Hematol., 11 (7), 649-660 (1983); Saito, et al., Jap. J. Med., 23 (1), 16-21 (1984); Nathan, et al., New. Eng. J. Med., 308 (9), 520-522 (1983); and various reference materials related to the studies mentioned in these works. The proposed polypeptides, including synthetic peptides that contain sequences of residues of the first detected EPO, also have extremely useful pure materials for the production of polyclonal antibodies and "banks" of monoclonal antibodies specific for different continuous and discontinuous EPO epitopes. As one example, a preliminary analysis of the amino acid sequences of Table VI in the sense of water cure according to Norr, et al., P.N.A.S. (U.S.A.), 78, pp. 3824-3828 (1981) and secondary structures according to Chou, et al., Ann. Rev. Biochem., 47, p. 251 (1978) found that synthetic peptides that duplicate continuous sequences of residues contracting positions 41-57, inclusive, 116-118, inclusive, and 144-166, inclusive, are likely to produce an extremely antigenic reaction and to produce useful monoclonal and polyclonal antibodies immunoreactive with both a synthetic peptide and a whole protein. Such materials are expected to be useful in the detection and related purification of EPO and related products.

Three synthetic peptides were illustratively prepared:

Preliminary immunization studies using the above polypeptides have revealed a relatively weak positive response to hEPO 41-57, an undetectable response to hEPO 116-128 and a strong positive reaction to hEPO 144-166, as determined by the ability of rabbit serum antibodies to immunoprecipitate human urine EPO labeled ¹²⁵ I. Preliminary studies of in vivo activity carried out on three peptides did not reveal significant activity, either individually or in combination.

Despite the fact that the deduced sequences of the amino acid residues of mammalian EPO, presented in the illustrative examples, essentially determines the primary structural conformation of the mature EPO, it should be understood that the specific sequence of 165 amino acid residues of the monkey EPO in Table V and 166 human EPO residues in Table VI does not limit the volume of useful polypeptides of the invention. The present invention covers those various freely occurring allelic forms of EPO that have in the past been included in studies of biologically active mammalian polypeptides, such as human γ interferon (Compare, for example, human immune interferon having an arginine residue at position No. 140 in EPO, about which reported in published patent application 0077670, and a species having glutamine at position 140, as reported in Gray, et al., Nature, 295, pp. 503-508 (1982). Both species differ in that they constitute the sequence " matured "γ inte Feron person). The allelic forms of the matured EPO polypeptides may differ from each other and from the sequences shown in Tables V and VI in terms of length and sequence and / or in relation to deletions, substitutions, insertions or additions of amino acids to the sequence along with the consequent variations in glycosylation ability . As noted above, one hypothesized allelic form of human EPO seems to include the methionine residue at position 126. Presumably, naturally occurring allelic forms of cDNA and EPO encoding genomic DNA sequences are likely to also occur, coding for the above types of allelic polypeptides or simply using different codons to denote the same polypeptides as indicated.

In addition to naturally occurring allelic forms of mature EPO, the present invention also includes other “EPO products” such as polypeptide analogs of EPO and fragments of a “mature” EPO. Following the procedures of the above published patent application Alton, et al. (WO / 83/04053), it is easy to calculate and produce genes encoding the microbial expression of polypeptides having a primary conformation that differs from the conformation indicated here for mature EPO in terms of the identity or location of one or more residues (e.g., substitution , final or intermediate additions and deletions). Alternatively, modifications to cDNA and genomic EPO genes can be easily implemented using well-known site-directed mutagenesis techniques and used to generate EPO analogues and derivatives. Such EPO products must have at least one of the biological properties of EPO, but may differ in other properties. As examples, the engineered EPO products of the present invention include those abbreviated by, for example, deletions of [Asn ² , des-Pro ² according to Ile ⁶ ] hEPO, [des-Thr ¹⁶³ no Arg ¹⁶⁶ ] hEPO and "Δ27-55hEPO", the latter has residues encoded by the deleted whole exon; or which are more resistant to hydrolysis (and, therefore, may have more pronounced or longer lasting effects than the naturally occurring EPO); or which have been modified to remove one or more potential glycosylation sites (which may result in high activity for products produced by yeast); or which have one or more cysteine residues removed or replaced by, for example, histidine or serine residues (such as the analogue of [His ⁷ ] hEPO), and are potentially more readily released in active form from microbial systems; or which have one or more phenylalanine substituted tyrosine residues (such as, for example, the analogues of [Phe ¹⁵ ] hEPO, [Phe ⁴⁹ ] hEPO and [Phe ¹⁴⁵ ] hEPO) and can bind more or less readily to EPO receptors on target cells. Polypeptide fragments that duplicate only part of the continuous amino acid sequence or secondary conformations within the mature EPO are also encompassed, and the fragments may have one EPO activity (e.g., receptor binding) and no other (e.g., erythropoietic activity). Particularly important in this regard are those potential fragments of EPO that are explained when considering the sequence of human genomic DNA shown in Table VI, i.e. "fragments" of the complete continuous EPO sequence, which is outlined by intron sequences and which can make up distinct "domains" of biological activity. Notably, the lack of in vivo activity for any one or more of the "EPO products" of the present invention as a whole does not interfere with therapeutic utility (see Weiland, et al., Supra) or utility in other contexts such as EPO trials or EPO antagonism . Erythropoietin antagonists can be very useful in the treatment of polycythemiasis or in cases of overproduction of EPO [see, for example, Adamson, Hosp.Practice, 18 (12), 49-57 (1983) and Hellmann, et al., Clin. Lab. Haemat., 5, 335-342 (1983)].

Another feature of the present invention is that the cloned DNA sequences described herein that encode human and monkey EPO polypeptides are very valuable for the information they offer regarding the amino acid sequence of mammalian erythropoietin, which has still not been available, despite decades of analytical processing of isolates of naturally occurring products. DNA sequences are also very valuable as products suitable for the implementation of large-scale microbial synthesis of erythropoietin using a variety of recombinant techniques. In addition, the proposed DNA sequences are useful in generating new and useful viral and circular plasmid DNA vectors, new and useful transformed and transfected microbial prokaryotic and eukaryotic host cells (including bacterial and yeast cells and mammalian cells grown in culture), as well as new and suitable methods of cultured growth of such microbial host cells capable of providing expression of EPO and EPO products. The DNA sequences of the invention are also very suitable materials for use as labeled probes in the isolation of EPO and its associated protein encoding cDNA and genomic DNA sequences of mammals other than humans and monkeys, specifically illustrated here. The degree of possible use of DNA sequences in various alternative methods of protein synthesis (for example, in insect cells) or in the genetic therapy of humans and other mammals cannot yet be determined. The proposed DNA sequences are expected to be useful in the development of transgenic mammalian species that can serve as eukaryotic "hosts" for the production of large quantities of erythropoietin and erythropoietin products. See mainly Palmiter, et al., Science, 222 (4625), 809-814 (1983).

In the light of the discussion here, a specific description of illustrative examples, of course, does not limit the scope of the present invention, and specialists in this field will be apparent numerous modifications and variants of its implementation. As one example, although the DNA sequences provided by the illustrative examples include cDNA and genomic DNA sequences, this application provides information on the amino acid sequence essential for constructing a DNA sequence, the invention also contemplates such DNA sequences that can be constructed based on knowledge of the amino acid sequences of EPO. They can encode EPO (as in Example 12), as well as fragments of EPO and polypeptide analogs of EPO (ie, “EPO products”), which may possess one or more of the biological properties of naturally occurring EPO, but not others ( or possess others to varying degrees).

The DNA sequences of the present invention, therefore, should encompass all DNA sequences suitable for use in providing expression in a prokaryotic or eukaryotic host cell of a polypeptide product having at least a portion of the primary structural conformation and one or more biological properties erythropoietin, and are selected from: (a) the DNA sequences shown in Tables V and VI; (b) DNA sequences that hybridize to the DNA sequences defined in (a), or fragments thereof; (c) DNA sequences which, due to degeneration of the genetic code, must hybridize to the DNA sequences defined in (a) and (b). In this regard, it is noteworthy, for example, that the existing human and monkey EPO allelic gene sequences and gene sequences of other mammalian species are expected to hybridize to the sequences in Tables V and VI or fragments thereof. In addition, due to degeneration of the genetic code, the SCEPO and ECEPO genes and the obtained or mutagenized cDNA or genomic DNA sequences encoding various fragments of EPO and its analogs must also hybridize to the above DNA sequences. Such hybridizations can be easily carried out under the hybridization conditions described here with respect to the initial isolation of DNA encoding human and monkey EPO, or more stringent conditions, if it is desired to reduce background hybridization.

Similarly, although the above examples illustrate the invention with respect to the microbial expression of EPO products in the context of mammalian cell DNA expression inserted into a hybrid vector of bacterial plasmid and viral genomic species, a wide variety of expression systems are within the scope of the invention. Especially covered expression systems, including vectors of homogeneous species related to the diversity of bacterial, yeast and mammalian cells in culture, as well as expression systems that do not include vectors (such as transfection of cells with calcium phosphate). In this regard, it will become clear that the expression of, for example, monkey DNA in monkey host cells in culture and human host cells in culture does constitute an example of the expression of “exogenous” DNA, since EPO, whose high level expression is being sought, will not be have their sources in the host genome. The expression systems of the present invention, in addition, suggest these actions resulting in the cytoplasmic formation of EPO products and the accumulation of glycosylated and non-glycosylated EPO products in the cytoplasm of the host cell or membranes (for example, accumulation in periplasm bacterial spaces), or in supernatant liquids of culture media, such as explained above, or in rather rare systems, such as P. aeruginosa expression systems (described in Gray, et al., Biotechnology, 2, pp. 161-165 (1984).

The improved hybridization methodologies of the present invention, although they have been illustratively applied above to screening for DNA / DNA hybridization, are equally applicable to screening for RNA / RNA and RNA / DNA. The techniques of mixed probes, as illustrated here, mainly comprise a number of improvements in hybridization processes, allowing faster and more reliable isolation of polynucleotides. These many individual processing enhancements include: improved colony transfer and conservation techniques; the use of nylon-based filters, such as GeneScreen and GeneScreen Plus, allowing re-sensing with the same filters and reuse of the filter, the use of new protease treatment methods [compare, for example, with Taub, et al., Anal. Biochem. 126, pp. 222-230 (1982)]; the use of very low individual concentrations (of the order of 0.025 picomole) of a large number of mixed probes (for example, more than 32); and conducting hybridization and post-hybridization cycles in strict temperature ranges that are close to approaching (i.e., within 4 ° C. and preferably within 2 ° C.) to the lowest calculated dissociation temperature of any of the mixed probes used. These improvements combine to produce results that were not to be expected. This is fully explained by the fact that the techniques of mixed probes, including a 4-fold number of probes, which, as previously reported, have always been successfully used even in cDNA sieves with relatively small numbers of informational DNA species, have been successfully applied to gene isolation of a unique sequence in screening a genomic library of 1,500,000 sterile spots. This was easily accomplished, essentially concurrently with the publication of the opinion of Andersen, et al., Above, that screening methods using mixed probes were "... impractical to isolate mammalian protein genes when the corresponding DNA is not available."

Distinctive features of the invention disclosed in the foregoing description, in the following claims and / or in the accompanying drawings, can separately and in any combination of them become material for carrying out the invention in its various forms.

Claims (22)

1. The selected DNA molecule encoding human erythropoietin, which is characterized by a nucleotide sequence corresponding to the amino acid sequence: Ala Pro Pro Arg Leu Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu Leu Glu Ala Lys Glu Ala Glu Asn Ile Thr Thr Gly Cys Ala Glu His Cys Scr Leu Asn Glu Asn Ile Thr Val Pro Asp Thr Lys Val Asn Phe Tyr Ala Trp Lys Arg Met Glu Val Gly Gln Gln Ala Val Glu Val Trp Gln Gly Leu Ala Leu Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu Leu Val Asn Scr Ser Gln Pro Trp Glu Pro Leu Gln Leu His Val Asp Lys Ala Val Ser Gly Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu Gly Ala Gln Lys Glu Ala Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala Pro Leu Arg Thr Ile Thr Ala Asp Thr Phc Arg Lys Leu Phe Arg Val Tyr Ser Asn Phe Leu Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala Cys Arg Thr Gly Asp Arg. 2. An isolated DNA molecule encoding human erythropoietin, which is characterized by a nucleotide sequence corresponding to the amino acid sequence: Glu Cys Pro Ala Trp Leu Trp Leu Leu Leu Ser Leu Leu Ser Leu Pro Leu Gly Leu Pro Val Leu Gly Ala Pro Pro Arg Leu Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu Leu Glu Ala Lys Glu Ala Glu Asn Ile Thr Thr Gly Cys Ala Glu His Cys Ser Leu Asn Glu Asn Ile Thr Val Pro Asp Thr Lys Val Asn Phe Tyr Ala Trp Lys Arg Met Glu Val Gly Gln Gln Ala Val Glu Val Trp Gln Gly Leu Ala Leu Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu Leu Val Asn Ser Ser Gln Pro Trp Glu Pro Leu Gln Leu His Val Asp Lys Ala Val Ser Gly Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu Gly Ala Gln Lys Glu Ala Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala Pro Leu Arg Thr Ile Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val Tyr Ser Asn Phe Leu Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala Cys Arg Thr Gly Asp Arg. 3. An isolated DNA molecule encoding human erythropoietin, which is characterized by a nucleotide sequence corresponding to the amino acid sequence: Met Gly Val His Glu Cys Pro Ala Trp Leu Trp Leu Leu Leu Ser Leu Leu Ser Leu Pro Leu Gly Leu Pro Val Leu Gly Ala Pro Pro Arg Leu Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu Leu Glu Ala Lys Glu Ala Glu Asn Ile Thr Thr Gly Cys Ala Glu His Cys Ser Leu Asn Glu Asn Ile Thr Val Pro Asp Thr Lys Val Asn Phe Tyr Ala Trp Lys Arg Met Glu Val Gly Gln Gln Ala Val Glu Val Trp Gln Gly Leu Ala Leu Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu Leu Val Asn Ser Ser Gln Pro Trp Glu Pro Leu Gln Leu His Val Asp Lys Ala Val Ser Gly Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu Gly Ala Gln Lys Glu Ala Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala Pro Leu Arg Thr Ile Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val Tyr Ser Asn Phe Leu Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala Cys Arg Thr Gly Asp Arg. 4. A DNA molecule complementary to a DNA sequence characterized in any one of claims 1 to 3. 5. The DNA molecule according to claim 4, characterized in that it is covalently linked to a defined label. 6. The DNA molecule according to claim 5, characterized in that the detected label is a radioactive label. 7. The DNA molecule according to claim 5 or 6, characterized in that it is a single-stranded DNA. 8. A DNA molecule that hybridizes under stringent conditions with a protein-coding region of the DNA sequence described in any one of claims 1 to 3. 9. The DNA molecule of claim 8, characterized in that it is covalently linked to a defined label. 10. The DNA molecule according to claim 9, characterized in that the detectable label is a radioactive label. 11. The DNA molecule according to claim 9 or 10, characterized in that it is a single-stranded DNA. 12. An expressing plasmid or viral DNA vector containing a DNA molecule, characterized in any one of claims 1 to 3. 13. The erythropoietin glycoprotein, which is the product of the expression of the exogenous DNA sequence in a mammalian cell and is characterized by the primary structure of human erythropoietin, having the following amino acid sequence: Ala Pro Pro Arg Leu Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu Leu Glu Ala Lys Glu Ala Glu Asn Ile Thr Thr Gly Cys Ala Glu His Cys Ser Leu Asn Glu Asn Ile Thr Val Pro Asp Thr Lys Val Asn Phe Tyr Ala Trp Lys Arg Met Glu Val Gly Gln Gln Ala Val Glu Val Trp Gln Gly Leu Ala Leu Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu Leu Val Asn Ser Ser Gln Pro Trp Glu Pro Leu Gln Leu His Val Asp Lys Ala Val Ser Gly Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu Gly Ala Gln Lys Glu Ala Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala Pro Leu Arg Thr Ile Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val Tyr Ser Asn Phe Leu Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala Cys Arg Thr Gly Asp Arg, a higher molecular weight as determined by SDS-PAGE than human erythropoietin isolated from urine and the ability to increase the production of reticulocytes and erythrocytes and enhance the synthesis of hemoglobin and iron consumption by bone marrow cells, or part of this glycoprotein, which retains the activity of a full-sized form. 14. The glycoprotein of claim 13, wherein said glycoprotein is a glycoprotein having an average carbohydrate content different from that of human erythropoietin excreted from urine. 15. A glycoprotein according to any one of claims 13-14, wherein said exogenous DNA sequence is contained in an autonomously replicated circular DNA plasmid or viral vector. 16. The glycoprotein according to any one of paragraphs.13-15, characterized in that it is a product of the expression of a DNA molecule, characterized in any one of claims 1 to 3, in a mammalian cell. 17. A method of producing an erythropoietin glycoprotein, comprising culturing in a suitable nutrient medium a mammalian host cell transformed with the expression vector of claim 12, under conditions in which said host cell is capable of expressing said polypeptide. 18. A pharmaceutical composition for treating anemia comprising an erythropoietin glycoprotein and a pharmaceutically acceptable diluent, adjuvant or carrier, characterized in that the erythropoietin glycoprotein contains the glycoprotein according to any one of claims 13-16. 19. The erythropoietin glycoprotein, which is the product of the expression of an exogenous DNA molecule in a mammalian cell, said DNA molecule being characterized by a nucleotide sequence corresponding to the following amino acid sequence: Ala Pro Pro Arg Leu Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu Leu Glu Ala Lys Glu Ala Glu Asn Ile Thr Thr Gly Cys Ala Glu His Cys Ser Leu Asn Glu Asn Ile Thr Val Pro Asp Thr Lys Val Asn Phe Tyr Ala Trp Lys Arg Met Glu Val Gly Gln Gln Ala Val Glu Val Trp Gln Gly Leu Ala Leu Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu Leu Val Asn Ser Ser Gln Pro Trp Glu Pro Leu Gln Leu His Val Asp Lys Ala Val Ser Gly Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu Gly Ala Gln Lys Glu Ala Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala Pro Leu Arg Thr Ile Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val Tyr Ser Asn Phe Leu Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala Cys Arg Thr Gly Asp Arg, and said erythropoietin glycoprotein is characterized by a higher molecular weight as determined by SDS-PAGE than human erythropoietin, and the ability to increase the production of reticulocytes and red blood cells and enhance hemoglobin synthesis and iron consumption by bone marrow cells, or part of the specified glycoprotein, preserving activity of the full-sized form. 20. A pharmaceutical composition for treating anemia comprising the erythropoietin glycoprotein of claim 19 and a pharmaceutically acceptable diluent, adjuvant or carrier. 21. The mammalian cell line COS-1 / pDSVL-gHuEPO is a producer of human erythropoietin. 22. Mammalian cell line CHO / pDSVL-gHuEPO - producer of human erythropoietin.

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