SK443885A3 - Dna sequence, a plasmid, vector, host cell, polypeptide of an erythropoietin-type, method of its production and pharmaceutical composition based thereon - Google Patents

Description

DNA sequence, plasmid, vector, host cell, erythropoietin-type polypeptide, process for its manufacture and pharmaceutical composition based thereon

Area; technique

The invention relates to a DNA sequence, a plasmid, a vector, a host cell, an erythropoietin type polypeptide, a process for its manufacture, and a pharmaceutical composition based thereon. In particular, the invention relates to a DNA sequence for use in securing / expressing a polypeptide product, the structure of which at least in part corresponds to the primary structure of erythropoietin to assure it.

"H ic · L biological properties, either prokaryorphic or eukaryotic". , host cells, prokaryotes and eukaryogenic host cells transformed with the DNA sequence, a plasmid or a vector comprising the DNA sequence, the above-described polypeptide, and a process for its manufacture, and a pharmaceutical composition based thereon. The term DNA sequence is used interchangeably with the terms DNA strand or DNA sequence.

BACKGROUND OF THE INVENTION

A. Handling of genetic materials

Genetic materials can be broadly defined; as those chemical structures that plan and carry out the production of cell and virus components. Long-chain polymeric substances, deoxyribonucleic acids (DNA) carry genetic material of all living cells and viruses, with the exception of some viruses whose genetic material is carried by ribonucleic acid (RNA). The repeating units in the polymeric DNA molecule are four different nucleotides, each consisting of a purine (adenine or guanine) or a pyrimidine (thymine or cytosine) bound to a phosphate-linked desoxyribose. Binding of nucleotides in linear polymeric form is effected by means of ϊ 5'-phosphate nucleotide on the one hand and via a 3'-hydroxyl group on the other. Functional DNA occurs in the form of double stable strands, which arise from single strands of nucleotides (called deoxyoligonucleotides), the strands being linked by a hydrogen bond between the purine and pyrimidine bases (ie, a complementary link between adenine (A) and thymine (T) or guanine ( G) and cytosine (C)). Nucleotides are commonly referred to as individual purine and pyrimidine bases, and the complementary association of nucleotides in double-stranded DNA (i.e., A-T and G-C) is called base pairs. A ribonucleic acid is a polynucleotide that contains adenine, guanine, cytosine, and a urine (U) thymine site bound to a ribose and phosphate moiety.

Very briefly, the DNA scheduling function is usually accomplished by a process that transcribes the nucleotide sequence (gene) into a relatively unstable mRNA. This mRNA serves as a template for the production of structural, control and catalytic proteins from amino acids. The translation process under mRNA efficiency involves the interactions of small RNA strands (tRNAs), which are key to the transfer of individual amino acids along the mRNA strand, so that polypeptides with the correct amino acid sequence can be produced. This DNA-derived mRNA message providing tRNA interaction and orientation of any of the twenty amino acids is the basis for triplet (codon) expression; sequences of three consecutive nucleotides. Protein formation is the ultimate form of expression of a planned genetic message that is hidden in the nucleotide sequence of a particular gene.

Promoters are sequences of DNA that are usually inserted upstream of a gene in a DNA polymer and represent the site at which transcription into mRNA begins. The DNA control sequence, usually also upstream of the gene in a given DNA polymer, includes a protein guideline that determines the frequency or rate of the transcription start.

Thus, a promoter / control sequence or control system alone is a sequence that is inserted upstream of a gene (or genes) in a functional DNA polymer and that determines whether a particular gene will be transcribed and optionally expressed. The DNA sequences that are located downstream of the gene (s) in the polymeric DNA provide a signal to terminate transcription into mRNA, i.e., termination sequences.

The focus of microbiologists in the last decade has been the industrial production of substances important from an industrial or pharmaceutical point of view, using organisms that did not initially have information in their genetic material regarding the desired product or (in the case of mammalian cells in tissue culture) there is no large-scale expression of the chromosomal gene.

In this case, a gene that is specific for the structure of the polypeptide of interest is either isolated from the organism containing the gene, or is produced chemically and then continuously incorporated into another organism, which is normally a normal single-cell multiplying organism, such as bacteria, yeast. or a mammalian cell in tissue culture.

Once this occurs, the normal operation of the existing cell mechanism in this transformed cell after transfection to construct the desired product occurs, whereby exogenous DNA is used as a template for transcription of mRNA followed by translation to produce a continuous sequence of amino acid residues.

A number of publications are known in the art regarding methods using recombinant DNA for the isolation, synthesis, purification and propagation of genetic material for use in transforming a selected host organism. U.S. Pat. No. 4,237,224 (Cohen et al.) Describes, for example, the transformation of a single-cell host by treatment with hybrid viral or circular plasmid DNA which comprises selected sequences of exogenous DNA; In carrying out the method encompassed by said patent, one proceeds by first obtaining a vector for transformation by enzymatic digestion of viral or circular plasmid DNA to form ^s linear DNA strands. Using similar enzymes, selected exogenous or heterologous chains are then prepared which usually contain the coding region for the desired product, also in linear form. The linear viral or plasmid DNA is then incubated with exogenous DNA in the presence of binding enzymes which allow the fragments to be recombined to produce hybrid vectors containing a selected segment of exogenous DNA that is inserted into the viral or circular plasmid DNA.

Transformation of a suitable one-cell host with the hybrid vector thus obtained results in the generation of multiple copies of exogenous DNA in the host cell population. In some cases, the desired result is a simple multiplication of the foreign DNA and the product obtained is thus the DNA itself. More often, however, the goal of the entire transformation is to express host-scale exogenous DNA on a larger scale so that it is possible to obtain isolable amounts of commercially significant fragments of proteins or polypeptides encoded by exogenous DNA. Similar procedures have also been described in other patents, for example U.S. Pat. No. 4,264,731, (Shine), 4,273,875 (Manis), 4,293,652 (Cohen), and in European Patent Publication No. 5,167,144.

619 of November 9, 1983.

Development of specific DNA sequences for incorporation into vectors

The DNA can be performed in a variety of ways, which largely depend on the donor's strangeness with respect to the putative host and the size of the polypeptide molecule to be expressed in the host organism. In a great simplification can be stated the basic three ways, namely

(1) the method of isolating a double-stranded DNA sequence from the donor genome;

2) a method comprising chemically producing a DNA sequence encoding a polypeptide of interest; and

3) synthesis of double-stranded DNA in vitro using an enzyme called so-called reverse transcription of mRNA that has been isolated from donor cells.

Methods that produce DNA that is complementary to the mRNA of interest are commonly referred to as cDNA methods.

The production of a DNA sequence is often a method of choice when the entire sequence of amino acid residues of the desired polypeptide is known. This procedure is described, for example, in U.S. Pat. No. 483,451 (Alton), filed April 15, 1983 and published November 24, 1983 as WO83 / 04053). This publication describes the means of achieving very good results, such as: the possibility of introducing alteration codons, which are commonly found in genes, especially those that are highly expressed in the host organism (for example, preferential codons for the use of certain a host, such as yeast or E. coli), further ensuring the absence of a non-transmitted intron, i. within a deposited sequence that is frequently and commonly present in mammalian genomic DNA and also in the respective mRNA and is not further processed by a prokaryotic host cell, further preventing expression of unwanted polypeptide leader sequences that are commonly present but are not readily cleaved from the resulting polypeptide by cells bacteria or yeast, further ensuring good incorporation of the DNA into a suitable expression vector in conjunction with the desired promoter / regulator system (control sequence), and a sequence for terminating the transfer and finally constructing genes encoding the polypeptide fragments and analogs of the polypeptides of interest.

If the entire sequence of amino acid residues in the desired polypeptide is known, it is not possible to produce the DNA sequence directly and then the method of choice is to isolate the DNA sequence coding for the desired polypeptide by using cDNA despite potential disadvantages. In this way, it is possible to obtain expression vectors ensuring a high level of microbial expression as described above. One standard procedure for isolating the cDNA sequence of the desired end product is to prepare cDNA plasmids obtained by reverse transcription of mRNA, which is found in large quantities in donor cells. For example, a whole group of cDNA samples derived from pituitary cells are expressed relatively when used. significant amounts of growth hormone as the final product. Where substantial portions of the amino acid residue sequence of a polypeptide are known, labeled single-stranded DNA sequences that link to sequences likely to be present in the target cDNA may be used. These sequences are then used for DNA / DNA hybridization procedures performed on cloned copies of cDNA denatured to a single stranded form. These processes have been described, for example, in U.S. Pat.

394,443 (Weisman et al.) And in a number of publications, for example, Wallace et al., Nuc. Acids. Res. 6, p. 3543-3557 (1979); Reyes et al., P.N.A.S. (USA) 79, p. 3270-3274 (1982) and Jaye et al., Nuc. Acids Res. 11, p. 2325-2335 (1983). Also, U.S. Pat. U.S. Patent No. 4,358,535 to Falkow et al. 70685 and 70687 disclose a method for labeling single-stranded polynucleotides. Davis et al. In A Manual for Genetic Engineering, Advanced Bacterial Genetics Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1980) p. 55-58 and 174-176 describe the hybridization of colonies and coatings and in the New England Nuclear brochure

- 7 (Boston, Mass.) For gene-finding membrane materials, instructions for transfer and hybridization of DNA and RNA can be found (Catalog No. NEF-972).

Among the most significant novelty in recombinant cloning hybridization procedures, for example, labeled mixed synthetic oligonucleotide samples, each of which is a potential complete complement to a specific DNA sequence in a hybridization sample, including a heterogeneous mixture of RNA or single stranded DNA, should be mentioned. In particular, these methods are useful in screening for cDNA clones derived from sources in which a very small amount of mRNA is available for the polypeptide of interest. The purpose of the procedure is to select limited hybridization conditions so that, as far as possible, non-specific binding may not occur with subsequent autoradiographic visualization of a specific cDNA clone upon hybridization of the target DNA to this single sample, which is a complete complication of the sequence. These procedures have been described in various publications, for example, Wallace et al., Nuc. Acids Res. 9, p. 879-897 (1981), Suggs et al., P.N.A.S. (USA) 78, p. 6613-6617 (1981); Choo et al., Nature 299, p.

178-180 (1982), Kurachi et al., P.N.A.S. (USA) 79, p.

6461-6464 (1982), Ohkubo et al., P.N.A.S. (USA) 80, p. 2196-2200 (1983) and Kornblihtt et al., P.N.A.S. (USA) 80, p. 3218-3222 (1983). In general, mixed sample procedures as described by Wallace et al. (1981) were further elaborated by various personnel to obtain satisfactory results in isolation of cDNA clones using a mixture of 32 strands of 16 bases in length. with a different base sequence along with a single 11 base chain to carry out a two-way confirmation of the presence of the desired cDNA as described by Singer-Sam and other PNASs (USA) 80, p. 802-806 (1983).

The use of DNA isolated from genomes is a common feature of at least the above three procedures for obtaining DNA sequences specific for use in recombinant procedures. This is particularly true in procedures designed to ensure ¹ microbial expression of mammalian polypeptides where the main problem is the complexity of the mammalian genome DNA. There are relatively suitable procedures for phage-borne mixtures of the DNA genome of human and other mammals, as described, for example, in Lawn et al., Cell 15, p. 1157-1174 (1978), which describes a procedure for obtaining a human genome library called Maniatis Library, Karn et al., PNAS (USA) 77, p. 5172-5166 (1980), the publication relates to a human genomic library based on endonuclease cleavage, and Blattner et al., Science 196, p. 161-169 (1977), which describes the construction of a bovine genome library. On the other hand, very few successful attempts have been made with hybridization procedures to obtain the DNA genome if the amino acid sequence or base sequence of the DNA in question was not well known in advance. An example is Fiddes et al., J. Mol. and Appl. Genetics 1, p. 3-18 (1981), which describes successful isolation of a gene encoding a subunit and a human pituitary glycoprotein (hormone) from the Maniatis Library using a 621 base pair full length sample from a pre-isolated cDNA sequence for the α subunit. Another example is Das et al.

A. S. (USA) 80, p. 1531-1535 (1983), which describes the isolation of human human HLA-DR genome clones using a 175 bp synthetic oligonucleotide. Finally, Anderson et al., P.N.A.S. (USA) 80, p. 6838-6842 (1983) describe the isolation of a clone from the pancreatic bovine trypsin inhibitor (BPTI) genome using a single 86 base pair sample, constructed according to a known amino acid sequence in BPTI. The authors report inadequate methods for isolating mRNA that would be suitable for obtaining a cDNA collection due to apparently too low levels of salivary gland and lung tissue mRNAs that were the source of this acid, and also describe genome tracking options using a mixture of labeled samples, that:

more generally, samples were used that contained a mixture of oligonucleotide sequences to isolate protein genes of unknown sequence from the cDNA mixture. These samples typically take the form of a mixture of 8 to 32 oligonucleotides with a length of 14 to 17 nucleotides, which represent the most diverse possible codon combinations for small sequences (5 to 6 residues) of amino acids. Under limited hybridization conditions that limit the formation of samples with incorrect base pairs, these samples can help to detect a specific sequence of the gene in a clone mixture that is not very complex. However, due to its small length and heterogeneity, the sample does not have the specificity that would be required to detect as complex sequences as those in the mammalian genome. In this way, this procedure is unsuitable for the isolation of mammalian protein genes when the corresponding mRNA cannot be obtained.

Thus, there is a continuing need to devise improved methods for performing rapid and efficient isolation of cDNA clones in cases where insufficient information is available regarding the amino acid sequence of a polypeptide for which the gene is to be encoded and where enriched tissues are not available. as a source of mRNA to construct a cDNA mixture. Such improved methods would be particularly useful if they could be applied to the isolation of genes in the mammalian genome for which only limited information is available regarding the amino acid sequences of the polypeptides encoded by the genes.

B. Erythropoietin as a target polypeptide

Erythropoiesis, ie. the production of red blood cells is carried out continuously throughout the life to replace cells that have been destroyed. It is a very well-controlled physiological process that allows sufficient red blood cells to be available in the blood to properly oxygenate the tissues, but the blood cells must not be enough to limit blood circulation. Red blood cells are formed in the bone marrow and their production is controlled by the hormone erythropoietin.

Erythropoietin is an acidic glycoprotein with a molecular weight of approximately 34,000 units and can occur in three forms: α, β, and asialo. Forms a and β differ somewhat in the carbohydrate component, but have the same potency, biological effect and molecular weight. The asialo form is a or β form from which the terminal carbohydrate group is removed. Erythropoietin is present in plasma at very low concentrations when tissues in good health are sufficiently aerated with the existing amount of erythrocytes. This normal low concentration is sufficient to stimulate red blood cell replacement that is normally subject to physiological breakdown during aging.

The amount of erythropoietin in the circulation increases when there is a lack of oxygen when the oxygen transfer of the red blood cells in the bloodstream is reduced. Hypoxia can be caused by the loss of large amounts of blood by bleeding, destruction of red blood cells' when exposed to radiation, reduced oxygen intake at high altitudes or due to long unconsciousness or various forms of anemia. In response to hypoxic tissue stress, erythropoietin will increase red blood cell production by stimulating the conversion of primitive germ cells into bone marrow into proerythroblasts that gradually mature, synthesize hemoglobin and are released into the bloodstream as red blood cells. If the circulating red blood cell count is greater than necessary for normal tissue oxygen requirements, the circulating erythropoietin level will decrease.

These ratios are described, for example, in Testa et al., Extp. Hematol., B (Supp. 8), 144-152 (1980); Tong et al., J. Biol. Chem. 256 (24), 12666-12672 (1981); Goldabsser, 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 (1983); Emannouel et al., Am. J. Physiol 247 (Ip 2) F 168-76 (1984).

Because erythropoietin is an essential factor in the production of red blood cells, this hormone is potentially useful for both diagnosis and treatment of blood disorders that are characterized by low or defective red blood cell production. This has been described in Pennartur-Das et al., Blood 63 (5), 1168-71 (1984) and Haddy, Am. J. Ped. Hematol./Oncol. 4, 191-196 (1982), publications refer to erythropoietin as a possible drug for a disease in which the red blood cells have a crescent shape and Esbach et al., J. Clin. Invest. 74 (2), p. 434-441 (1984), which describes a treatment method in uremic sheep and the effect of erythropoietin-rich plasma infusions, suggesting doses of 10 erythropoietin (EPO) / kg per day for 15 to 40 days to correct anemia that occurs in chronic kidney failure. A similar problem is also discussed by Krane, Henry Ford Hosp. Med.

J., 31 (3), 177-181 (1983).

Recently, it has been shown that if erythropoietin was available in sufficient quantities, anemia of 1,600,000 patients could only be treated annually in the US, as described in Morrison, Bioprocessing in Space

- 12 - an overview, p. 557-571 in The World Biotech. Report 1984, Volume 2, USA (Online Publications, New York, N.Y., 1984). Recently, studies have been the basis for estimating the efficacy of erythropoietin in various disease states, disorders and haematological irregularities, as described in Vedovato et al., Acta Haematol. 71, 211-213 (1984) (β-tlassemia), Vichinsky et al., J. Pediatr. 105 (1), 15-21, 1984 (cystic fibrosis), Cotes et al., Brit.

J. Obstet. Gyneacol. 90 (4), 304-311 (1983) (abusiveness, menstrual distress), Haga et al., Acta Pediatr. Scand. 72.

827-831, 1983 (early anemia of immaturity), Claus-Walker et al., Ar. Phys. Med. Rehab. 65, 370-374 (1984) (injuries to the palate), Dunn et al., Eur J. Appl. Physiol. 52, 178-182, 1984 (space flights), Miller et al., Brit. J. Haematol. 52, 545-590, 1982 (acute blood loss), Udupa et al., J. Lab. CIIN. Med. 103 (4), 574-580 and 581-588 (1984), and Lipschitz et al., Blood, 63 (3), 502-509, 1983 (aging) and Daniak et al., Cancer 51 (6), 1101-1106 (1983) and Schwartz et al., Otolaryng. 109, 269-272 (1983) (various neoplastic diseases associated with abnormal erythropoiesis).

Previous attempts to recover erythropoietin in good plasma or urine yields have been relatively unsuccessful. A complicated laboratory equipment is needed, and even in this case it is usually possible to obtain only small amounts of impure and unstable extract containing erythropoietin.

U.S. Pat. No. 3,033,753 discloses a method for partially purifying erythropoietin from sheep blood plasma. In this way, a crude solid extract containing erythropoietin can be obtained in a small yield.

Initial attempts to isolate erythropoietin from urine have led to the recovery of unstable, non-biologically inactive formulations of the hormone. U.S. Pat. No. 3,865,801 discloses a method of stabilizing the biological activity of a crude material comprising urinary erythropoietin. The resulting crude product of this process contains 90% erythropoietin activity and is stable

Another method for purifying human erythropoietin from the urine of patients with aplastic anemia is described in Miyake et al., J. Biol. Chem. Vol. 252, no. 15 (August 10, 1977), p. The process is carried out in seven stages, including ion exchange chromatography, ethanol precipitation, gel filtration, adsorption chromatography, and yields pure erythropoietin with an efficiency of 70,400 units / mg protein in 21% yield.

s

U.S. Pat. No. 4,397,840 (Takezawa et al.) Discloses a process for producing an erythropoietin product from the urine of healthy humans using weakly alkaline ion exchangers, and it is reported that the low molecular weight products obtained have no inhibitory effects on erythropoietin.

In British Pat. No. 2,085,887 to Sugimoto et al., Issued May 6, 1982, discloses a method for producing human hybrid lymphoblastoid cells that can produce 3-440 erythropoietin units in a 1 ml cell suspension in culture after multiplication of mammalian host cells per culture. 10 cells / ml. The highest production rate can be expressed as a form of erythropoietin 40-4000 units / 10 ⁶ cells / 48 hours in culture in vitro for transferring the living cells. Reference may also be made in this regard to U.S. Pat. A number of methods have been proposed for isolating erythropoietin from tissues, including the use of tumor cells, but yields were very low, as is evident from, 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).

Another possible method for isolating purified erythropoietin

I are immunological procedures. This is done by injecting human erythropoietin into animals, preferably rats or rabbits, to induce serum antibody formation against human erythropoietin. Human erythropoietin, injected into an injection animal, is recognized as a foreign substance, i. as an antigen for the animal's immune system and thus elicits antibodies against the antigen. Various cells that respond to antigen stimulation produce antibodies that differ slightly from each other. When blood is drawn, the antibody remains in the blood serum. Both unclean serum and purified antibody, such as serum immunoglobulin G fraction, can be used to detect and complex with human erythropoietin, but these materials have the major drawback of being polyclonal antibodies derived from different cells and therefore bind in addition to to erythropoietin to other ingredients of the crude extracts.

Also of interest are current attempts to develop continuous cell cultures capable of producing a homogeneous antibody that reacts specifically with a single antigen. This procedure has been generally described in Chisholm, High Technology Vol.

3, no. 1, p. 57-63 (1983). Attempts have also been made to use cell fusion and hybridization to obtain monoclonal antibodies against erythropoietin and to use these antibodies to isolate and quantitate human erythropoietin. By way of example, the successful development of mouse hybridoma cell lines that secrete monoclonal antibodies against human erythropoietin as described in Lee-Huang, Abstract no. 1463 in Fed. Proc. 41, 520 (1982). Another example may be a detailed description of the preparation and use of a monoclonal antibody against human erythropoietin according to Weiss et al., P.N.A.S. (USA) 79, 5465-5469 (1982) and also in Sasaki, Biomed. Biochim. Acta 42 (11/12), p. 202-206 (1983); Yanagawa et al., Blood, 64 (2), 357-364 (1984); Yanagawa et al., J. Biol. Chem. 259 (5), 2707-2710 (1984) and U.S. Pat. 4,465,624.

Also of interest are all reports regarding the immunological efficacy of synthetic peptides that essentially mimic the amino acid sequence that exists in naturally occurring proteins, glycoproteins and nucleoproteins. It is noted that lower molecular weight proteins are involved in immunological responses that are similar in magnitude and duration to physiologically important proteins, for example, viral antigens, polypeptide-like hormones, and the like. Immunological reactions of these polypeptides also include induction of specific antibody production in animals, as described, for example, in Lerner, et al., Cell, 23, 309-310, 1981, Ross et al., Nature 294, p. 654-656, 1981, Walter et al., PNAS (USA) 77, p. 5197-500, 1980, Lerner et al., PNAS (USA), 78,

3403-3407 (1981), Walter et al., PNAS (USA) 78, 4882-4886, 1981, Wong et al., PNAS (USA), 78, 7412-7416,

1981, Green et al., Cell, 28, 477-487, 1982; Nigg et al., PNAS (USA) 79, 5322-5326 (1982), Baron et al., Cell 28, 395-404 (1982), Dreesman et al. Nature 295: 158-160 (1982); Lerner, Scientific American 248, no. 2. 66-74 (1983). A similar procedure is also described in Kaiser et al., Science 223, p. 249-255 (1984), in particular the biological and immunological efficacy of synthetic peptides having approximately the same secondary structures with peptide hormones but need not have the same primary structure. However, the above studies mostly relate to amino acid sequences of proteins other than erythropoietin, for which no relevant information regarding the amino acid sequence of this substance has been published yet. U.S. Pat. No. 463,724, filed Feb. 4, 1983 (J. Egrie), published May 22, 1983;

August 1984 as European application no. 0 116 446 describes a mouse hybridoma cell line (ATCC No. HB 8209) that produces a highly specific type of antibody that is also specifically reactive to a polypeptide consisting of 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.

This sequence corresponds to that envisaged for the first twenty amino acids of full human erythropoietin, isolated by the method of Miyake et al., J.

Biol. Chem. 252, 5558-5564 (1977), wherein amino acid sequence analysis was performed in gas phase (Applied Biosystems Inc.) according to the method of Hewick M. et al., J. Biol. Chem. 256, 7990-7997, (1981). A similar procedure is also described in Sue et al., Proc. Natl. Acad. Sci. USA 80, p. 3651-3655 (1983), the publication of which relates to the generation of polyclonal antibodies against a synthetic sequence of 26 amino acids, and Sytowski et al., J. Immunol. Methods 69, p. 181-186 (1984).

The polyclonal and monoclonal antibodies described above are highly useful materials for use in immunology for the detection and quantitative determination of erythropoietin and may also be useful for affinity purification of erythropoietin, but it is unlikely that these materials can be used to isolate large amounts of erythropoietin from mammals in amounts , sufficient for further analysis, clinical trials and therapeutic use of the compound, for example, in the case of chronic kidney diseases in which the tissue is not sufficient to maintain sufficient erythrocyte production. Consequently, it is believed that the best prospects for fully characterizing mammalian erythropoietin and for obtaining large amounts of this substance for potential diagnostic and clinical uses have been successfully applied by recombinant techniques to ensure microbial synthesis of said material in large quantities.

Great efforts have been made to isolate, a DNA sequence that encodes human erythropoietin and the like in other mammals, but has so far been unsuccessful. This failure is likely to be due to the very small amount of tissue available as a source of this substance, especially in humans, especially mRNA-enriched materials that could allow the construction of a series of cDNAs, from which the sequence could then be isolated , which is the code for erythropoietin. In addition, the continuous sequence of amino acids in erythropoietin is so little known that it is not possible, for example, to construct longer samples of polynucleotides that could further be used in DNA / DNA hybridization using cDNA and in particular to obtain a genome assembly. For example, the aforementioned sequence of twenty amino acids used to obtain the monoclonal antibody produced by ATCC no. HB 8209 does not permit the construction of a 60 base oligonucleotide sample as described in Anderson et al., Supra. It is likely that the human erythropoietin gene may occur as a single copy of the gene in the human genome, and in any case the human material that encodes the human erythropoietin accounts for less than 0.00005% of the total DNA of the human genome.

Even the best results so far achieved using recombinant methods to obtain DNA sequences in which expression in a microorganism could be obtained with isolable amounts of mammalian erythropoietin were far from being successful. For example, Farber et al., Exp. Hematol. 11, Suppl. 14, Abstract 101 (1983) describes the extraction of mRNA from baboon kidney tissue after phenylhydrazine treatment followed by injection of mRNA into Xenopus laevis oocytes, providing in vitro transient formation of a mixture of translation products having, inter alia, some erythropoietin-like activity. In Farber et al., Blood 62, no. 5, Supp. no. 1, Abstract 392 at p. 122a (1983) describes the translation of mRNA from the human kidney into frog oocytes. The resulting translation product mixture contained 220 m units of translation product with erythropoietin activity per mg mRNA. This level of translation of the exogenous mRNA code for erythropoietin in vitro is very low (even compared to the previously reported baboon mRNA translation results), but the results seem to confirm that the human kidney is a site where erythropoietin is expressed, so it should be possible obtain a cDNA assembly enriched for the gene of interest from the kidney (Farber, Clin. Res. 31 (4), 769A (1983)).

Pending the filing of U.S. Pat. Only one report on cloning and expression of human erythropoietin cDNA in E. coli was known from 561,024 and 582,185. A plurality of cDNA clones were incorporated into E. coli plasmids and the β-lactamase fusion products were immunoreactive with a monoclonal antibody to a non-specific epitope of human erythropoietin, as described by Lee-Huang, Proc. Natl. Acad. Sci. USA 81, p. 2708-2712 (1984).

SUMMARY OF THE INVENTION

The following aspects are within the scope of this invention:

A DNA sequence for use in providing expression of a polypeptide product whose structure at least partially corresponds to the primary structure of erythropoietin to provide its biological properties consisting in increasing reticulocyte and red blood cell production in bone marrow cells and enhancing hemoglobin synthesis or iron uptake in prokaryotic or eukaryotic cells, wherein the DNA sequence is selected from the group consisting of (a) the DNA sequences set forth in Tables V and VI and their complementary strands;

(b) DNA sequences hybridizing under stringent conditions to the DNA sequences defined in (a) or fragments thereof; and (c) DNA sequences that would hybridize to the DNA sequences defined in (a) or (b) if there were no degeneracy of the genetic code.

2. The DNA sequence of aspect 1 encoding human erythropoietin.

3. The DNA sequence of aspect 1 encoding monkey erythropoietin.

4. The DNA sequence of aspect 3 comprising the protein coding region set forth in Table V.

5. The DNA sequence of aspect 1 or 2, which is a genomic DNA sequence.

6. The DNA sequence of aspect 5 encoding human erythropoietin.

7. The DNA sequence of aspect 6 comprising the protein coding region set forth in Table VI.

8. The DNA sequence of aspect 1 or 2, which is covalently linked to a detectable marker.

9. The DNA sequence of aspect 8, wherein the detectable label is radioactive.

10. The DNA sequence of aspect 8 or 9, which is single stranded.

11. The DNA sequence of aspect 1 encoding [Phe ¹⁵ ] hEPO, [Phe ⁴⁹ ] hEP0, [Phe ¹⁴⁵ ] hEP0, [His ⁷ ] hEP0, [Asn ² -des-Pro ² to Ile ⁶ ] hEPO, [des-Thr ¹⁶³ to Arg ¹⁶⁶ ] hEP0 or [delta 27-55] hEPO.

12. A prokaryotic or eukaryotic host cell transformed or transfected with the DNA sequence of any one of aspects 1, 2, 3, 6, 7, and 8 in a manner allowing said host cell to express said polypeptide product.

13. The prokaryotic or eukaryotic host cell of aspect 13, which is capable of glycosylating said polypeptide.

14. The prokaryotic or eukaryotic host cell of aspect 13, which is a mammalian cell.

15. The prokaryotic or eukaryotic host cell of aspect 13, which is a COS cell.

16. The prokaryotic or eukaryotic host cell of aspect 13, which is a CHO cell.

17. A biologically functional circular plasmid or viral DNA vector comprising the DNA sequence of any one of aspects 1;

2, 3, 5, 6, 7 or 11.

18. A prokaryotic or eukaryotic host cell stably transformed or transfected with the DNA vector of aspect 17.

19. A recombinant polypeptide having all or part of the primary structural conformation of human or simian erythropoietin as shown in Table VI or V, or any portion thereof, or any derivative thereof, or a derivative thereof exhibiting biological activity by increasing reticulocyte and red blood cell production in bone cells. and increase hemoglobin synthesis or iron uptake. This polypeptide is the product of eukaryotic expression of an exogenous DNA sequence and has a molecular weight by SDS-PAGE higher than erythropoietin isolated from urine.

20. The polypeptide of aspect 19, which is a glycoprotein whose average carbohydrate composition differs from human erythropoietin isolated from urine.

21. The polypeptide of aspect 19 or 20, wherein the exogenous DNA sequence is a cDNA sequence.

22. The polypeptide of aspect 19 or 20, wherein the exogenous DNA sequence is a genomic DNA sequence.

I

23. The polypeptide of aspect 19 or 20, wherein the exogenous DNA sequence is part of an autonomously replicating circular DNA plasmid or viral vector.

24. The polypeptide of any one of aspects 19 to 23, which is covalently attached to a detectable label.

25. The polypeptide of aspect 24, wherein the detectable label is radioactive.

26. A polypeptide product obtainable by expressing the DNA sequence of any one of aspects 1, 2, 3, 5, 6, and 7 in a eukaryotic host cell.

27. A method of producing a polypeptide having at least a portion of the primary structural conformation of erythropoietin to enable biological efficacy by increasing the production of re22 ticulocytes and red blood cells in bone marrow cells and increasing the synthesis of hemoglobin or iron uptake by procaryotic or eukaryotic host cell. transformed or transfected with the DNA sequence of any one of aspects 1, 2, 3, 5, 6 and 7 in a manner allowing the host cell to express the polypeptide is cultured under appropriate nutrient conditions and optionally the desired polypeptide product of expression of the DNA sequence is isolated.

28. The method of aspect 27, wherein the host cell of any one of aspects 12 to 16 is cultured.

I

29. The method of aspect 27 or 28 for producing a polypeptide according to any of aspects 19 to 23 and 26, wherein the process is conducted under conditions resulting in said polypeptides.

I

30. A pharmaceutical composition comprising a polypeptide obtainable by the method of aspect 27, 28 or 29 and a pharmaceutically acceptable diluent, excipient or carrier.

31. The pharmaceutical composition of aspect 30, comprising the polypeptide of any of aspects 19 to 23 and 26.

Vertebrates (e.g., COS-1 and CHO) have provided the first ever cell that can be propagated continuously in vitro and which, when grown in culture, are capable of producing into an environment in which they grow more than 100 units (preferably more than 500 units, in particular 1000 to 5000 units) of erythropoietin per 10 ⁶ cells over 48 hours as determined by radioimmunoassay.

It is also possible with the method according to the invention to obtain synthetic polypeptides which, wholly or partially, by virtue of their structure, lead to a continuous sequence of amino acid residues of erythropoietin, which were thus illuminated for the first time. These sequences, which may have common primary, secondary or tertiary structural properties with naturally occurring erythropoietin, may also have biological activity and / or immunological properties common to the naturally occurring product, so that they may be used as biologically or immunologically effective substitutes for erythropoietin for medical and immunological purposes. Correspondingly, it is also possible to obtain monoclonal and polyclonal antibodies which are immunoreactive to the aforementioned polypeptides and preferably also immunoreactive to naturally occurring erythropoietin.

s

Examples of substances obtained by the method of the invention may be cloned DNA sequences of human or monkey origin and polypeptide sequences obtained using these materials, which are the first confirmation of the structure of erythropoietin in monkeys and humans.

It is also possible with the method of the invention to provide novel biologically functional viral and circular vectors for plasmid DNA into which the DNA sequences of the invention and microbial (e.g. bacterial, yeast and mammalian) cells as host organisms stably transformed or transfected using the above can be incorporated. of the vectors.

In this way, it is possible according to the invention to obtain valuable polypeptides by culturing in a culture a transformed microbial host under conditions which allow or facilitate expression of exogenous, vector-incorporated DNA in large quantities, followed by isolation of the desired polypeptides from growth media, cell lysate or cell fractions membrane.

Isolation and purification of polypeptides obtained by microbial expression can be accomplished by conventional means, including, for example, preparative separation chromatography and immunological separation using monoclonal and / or polyclonal antibodies.

After illuminating the erythropoietin amino acid sequence, it is also possible to construct a complete and / or partial DNA sequence coding for erythropoietin, including the beneficial properties of this sequence, such as the incorporation of codons that are advantageous to achieve expression using a non-mammalian host, restriction endonuclease digestion and addition of initial terminal or restricted DNA sequences that may facilitate construction or facilitate expression of the vector. By the methods described in the Examples, it is possible to obtain (and further develop by specific mutagenesis of cDNA and DNA genome) DNA sequences coding for microbial expression of polypeptide analogs or erythropoietin derivatives that differ from the naturally occurring form by the identity or location of one or more amino acid residues, i.e., analogs resulting from the deletion of less than all erythropoietin-specific residues (EPOs) and / or from substitution in which one or more residues are replaced by other residues and / or by an addition in which one or more amino acids are added. a plurality of amino acid residues to the terminal or media portion of the polypeptide, all of which forms may have part or all of the properties of naturally occurring forms.

Polypeptides resulting from the above process may be labeled by binding a detectable material to them by covalent bonding. For example, it may be radiolabelled with the ¹²⁵ I isotope to provide a reagent that can be used to detect and quantify erythropoietin in solid tissues as well as in fluid samples such as urine or blood. The DNA of the invention may also be radiolabeled or biotin-labeled and then used in hybridization to determine the position of the erythropoietin gene and / or the position of any related gene in humans, monkeys and other mammals in their chromosomal map. In this way, it is also possible to demonstrate DNA erythropoietin gene disorders or related gene disorders.

As will be described in more detail below, the invention means substantially improving the detection of a specific single-chain polynucleotide of unknown sequence in a heterogeneous cellular or viral sample, including multiple single-chain polynucleotides by

(a) prepare a mixture of labeled single-stranded polynucleotide samples with uniformly varying base sequences, each of which is potentially specifically complementary to the sequence that occurs in the sample and belongs to the polynucleotide to be detected;

(b) the sample is fixed to a solid substrate;

c) the substrate with the fixed sample is treated so as to reduce the additional possibility of binding to the polynucleotides, except for hybridization to the polynucleotide of said sample,

(d) contacting the treated substrate with the fixed sample temporarily with a mixture of labeled samples under conditions which facilitate hybridization only between completely complementary polynucleotides; and

(e) the specific polynucleotide is detected by detecting the presence of a hybridization reaction of the polynucleotide with a complete complementary sample from the labeled sample, as evidenced by the presence of greater density of labeled material on the substrate at the specific polynucleotide samples per substrate.

This method is particularly effective in situations where 64, 128, 256, 512, 1024 or more polynucleotide samples of 17 to 20 bases in length need to be used for DNA / DNA hybridization,

RNA / RNA or DNA / RNA.

As mentioned above, this procedure allowed the identification of DNA clones coding for monkey erythropoietin from a sequence of sequences prepared from mRNA of kidney anemic monkey cells. In this case, 128 uniform variable samples of 20 nucleotides in length were used according to the information and amino acid sequence of the fractions of human erythropoietin fractions in colony hybridization, thus demonstrating seven positive monkey cDNA clones out of a total of 200,000 colonies. Furthermore, it was possible to rapidly isolate three positive clones from 1,500,000 phage coatings of the human genome. Again, a mixture of 128 samples of 20 base polynucleotides was used along with a second set of 128 nucleotides of 17 bases according to amino acid analysis in another continuous sequence of human erythropoietin.

The above procedures involve the first use of mixed oligonucleotide samples for DNA / DNA hybridization to isolate mammalian genome clones, wherein a mixture of more than 32 oligonucleotide samples for the first time is used to isolate cDNA clones.

Further advantages of the process of the invention will be apparent from the following detailed description of preferred embodiments of the process.

By the method of the invention, it is possible to isolate and characterize DNA sequences which encode a portion or a complete polypeptide sequence of human and monkey erythropoietin. Furthermore, it was possible to express human and monkey DNA in eukaryotic and prokaryotic cells to obtain isolable amounts of polypeptides with the biological (e.g., immunological) efficacy of naturally occurring EPO both in vivo and in vitro.

The monkey DNA was isolated from a cDNA group that was generated by mRNA from kidney tissue of a chemically induced monkey, the serum of the monkey contained a high level of

EPO compared to normal monkey serum. Isolation of the desired cDNA clones containing the DNA encoding erythropoietin was performed using colony DNA / DNA hybridization using a mixture of 128 radiolabeled oligonucleotides with 20 bases with a rapid evaluation of 200,000 colonies formed. The construction of the oligonucleotide samples was determined according to the amino acid sequence of enzymatic fragmentation and the sequence determination in a small portion of human EPO.

Human DNA was isolated from the human genome. Isolation of EPO-encoded DNA clones was performed by hybridization of coatings using the above mixture of 128 base 20 oligonucleotides and a second mixture of 128 radiolabeled 17 base oligonucleotides according to the amino acid sequence from another enzymatically obtained fragment of human EPO .

i I

Positive clones and coatings were verified by detecting the DNA sequence of the clone using a 16-sequence subassembly for a 20 base sample mix, and selected clones were subjected to a nucleotide sequence resulting in the deduction of the primary structure of EPO-type polypeptides for which this DNA code was coded. The polypeptide sequence thus demonstrated was highly homologous to a partial amino acid sequence as determined by amino acid analysis of human EPO fragments.

The selected monkey cDNA positive clone and the selected human cDNA positive clone were incorporated into a DNA transfer vector that was amplified in E. coli and then used to transfect mammalian cells in culture. These cells were then cultured, and up to 3000 EPO units per ml could be detected in the culture supernatant.

The invention will be illustrated by the following examples, and the specific examples are specifically directed to procedures that have been performed prior to the identification of cDNA clones that encode EPO monkeys and human genome clones, procedures that have been identified and identified, and the development of an expression system and to immunologically verify EPO expression in these systems.

PirrL.lú.cíy us'k'U / oČhej'i / G. tynokiH

Example 1 is aimed at determining the amino acid sequence in human EPO fragments and constructing a mixture of radiolabeled samples based on the sequence determination results.

Example 2 is directed to procedures leading to the identification of positive monkey cDNA clones and provides information for animal treatment and preliminary radioimmunoassay (RIA) of animal sera.

Example 3 is directed to the preparation of a cDNA mix and assembly, to colonization, search for positive colonies, and to verify positive clones, to detect DNA sequence in a positive cDNA clone, and to confirm the primary structure of an EPO monkey (amino acid sequence determination).

Example 4 is directed to procedures involving the identification of positive clones of the human genome and provides information about the genomic library source, hybridization of coatings, and verification of positive clones.

Example 5 is aimed at detecting the DNA sequence in a positive clone of the genome and obtaining information about the amino acid sequence in human EPO, including its comparison with the monkey EPO sequence.

Example 6 shows the procedures for constructing a vector incorporating DNA encoding EPO derived from a positive cDNA monkey clone, using the vector to transfect COS-1 cells, and culturing the cells after transfection.

Example 7 is directed to the construction of a vector incorporating DNA encoding EPO derived from a positive clone of the human genome, using the vector to transfect COS-1 cells and culturing these cells after transfection.

Example 8 relates to immunological procedures in which the supernatant from the transfected cell cultures according to Examples 6 and 7 was used.

Example 9 is aimed at monitoring the biological activity in vivo and in vitro for EPO, obtained by microbial expression in Examples 6 and 7.

Example 10 relates to the development of mammalian expression systems for EPO monkey cDNA and human genome DNA, including the use of Chinese Hamster Ovary (CHO cells) as well as immunological and biological monitoring of the efficacy of the expression products and the properties of these products.

Example 11 is directed to the production of genes that encode human EPO and analogs thereof, which genes may contain a number of preferential codons that allow or facilitate expression in E. coli or yeast. The example also relates to appropriate systems for the expression of these genes.

Example 12 refers to the immunological and biological activity of the products obtained by the expression method of Example 11.

The invention will be illustrated by the following examples.

Example 1

A. Determination of amino acid sequence in human erythroJ fragment of poetin,

Human EPO was isolated from urine and digested with trypsin to produce and isolate 17 separated fragments in amounts of about 100 to 150 pmoles.

These fragments were bit-numbered and then analyzed for amino acid sequence by sequence microanalysis using a gas-phase apparatus (Biosystems Applied) to obtain the information shown in Table I. In this table, the individual letters are used as codes and X is a residue that has not been clearly identified.

Table I

Sample no. Sequence analysis result T4A A-P-P-R T4b G-K-L-K T9 A-L-A-G-Q-K T13 W-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-C-A-R T27 T-I-T-A-T-D-F-R T28 E-A-I-S-P-P-D-A-A-A-M-A-P-L-R T30 E-A-E-X-I-T-T-G-X-A-H-E-X-S-L-N-E-X-I-T-V-L T31 W-Y-S-C-F-L-R T33 S-L-T-T-L-L-R T35 The F-N-Y-A-N-C T38 G-Q-L-L-W-X-S-S-Q-P-W-E-L-L-Q-L-H-W-D-K

B. Composition and construction of a mixture of oligonucleotide samples

The amino acid sequences shown in Table I were used with respect to the possible degeneracy of the genetic code to determine whether a mixed DNA / DNA hybridization procedure could be used for cDNA and / or DNA from a genome library. This analysis revealed that in fragment no. T35 was a sequence of seven amino acid residues (Val-Asn-PheTyr-Ala-Trp-Lys) for which one of the 128 possible 20 bp DNA sequences could be coded. Therefore, a first summary of 128 sequences with 20 base pairs was constructed using the standard phosphoamidite method of Beaucage et al., Tetrahedron Letters 22, p. 1859-1862 (1981) on a solid support according to the following Table II.

Table II

The rest - Val - Asn - Phe - Tyr - Ala  Trp - Lys 3 ’ CAA TTG AAG ATG CGA AAC TT -5 T A A A T

G G

C C

Further analysis showed that in fragment no. T38 exists a sequence of six amino acid residues (Gln-ProTrp-Glu-Pro-Leu), based on which sequence it was possible to prepare a mixture of 128 oligonucleotides with 17 base pairs according to the following Table III.

Table III

The rest - Gin - Pro - Trp - Glu - Pro - Leu

3 'GTT GGA ACC CTT GGA GA -5'

Oligonucleotide samples were labeled at the 5'-end with ³² P-ATP, 7500 to 8000 Ci / mol (ICN) using T ₄ polynucleotide kinase (NEN).

Example 2

A. Monkey pretreatment and RIA

Macac fascicularis monkeys weighing 2.5 to 3 kg aged 1.5 to 2 years were treated with a pH 7 solution containing 12.5 mg / kg of phenylhydrazine hydrochloride on days 1, 3, and 5 subcutaneously. hematocrit was controlled. On day 7 or at any time when the hematocrit value dropped to 25% of the original value, blood serum and kidney were isolated after administration of 25 mg / kg ketamine hydrochloride. The material was immediately frozen in liquid nitrogen and stored at -70'C.

B. RIA for EPO

Radioimmunoassays for the quantitative determination of erythropoietin in samples were performed as follows:

The erythropoietin standard or unknown sample was incubated with the antiserum for two hours at 37 ° C. After two hours of incubation, the tubes with ice samples were cooled, erythropoietin was added, labeled, and the tubes were incubated at 0 ° C for at least 15 hours. Each tube contained 500 μΐ incubation mixture consisting of 50 μΐ of diluted immune serum, 10,000 cpm -erytropoietínu ¹² ^ 5 μΐ trasylol and 0-250 μΐ EPO standard or unknown sample, the volume of the residue was added with PBS containing 0.1% BSA. The antiserum used was from a second experimental blood sample of rabbits immunized with 1% pure preparation of erythropoietin from human urine. The final dilution of the antiserum was adjusted during the experiment so that the amount of ¹²⁵ I-EPO bound to the antibody was at most 10-20% of the possible input pulses, which is generally the final dilution of the antiserum in the range of 1: 50,000 to 1: 100,000.

The ¹²⁵ I-erythropoietin-bound antibody was precipitated by the addition of 150 μΐ Staph A. Pp for 40 minutes incubation, the samples were centrifuged and the pellets were washed twice with 0.75 ml of 10 mM TrisHCl pH 8.2 containing 0.15 M sodium chloride, 2. mM EDTA and 0.05% Triton X-100. The number of pulses was determined in the washed pellets to determine the percentage of bound labeled erythropoietin. Pulses obtained for serum prior to immunization were subtracted from the values obtained to exclude the effect of non-specific precipitation. The amount of erythropoietin in the unknown sample was calculated by comparison with a standard curve.

The above procedure was applied to the serum of monkeys obtained in Part A and to the serum of healthy monkeys. Normal serum contained approximately 36 mJ / ml, while serum from monkeys pretreated contained between 1000 and 1700 mJ / ml.

Example 3

A. Construction of monkey cDNA library

MRNA was isolated from the kidneys of normal and anemic monkeys using guanidinium thiocyanate according to Chirgwin et al., Biochemistry 18, p. 5294 (1979) and poly (A) ⁺ mRNA was purified by double-pass the oligo (dT) cellulose packed column according to the method of Maniatis et al., Molecular Cloning, and Laboratory Manual (Cold Springs Harbor Laboratory, Cold Springs Harbor, NY 1982). p. The cDNA library was constructed according to a modification of the general procedure described by Okayama et al., Mol. and Cell. Biol. 2, p. 161-170 (1982). The key steps of the preferred process are as follows:

1) pUC8 digested with PstI followed by oligo dT with a length of 60 to 80 bases is used as the sole vector,

2) the enzyme Hinc II is used to remove oligo dT from one end of the vector,

3) first chain synthesis and oligo dT binding were performed ¹ according to said publication,

4) BamHI digestion was used to remove oligo dG from one end of vector a

5) Replacement of the RNA strand with the DNA strand was performed in the presence of two helper binding chains, GATCTAAGACCGTCCCCCCCCC and ACGGTCTTTA, in triple molar excess, based on the oligo dG-terminated vector.

B. Colonization Hybridization for Sequence Sequence in Monkey ‘'i'

The transformed E. coli microorganism was plated at a density of 9000 colonies on 10 x 10 cm culture plates containing 50 micrograms of ampicillin. GeneScreen filters (New England Nuclear Catalog No. Nef-972) were pre-wetted on BHI-CAM plates (Bacto brain heart infusion 37 g / l, 2 g / l casamino acid and 15 g / l agar containing 500 micrograms of chloramphenicol) and then used on removing colonies from the plate.

Colonies were grown in the same medium for 12 hours or longer to obtain multiple copies of the plasmid. Thereafter, the colonies (colonies upwards) were treated by sequentially depositing the filters on two layers of Whatman 3 MM paper, saturated with the following solutions:

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

2) 0.5 M NaOH for ten minutes and then

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

The filters were then air dried under vacuum at 80 ° C for two hours.

Furthermore, the filters were digested with proteinase K with a solution containing 50 micrograms / ml of this enzyme in K buffer [0.1 M Tris-HCl pH 8.0, 0.15 M NaCl, 10 mM EDTA pH 8.2 and 0.2% SDS]. 5 ml of the solution are added to each filter and the cleavage is carried out at 55 ° C for 30 minutes, then the solution is removed.

4 ml of prehybridization buffer (5x SSPE, 0.5% SDS, 100 micrograms / liter SS E. coli DNA and 5x BFP) are then added to the filters. The buffer is allowed to act; at 55 ° C, typically 4 hours or longer and then discarded.

The hybridization is then performed as follows:

Add 3 ml of hybridization buffer (5x SSPE, 0.5% SDS, 100 micrograms / liter of yeast tRNA) containing 0.025 picomoles of each of the 128 samples listed in Table II (referred to as EPV mixture) to each filter, and then the filters are kept at 48 ° C for 20 hours. This temperature is 2 ° C lower than the lowest calculated dissociation temperature (Td) for each of the samples.

After hybridization, the filters were washed three times on a shaker for 10 minutes each using 6x SSC and 0.1% SDS at room temperature, then washed two to three more times 6x SSC with 1%.

SDS at 48 ° C hybridization temperature.

It was shown by autoradiography of the filters that out of the original 200,000 colonies, seven were positive.

The sequence of one of the monkey cDNA clones (designated as clone 83) was then analyzed by modification of the procedure of Wallace et al., Gene 16, p. 21-26 (1981). The procedure was carried out by plasmid DNA of monkey clone 83 cDNA linearized with EcoRI and then denatured by heating in a boiling bath. The sequence of desoxynucleotides was determined by the method of Sanger et al., P.N.A.S. (United States) 74, Str. 5463-5467 (1977). A subset of the EPV mixture consisting of 16 sequences was used as a primer for this reaction. and

J ''

C. Determination of monkey cDNA sequence for EPO

Nucleotide analysis of clone 83 was performed according to Messing, Methods in Enzymology 101, p. 20-78 (1983). In Table IV, restriction enzyme analysis of approximately 1600 base pairs of the fragment of clone 83, digested with EcoRI / Hind III, is summarized. The approximate placement of the restriction endonuclease sites was determined by determining the number of bases from the 3 'end to the EcoRI site near the 5' end of the fragment. Nucleotide sequence analysis was performed by determining the sequence of individual fragments after treatment with individual restriction enzymes so that fragments that overlap could also be detected. Thus, for example, an overlap was detected in the nucleotide sequence analysis for the restriction fragment designated C113 (Sau3A at about 11 / SmaX at about 324) and the reverse sequence for the fragment designated C73 (Alu1 at about 424 / BstEII at about 203).

Table IV shows the approximate site of action of each restriction endonuclease.

Table IV

Restriction enzyme Approximate location

Place of action

EcoRI 1 Sau3A 111 Smal 180 BstEII 203 Smal 324 KpnI 371 RsaI 372 alul 424 Pst 426 alul 430 HpaI 466 alul 546 Pst 601 PvuII 604 alul 605 alul 782 alul 788 RsaI 792 Pst 807 alul 841 alul 927 ncol 946 Sau3A 1014

alul 1072 alul 1115 alul 1223 Pst 1301 RsaI 1343 alul 1384 Hind III 1449 alul 1450 Hind III 1585

The determination of the 1342 base pair sequence (in the region from the Sau3A site near the 3'-end to the EcoRI and HindIII sites) and the subtraction direction analysis allowed to obtain information about the amino acid sequence and the corresponding DNA as shown in Table V In this table, the initial amino acid of full EPO (as verified by correlation with the above 20 amino acid terminal sequence) is indicated by +1. The presence of a methionine-specific ATG codon (designated by -27) upstream of the terminal alanine residue as the first residue for full EPO suggests that EPO expression occurs first in the cytoplasm as a precursor containing a 27 amino acid leader region, which cleaves before EPO enters the bloodstream. Potential glycosylation sites are indicated by asterisks in the polypeptide. The determined molecular weight of the transferred region was determined to be 21 117 units and the molecular weight of 165 polypeptide residues constituting the complete EPO monkey was determined to be 18 236 units.

Table V

Translation of monkey cDNA for EPO

Sau3A

GATCCCGCGCCCCCTGGACAGCCGCCCTCTCCTCCAGGCCCGTGGGGCTGGCCCTGCCC

CGCTGAACTTCCCGGGATGAGGACTCCCGGTGTGGTCACCGCGCGCCTAGGTCGCTGAG

-27 -20

Met GGACCCCGGCCAGGCGCGGAGATG Gly Val His GGG GTG CAC Glu GAA Cys For TGT CCT Ala GCC Trp TGG Leu Trp Leu Leu Leu Ser -10 Leu Val Ser Leu Pro Leu Gly Leu Pro CTG TGG CCT CTG TCT CTC GTG TCG CTC CCT CTG GGC CTC CCA -1 * 1 Gly Ala Pro Pro Arg Leu White Cys Asp Ser 10 Arg Val Leu GTC CCG CGC CTC ATC TGT GAC AGC CGA GTC CTG 1 Glu Arg Tyr Leu Leu Glu 20 Ala Lys Glu Ala * Glu Asn wall Thr Met GAG AGG TAC CTC TTG GAG GCC AAG GAG GCC GAG AAT GTC ACG ATG 30 Gly Cys ^- Glu Ser Cys Ser Leu Asn Glu ♦ Asn ile 40 Thr Val Pro GCG TGT TCC GAA AGC TGC AGC TTG AAT GAG AAT ATC ACC GTC CCA Asp Lys Val Asn Phe 50 Tyr Ala Trp Lys Arg Met Glu Val Gly GAC ACC AAA GTT TAT GCC TGG AAG AGG ATG GAG GTC GGG 60 Gin Gin Ala Val Gin Gly Leu Ala Leu 70 Leu Ser Glu CAG CAG GCT GAA GTC TGG CAG GGC CTG GCC CTG CTC TCA GAA Ala Val Leu 80 Ala Val Leu Ala * Asn Ser Ser Gin Pro GCT GTC GGC CAG GCC GTG TTG GCC AAC TCT TCC CAG CCT 90 Phe Glu For Leu Gin Leu His Met Asp Lys Ala ile 100 Ser Gly Leu TTC GAG CCC CTG CAC ATG GAT AAA GCC ATC AGT GGC CTT Arg Ser ile Thr Thr Leu 110 Leu Arg Ala Leu Gly Ala gin Glu Ala CGC AGC ATC ACC ACT CTT CGG GCG CTG GGA GCC CAG GAA GCC 120 Ser Leu Pro Asp Ala Ala Ser Ala Pro Leu 130 Arg Thr ile ATC TCC CTC GCA GAT GCG GCC TCG GCT GCT CCA CTC CGA ACC ATC Thr Ala Asp Thr Phe Cys 140 Lys Leu Phe Arg Val Tyr Ser Asn Phe ACT GCT ACT TTC TGC AAA CTC TTC CGA GTC TAC TCC AAT TTC

Table V - continued

150 Gly GGG 160 Glu Ala Cys Arg Arg Leu Arg Gly Lys Leu Lys Leu Tyr CTC CGG GGA AAG CTG Thr ACG GAG GCC TGC AGG AGA 165 Gly Asp Arg OP GGG GAC AGA TGA CCAGGTGCGTCCAGCTGGGCACATCCACCACCTCCCTCACCAACA

CTGCCTGTGCCACACCCTCCCTCACCACTCCCGAACCCCATCGAGGGGCTCTCAGCTAAG

CGCCAGCCTGTCCCATGGACACTCCAGTGCCAGCAATGACATCTCAGGGGCCAGAGGAAC

TGTCCAGAGCACAACTCTGAGATCTAAGGATGTCGCAGGGCCAACTTGAGGGCCCAGAGC

AGGAAGCATTCÁGAGAGCAGCTTTAAACTCAGGAGCAGAGACAATGCAGGGAAAACACCT

GAGCTCACTCGGCCACCTGCAAAATTTGATGCAGGACACGCTTTGGAGGCAATTTACCTG

TTTTTGCACCTACCATCAGGGACAGGATGACTGGAGAACTTAGGTGGCAAGCTGTGACTT

CTCAAGGCCTCACGGGCACTCCCTTGGTGGCAAGAGCCCCCTTGACACTGAGAGAATATT

TTGCÄATCTGCAGCAGGAAAAATTACGGACAGGTTTTGGAGGTTGGAGGGTACTTGACAG

GTGTGTGGGGAAGCAGGGCGGTAGGGGTGGAGCTGGGATGCGAGTGAGAACCGTGAAGAC

AGGATGGGGGCTGGCCTCTGGTTCTCGTGGGGTCCAAGCTT

Hind III

The polypeptide sequence set forth in Table V can readily be assayed for the presence of highly hydrophilic regions and / or for secondary structural properties, which are also, for example, potential highly immunogenic regions, for example by the method of Hopp et al., PNAS (USA) 78, p. 3824-3828 (1981) and Kyte et al., J. Mol. Biol. 157, p. 105-132 (1982) and / or Chou et al., Biochem. 13, p. 222-245 (1974) and Advances in Enzymology 47, p. 45-47 (1978). Computer-aided analysis can be performed using PEP Reference Section 6.7, Intelligenetics Inc., 124 University Avenue, Palo Alto, California, USA (method by Hoppe et al.).

Example 4

A. Human genome assembly

The human fetal liver genome assembly, carried by the phage Ch4A, was obtained as described by Lawn et al., Cell 18, p. 533-543 (1979), the assembly was then maintained for use in coating hybridization.

B. Hybridization of Coatings as a Method for Sequencing Sequences in the Human Genome Assembly

The phage particles were disrupted and the DNA was fixed to the filters in an amount of 50,000 filter coatings as described in Woo, Methods in Enzymology 68, p. 389-395 (1979) except that GeneScreen Plus filters (New England Nuclear Catalog No. NEF-976) and NZYAM plates (NaCl 5 g, MgCl ₂ .6 H ₂ O 2 g, NZ-Amin A 10) were used. g, yeast extract 5 g, casamino acid 2 g, maltose 2 g and agar 15 g per liter).

The air-dried filters were heated at 80 ° C for 1 hour and then digested with proteinase K according to Example 3, Part B. Preliminary hybridization was performed using 1 M NaCl, 1% SDS buffer at 55 ° C for 4 hours. or slightly longer, and then the buffer was removed. Hybridization and post-hybridization treatment was carried out in a similar manner to Example 3, Part B. Both a mixture of 128 samples with 20 base pairs, designated EPV, and a mixture of 128 samples with 17 base pairs from Table III, referred to as EPQ, were used. Hybridization was carried out using an EPV mixture at 48 ° C and an EPQ mixture at 46 ° C; 4 degrees below the lowest calculated Td for the components of the mixture. Removal of the hybridized sample for rehybridization was performed by boiling with 1x SSC, 0.1% SDS for two minutes. Filter autoradiography showed the presence of three positive clones (which reacted with both samples) from the original 1 500 000 phage group. It was then verified that these positive clones code for EPO by determining the DNA sequence and in an electron microscope where the monkey cDNA structure of Example 3 could be demonstrated. This procedure also demonstrated the presence of introns in the DNA genome sequence.

Example 5

Nucleotide sequence analysis was performed on one of the positive clones (designated lambdahE1). The results are shown in Table VI below.

Table VI aagcttctgggcttccagacccagctactttgcggaactcagcaacccaggcatctctgagtctccgccca

AGACCGGGATGCCCCCCAGGGGAGGTGTCCGGGAGCCCAGCCTTTCCCAGATAGCACGCTCCGCCAGTCCC

AAGGGTGCGCAACCGGCTGCACTCCCCTCCCGCGACCCAGGGCCCGGGAGCAGCCCCCATGACCCACACGC

ACGTCTGCAGCAGCCCCGCTCACGCCCCGGCGAGCCTCAACCCAGGCGTCCTGCCCCTGCTCTGACCCCGG

GTGGCCCCTACCCCTGGCGACCCCTCACGCACAC AGCCTCTCCCCCACCCCC ACCCGCGCACGCACACATG

CAGATAACAGCCCCGACCCCCGGCCAGAGCCGXAGAGTCCCTGGGCCACCCCGGCCGCTCGCCTGCCGCTG

CGCCGCACCGCGCTGTCCTCCCGGAGCCGGACCGGGGCCACCGCGCCCXGCTCTGCTCCGACACCGCGCCC

CTTGGACAGCCGCCCTCTCCTCTAGGCCCGTGGGGCTGGCCCTGCACCGCCGAGCTTCCCGGGATGAGGXX

-27 -24

Met Gly Val His

CCCGGTGACCGGCGCGCCCCAAGTCGCTGAGGGACCCCGGCCAAGCGCGGAG ATG GGG GTG CAC G

GTGAGTACTCGCGGGCTGGGCGCTCCCGGCGGCCGGGTTCCTGTTTGAGCGGGGATTTAGCGCCCCGGCT

Table VI - continued

ATTGGCCAAGAGGTGGCTGGGTTCAAGGACCGGCGACTTGTCAAGGACCCCGGAAGGGGGAGGGGGGTGGG gcagcctccacgtgccgcggggacttgggggagttcttggggatggcaaaaacctggcctgttgaggggca

CAGTTTGGGGTTGGGGAGGAGGTTTGGGGTTCTGCTGTGCAGTTGTGTCGTTGTCAGTGTCTCGÍI.S.T

TTGCACACGCACAGATCAATAAGCCAGAGGCAGCACCTGAGTGCTTGCATGGTTGGGACAGGAAGGACGAG

CTGGGGCAGAGACGTGGGGATGAAGGAAGCTGTCCTTCCACAGCCACCCTTCTCCCCCCCCGCCTGACTCT

CAGCCTGGCTATCTGTTCTAG -23 Glu Cys Pro -20 Ala GCC Trp Leu Trp Leu Leu Leu Ser Leu AA TGT CCT TGG CTG TGG CTT CTC CTG TCC CTG -10 -1 +1 Leu Ser Leu For Leu Gly Leu for wall Leu Gly Ala for for Arg Leu Ile Cys CTG TCG CTC CTC CCA GTC CTG GGC GCC CCA CCA CGC CTC ATC TGT 10 ’ 20 « Asp Ser Arg Val Leu Glu Arg Tyr Leu Leu Glu Ala Lys Glu Ala Glu same time Ile GAC AGC CGA GTC AGG TAC CTC TTG GAG GCC AAG GAG GCC GAG AAT ATC

Thr

ACG GTGAGACCCCTTCCCCAGCACATTCCACAGAACTCACGCTCAGGGCTTCAGGGAACTCCTCCCAGAT

CCAGGAACCTGGCACTTGGTTTGGGGTGGAGTTGGGAAGCTAGACACTGCCCCCCTACATAAGAATAAGTC

TGGTGGCCCCAAACCATACCTGAAACTAGGCAAGGAGC'AAAGCCAGCAGATCCTACGCCTGTGGGCCAGGG 27 30

CCAGAGCCTTCAGGGACCCTTGACTCCCCGGGCTGTGTGCATTTCAG Thr ACG Gly GGC Cys TGT Ala GCT Glu GAA * 40 His Cys Ser Leu Asn Glu Asn White Thr Val Pro Asp Thr Lys wall same time Phe Tyr CAC TGC AGC TTG AAT GAG ACC AAA GTT AAT TTC TAT 50 55 Ala Trp Lys Arg Het Glu GCC TGG AAG AGG ATG GAG GTGAGTTCCTTTTTTTTTTTTTTTCCTTTCTTTTGGAGAATCTCATT

TGCGAGCCTGATTTTGGATGAAAGGGAGAATGATCGGGGGAAAGGTAAAATGGAGCAGCAGAGATGAGGCT

GCCTGGGCGCAGAGGCTCACGTCTATAATCCCAGGCTGAGATGGCCGAGATGGGAGAATTGCTTGAGCCCT

GGAGTTTCAGACCAACCTAGGCAGCATAGTGAGATCCCCCATCTCTACAAACATTTAAAAAAATTAGTCAG

GTGAAGTGGTGCATGGTGGTAGTCCCAGATATTTGGAAGGCTGAGGCGGGAGGATCGCTTGAGCCCAGGAA

TTTGAGGCTGCAGTGAGCTGTGATCACACCACTGCACTCCAGCCTCAGTGACAGAGTGAGGCCCTGTCTCA

Table VI - continued

AAAAAGAAAAGAAAAAAGAAAAATAATGAGGGCTGTATGGAÁTACATTCATTATTCATTCACTCACTCACT

ČACTCATTCATTCATTCATTCATTCAACAAGTCTTATTGCATACCTTCTGTTTGCTCAGCTTGGTGCTTGG

GGCTGCTGAGGGGCAGGAGGGAGAGGGTGACATGGGTCAGCTCGACTCCCAGAGTCCACTCCCTGTAG

56 wall Gly gin gin 60 Ala wall Glu wall Trp gin Gly Leu Ala Leu 70 Leu Ser Glu Ala GTC GGG CAG CAG GCC GTA GAA GTC TGG CAG GGC CTG GCC CTG CTG TCG GAA GCT wall Leu Arg Gly gin Ala 80 Leu Leu wall * same time Ser Ser gin for Trp Glu 90 for Leu GTC CTG CGG GGC CAG GCC CTG TTG GTC AAC TCT TCC CAG CCG TGG GAG CCC CTG gin Leu His wall Asp Lys Ala wall 100 Ser Gly Leu Arg Ser Leu Thr Thr Leu Leu CAG CTG CAT GTG GAT AAA GCC GTC AGT GGC CTT CGC AGC CTC ACC ACT CTG CTT

110 115

Arg Ala Leu Gly Ala Gin

CGG GCT CTG GGA CCC GTGAGTAGGAGCGGACACTTCTGCTTGCCCTTTCTGTAAGAAGGGGA

GAAGGGTCTTGCTAAGGAGTACAGGAACTGTCCGTATTCCTTCCCTTTCTGTGGCACTGCAGCGACCTCCT

GTTTTCTCCT.TGGCAG 116 Lys AAG Glu Ala 120 Ser Pro Pro Asp Ala Ala Ser Ala Ala GCT GAA GCC ATC TCC CCT CCA GAT GCG GCC TCA GCT 130 140 For Leu Arg Thr White Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg wall Tyr Ser CCA CTC AGA ATC ACT GCT GAC ACT TTC CGC AAA CTC TTC CGA GTC TAC TCC 150 160 Asn Phe Leu Arg Lys Leu Lys Leu Tyr Thr Gly Glu Ala Cys Arg Thr Gly AAT TTC CTC CGG GGA AAG CTG AAG CTG TAC ACA GGG GAG GCC TGC AGG ACA GGG

166

Asp Arg OP

GAC AGA TGA CCAGGTGTGTCCACCTGGGCATATCCACCACCTCCCTCACCAACATTGCTTGTGCCACA

CCCTCCCCCGCCACTCCTGAACCCCGTCGAGGGGCTCTCAGCTCAGCGCCAGCCTGTCCCATGGACACTCC

AGTGCCAGCAATGACATCTCAGGGGCCAGAGGAACTGTCCAGAGAGCAACTCTGAGATCTAAGGATGTCAC

AGGGCCAACTTGAAGGGCCCAGAGCAGGAAGCATTCAGAGAGCAGCTTTAAACTCAGGGACAGAGCCATGC

TGGGAAGACGCCTGAGCTCACTCGGCACCCTGCAAAATTTGATGCCAGGACACGCTTTGGAGGCGATTTAC

CTGTTTTCGCACCTACCATCAGGGACAGGATGACCTGGAGAACTTAGGTGGCAAGCTGTGACTTCTCCAGG

TCTCACGGGCATGGGCACTCCCTTGGTGGCAAGAGCCCCCTTGACACCGGGGTGGTGGGAACCATGAAGAC

AXGATXGGGGCTGGCCTCTGGCTCTCATGGGGTCCAAGTTTTGTGTATTCTCAACCTATTGACAGACTGAA

ACACAATATGAC

In Table VI, the initial continuous DNA sequence is the 620 bases upper sequence, which is apparently a non-transferred sequence, immediately preceding the transferred portion of the human EPO gene. The sequence appears to contain the 5'-end of the gene and then the transferred DNA region, which encodes the first four amino acids (-27 to -24) of the leader or preliminary sequence. The four base pairs in sequence before the leader sequence code were not unambiguously determined and are therefore designated X. This is followed by an intron of 639 base pairs (439 base pairs from this sequence have been shown, the remaining 200 are labeled IS) and then a glutamine codon that was designated at position -23 in the transferred polypeptide. The exon sequence that immediately follows is the code for amino acid residues from the alanine residue (designated as +1 residue of the full length EPO amino acid sequence) to the threonine codon at position +26, followed by a second intron consisting of 256 bases as indicated. This intron is followed by an exon for amino acid residues at positions 27-55 and then by a third intron with 612 base pairs. The following exon codes for residues 56-115 of human EPO, followed by a fourth intron of 134 bases as indicated. It is followed by an exon that encodes residues 116-166 and a stop codon (TGA). Table VI shows the sequence of 568 base pairs, which appears to contain the untranslated 3'-region of the human EPO gene and the two base pairs in this gene (X) have not yet been fully identified.

Thus, Table VI serves to identify the primary structure (amino acid sequence) of full human EPO, including 166 fully specified amino acid residues (MW 18 399). As shown in the table, the DNA sequence is a code for a 27-residue signal sequence along with 5'- and 3'-DNA sequences that may be significant for the promoter / operator function of the human gene. The sites for potential glycosylation of the full length human EPO polypeptide are indicated by asterisks in the table.

It should be noted that the specific amino acid sequence shown in Table VI is likely to be the sequence of the naturally occurring allele form of human erythropoietin. Support for this claim can be found. in the results of continuous studies of urine-isolated human erythropoietin sequences that have shown that a significant amount of erythropoietin molecules contain methionine at position 126, unlike the serine shown in the table.

Table VII shows the extent of sequence homology between human and monkey erythropoietin. In the upper continuous line of the table, individual letters are used to indicate the sequence of the human polypeptide after translation, beginning at position -27, the lower continuous lines indicating the sequence for monkey erythropoietin, starting at the position indicated by position -27 again. Asterisks are used to illuminate the homology of individual sequences. It should be noted that sequences of human and simian EPO show an additional lysine (K) residue at position 116 of human erythropoietin. By comparison with Table VI, it is evident that this residue lies at the junction boundary in the genome sequence (mRNA). The presence of the lysine residue in the human polypeptide sequence was further verified by detecting the human cDNA sequence in the clone obtained by mRNA isolated from COS-1 cells transformed with the human genome DNA as set forth in Example 7, which will be presented below.

Table VII

Comparison of human and monkey EPO

IN , ,. -20 -10 +1 10 20 30 40

Inhuman MGVHECPAWLHLLLSLLSLPLGLPVLGAPPRLlCOSRVLERYLLEAKEAENITTGCAEHCSLNENirVPDTK '«*« «» · «*« «·» «« · ·

Monkey MGVHECPAWLWLLLSLVSLPLGLPVPGAPPRLICOSRVLERYLLEAKEAENVTMGCSESCSLNENITVPOTK _v . 50 60 70 80 90 100 110

I | dsky VNPYAWKRMEVGQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLOLHVDKAVSGLRSLTTLLRALGAqKE «* ·« «* · * ·· ** ·« «« «· ·« «« ««

Monkey VNFYAHKRMEVGqQAVEVWQGLALLSEAVLRGQAVLANSSQPFEPLQLHMOKAISGLRSITTLLRALGAQ-E í

V, 120 130 140 150 160

IÄdSfcy AISPPDAASAAPLRTITAOTFRKLFRVYSNFLRGKLKLYTGEACRTGOR and «« ee «« eoee «« e «e« «ee« ««

Monkey aislpoaasaaplrtitaotfcklfrvysnflrgklklytgeacrrgor

Example 6

The expression system chosen for the initial attempts to perform microbial synthesis of isolatable amounts of erythropoietin coded for by the corresponding cDNA and shown in Example 3 was a system in which mammalian cells (i.e., COS-1 cells, ATCC No. CRL-1650) These cells were transfected with a vector that allowed both autonomous replication in E. coli as a host (due to the presence of pBR322-derived plasmid) and replication in mammalian cells (due to the presence of virus-derived DNA) SV40).

In more detail, the expression vector was constructed as follows. The plasmid clone 83 in Example 3 was subjected to amplification in E. coli with approximately 1.4 kilobase DNA encoding monkey erythropoietin and isolated by digestion with EcoRI and HindIII. Separately, a fragment of approximately 4.0 kilobases was isolated from pBR322 using the HindIII and SalI enzymes. A binding fragment of approximately 30 base pairs was obtained from M13mp10 RF DNA (P and L Laboratories) by EcoRI and SalI. This fragment contained sequentially Ecolable digestion sites capable of re-binding, followed by SstI, SmaI, BamHI and XbaI digestion sites, and SalI digested sites. All three of the above fragments were ligated, yielding an intermediate plasmid (pERS) of approximately 5.4 kilobases, in which erythropoietin DNA was flanked by a group of sites for the action of various restriction enzymes. Plasmid pERS was then digested with HindIII and SalI to obtain the DNA for EPO and the EcoRI-SalI binding chain (M13mp10). The 1.4 base pair fragment was ligated to the 4.0 kilobase plasmid pBR322 fragment after digestion with BamHI and SalI, and to another binding strand obtained by cleavage of M13mp10 RF enzymes with HindIII and BamHI of approximately 30 base pairs. This second binding chain of M13 had a HindIII digestion site at one end, followed by a PstI, SalI, XbaI restriction site, followed by a BamHI digestion site. The binding product was again a useful plasmid (pBR-EPO) containing DNA for erythropoietin, which was flanked on both sides by chains with a variety of restriction enzyme sites.

The vector that was used to express erythropoietin DNA in COS-1 cells (pDSVL1) was constructed to allow selection and autonomous replication in E. coli. These properties of the vector are assured by the origin of replication and the DNA sequence coding for ampicillin resistance that is deposited between nucleotides 2448 and 4362 in the sequence of plasmid pBR322. This sequence was structurally modified by the addition of a helper binding chain to provide a site for the action of the HindIII restriction enzyme immediately adjacent to nucleotide 2448 prior to incorporation into the vector. Among the useful properties of the vector selected, the ability to replicate autonomously in COS-1 cells and the presence of a viral promoter sequence functional in mammalian cells should be mentioned. These properties are assured by the DNA sequence for the origin of replication and the sequence of the viral promoter, which is 342 base pairs in length and extends from position 5171 of the sequence to position 270 of the SV40 genome. The vector has a single restriction enzyme site (BamHI) and a commercially available binding sequence (Collaborative Research) is coupled in close proximity to the viral promoter. Also included in the vector is a 237 base pair sequence (nucleotide position 2553 to 2770 of SV40) containing viral polyadenylation signal mRNA (commonly referred to as a transcription terminator). This fragment was inserted into the vector in the correct orientation relative to the viral promoter using a single restriction site. of the BamHI enzyme. Also present in the vector is another mammalian gene at a position that is not critical to the potential transcription of a gene incorporated at a single BamHI site between the viral promoter and the termination sequence (this gene was a gene of approximately 2500 base pairs for mouse dihydrofolate reductase (DHRF) isolated from plasmid pMG-1 as described by Gasser et al., PNAS (USA)

79, p. 6522-6526 (1982)). The main operative components of plasmid pDSVL1 are at nucleotides 2448 to 4362 of plasmid pBR322 together with nucleotides 5171 to 270 (342 base pairs) and 2553 to 2770 (237 base pairs) of SV40 DNA.

The DNA coding for erythropoietin from plasmid pBR-EPO as a fragment after digestion with BamHI was isolated according to the procedures described, for example, in Maniatis et al., And then inserted into plasmid pDSVL1 after digestion with BamHI. Restriction enzyme analysis was used to confirm the incorporation of the erythropoietin gene in the correct orientation in two of the resulting cloned vectors (vectors H and L). These facts are also evident from FIG. 2, where plasmid pDSVL-MkE is shown. Vectors with EPO genes in the wrong orientation were retained as negative controls for transfection experiments to determine the level of expression of EPO in a host transformed with vectors containing erythropoietin DNA in the correct orientation.

Example 7

A. Initial EPO Expression System, COS-1 Cells

The system that was chosen for initial experiments with microbial synthesis of isolable amounts of human erythropoietin for which the DNA of the human genome is encoded also included expression in mammalian cells as host cells (i.e., COS-1 cells, ATCC No. CRL-1650). The human EPO gene was first subjected to initial cloning in a vector capable of autonomous replication in both E. coli hosts (both due to the presence of DNA from plasmid pBR322) and the mammalian cell line COS-1 (due to the presence of SV40-derived DNA) ). This erythropoietin gene containing vector was then introduced into COS-1 cells. The EPO-type polypeptide material was then produced and secreted by the cells after transfection into the environment in which the cells were grown.

Thus, the expression vector was constructed as follows: DNA isolated from the lambda clone A. hEl containing the human genome EPO gene was digested with the restriction enzymes BamHI and HindIII, and a 5.6 kb fragment known to contain a fragment was isolated. the complete erythropoietin gene. This fragment was ligated to the bacterial plasmid pUC8 (Betesda Research Laboratories Inc.), which was digested in a similar manner to give an intermediate plasmid (pUC8-HuE, which thus became a suitable source of said restriction fragment.):

The vector that was chosen for the expression of EPO DNA in COS-1 cells (pSV4SEt) was constructed previously. It is a plasmid that contains a DNA sequence allowing selection and autonomous replication in E. coli cells. These properties are provided by the DNA sequences for the origin of replication and for ampicillin resistance present in the region that extends from the nucleotide at position 2448 to the nucleotide at position 4362 of the bacterial plasmid pBR322, which sequence was then structurally modified by adding a linker with a site The HindIII restriction enzyme immediately near position 2448. Plasmid pSV4SEt is also capable of autonomous replication in COS-1 cells. This property is provided by a 342 base pair fragment containing an origin of replication from the SV40 virus (nucleotides at positions 5171 to 270). This fragment was then modified by adding the EcoRI restriction site in the immediate vicinity of nucleotide 270 and the SalI binding site in the immediate vicinity of nucleotide 5171. Also included in this vector is a 1061 base pair fragment from SV40 ( nucleotides 1711 to 2772 with a binding chain having a SalI site in the immediate vicinity of nucleotide 2772). In this fragment there is a single BamHI restriction enzyme site. Overall, the plasmid pSV4SEt contains a single BamHI and HindIII site and thus allows the insertion of the human erythropoietin gene, sequences allowing replication and selection in E. coli, and sequences allowing replication in COS-1 cells.

In order to incorporate the human erythropoietin gene into plasmid pSV4SEt, the plasmid pUC8-HuE was digested with BamHI and HindIII and the 5.6 kb DNA fragment for human erythropoietin was isolated. Plasmid pSV4SEt was also digested with BamHI and HindIII, and a 2513 base pair main fragment containing all the necessary functions was isolated. These fragments were mixed and bound together to give the resulting vector pSVgHuEPO as shown in FIG.

3. This vector was propagated in E. coli and the DNA of the vector was isolated. Restriction enzyme analysis was then used to confirm the incorporation of the EPO gene.

Plasmid pSVgHuEPO was used to express human EPO in COS-1 cells. The DNA of this plasmid was combined with carrier DNA and transfected in triplicate on 60 mm COS-1 cells each time. Carrier DNA was also used as a control and transfected into COS-1 cells. The culture medium was harvested after five and seven days and examined for the presence of polypeptides with properties of naturally occurring human EPO, particularly for immunological properties.

B. Second EPO Expression System Using COS-1 Cells

Yet another system for the improved production of human erythropoietin has been constructed for which it encodes DNA for the EPO of the human genome in COS-1 cells (ATCC No. CRL-1650).

In the previous system, EPO expression was achieved in COS-1 cells using its own promoter, located in the restriction fragment region after treatment with the BamHI and HindIII enzymes of 5.6 kbases. In the following construction, the EPO gene has been altered so that it is expressed using the SV40 virus promoter.

The cloned 5.6 base human EPO restriction fragment of BamHI to HindIII was digested as follows. The pUC8-HuE plasmid described above was digested with BamHI and BstEII. The restriction enzyme BstEII cleaves the 5.6 kb EPO fragment at a position that is 44 base pairs 5 'to the start of the ATG code for the preliminary peptide and approximately 680 base pairs 3' to the restriction enzyme HindIII cleavage site. A fragment of approximately 4900 base pairs was isolated.

A binding fragment with ends capable of binding at SalI and BstEII cleavage sites with a BamHI cleavage site within the fragment was also synthesized and purified. Both fragments were mixed and ligated to the plasmid pBR322, which had been digested with the restriction enzymes SalI and BamHI to form the intermediate plasmid pBRgHE. The human genome EPO gene was then isolated as a 4900 base pair fragment after BamHI digestion, containing the complete structural gene with a single ATG at 44 base pairs 3 'of the BamHI site near the end of the amino-terminus coding region.

This fragment was isolated and incorporated as a fragment capable of binding after BamHI digestion into the plasmid pDSVL1 described in Example 6 as an expression vector digested with BamHI. The resulting plasmid pSVLgHuEPO, which is shown in FIG. 4 was used to express EPO-type polypeptide material in COS-1 cells according to Examples 6 and 7A.

Example 8

The culture medium in which COS-1 cells (six cultures after transfection according to Example 6) were grown was analyzed by radioimmunology according to Example 2, part. B. Individual samples contained 250, 125, 50 and 25 μΐ of the medium. Culture supernatants containing vectors with incorrect orientation of the EPO gene were clearly negative. For samples of two supernatants from cultures of COS-1 cells transfected with vectors (H and L) containing DNA for EPO in the correct orientation, the percent inhibition of ^antibody -bound ^XZ3 I-EPO ranged from 72 to 88%, thus all values were at the top of the standard curve. The exact concentration of EPO in the culture could not be easily determined. From a sample containing 250 μΐ of supernatant, it was estimated that the EPO content was at least 300 units / ml.

The supernatant obtained from the culture of Example 6 and the five and seven day old culture of Example 7A were examined by RIA to compare the efficacy of recombinant monkey and human EPO and naturally occurring EPO as a standard, the results are shown in graphical form in FIG. As expected, recombinant simian EPO was competed against the anti-human EPO antibody, although inhibition was incomplete under the experimental conditions. However, the maximum percent inhibition values for recombinant human EPO were close to those for standard human EPO. Because dose and response curves are parallel, it is possible to judge the immunological identity of sequences (epitopes). The original results for monkey EPO were re-evaluated and values ranging from 2.91 to 3.12 units / ml were obtained. The observed amounts of human EPO were in the sample after a five-day cultivation of 392 m units / ml and 567 m units / ml in a sample that was grown for 7 days. The determined amounts of monkey EPO according to Example 7B were of the same order or slightly higher.

Example 9

The culture medium from the cultures prepared according to Examples 6 and 7 was subjected to in vitro assays for EPO efficacy as described by Goldwasser et al., Endocrinology 97. 2, p. 315-323 (1975). The values determined for monkey EPO in the culture medium of the cultures studied ranged from 3.2 to 2.0

4.3 units / ml. The culture medium of cultures containing human EPO was also effective in this study and, in addition, the efficacy of these environments could be neutralized by an anti-EPO antibody. The culture medium, from recombinant monkey EPO cultures according to Example 6, was examined for in vivo biological activity as described in Cotes et al.

191. p. 1065-1067 (1961) and Hammond et al., Ann. N.Y. Acad, Sci. 149, p. 516-527 (1968) and found to be 0.94-1.24 units / ml.

Example 10

In the previous examples, recombinant human alephyo monkey EPO-type material was obtained using vectors ¹ used to transfect COS-1 cells. These vectors replicate in COS-1 cells due to the presence of the SV40 T antigen in the cell and the SV40 origin of replication in the vector. Although usable amounts of EPO from COS-1 cells can be obtained using these vectors, this expression is only transient (7 to 14 days) because the vector is lost over time. In addition, only a small percentage of COS-1 is \

- 56 transfection capable of producing the relevant material. An ovarian cell expression system [CHO] will now be described

DHFR ^- Chinese Hamster and Selectable Labeling, DHFR.

A description of related expression systems is given in U.S. Pat. And U.S. Pat. 117 057, 117 059 and 117 060, published August 29, 1984.

CHO DHFR cells [cells (DuX-B11) CHO K1] have been described by Urlaub et al., Proc. Nat. Acad. Sci. USA, Vol.

77, 4461 (1980), these cells lack the enzyme dihydrofolate reductase (DHFR) due to structural gene mutations and therefore require the presence of glycine, hypoxanthine and thymidine in the culture medium. Plasmids pDSVL-MkE (Example 6) or pDSVL-gHuEPO (Example 7B) were transfected together with carrier DNA into CHO DHFR 'cells grown in hypoxanthine, thymidine and glycine containing media on 60 mm plates. Plasmid pSVgHuEPO (Example 7A) was mixed with plasmid pMG2 containing the mouse dihydrofolate reductase gene cloned in the bacterial plasmid pBR322 as a vector (according to Gasser et al., Supra). The mixture of plasmid and carrier DNA was transferred to CHO DHFR cells (cells containing this plasmid usually also contain a second plasmid). After three days, cells were dispersed by trypsin treatment on several 100 mm plates in a hypoxanthine and thymidine free environment. Only those cells that have been permanently transformed with the DHFR gene and hence the EPO gene survive in this environment. After 7 to 21 days, colonies of surviving cells can be observed. These colonies of transformants can be continuously grown in hypoxanthine and thymidine free media after dispersion by trypsinization to yield new cell strains (e.g. CHO pDSVLMkEPO, CHO pSVgHuEPO, CHO-pDSVL-gHuEPO).

Liquid material from culturing the above-mentioned cell strains was assayed by RIA for the presence of recombinant monkey or human EPO. The environment for the CHO strain pDSVL-MkEPO contained57 EPO with immunological properties similar to that obtained from COS-1 cells after transfection with the plasmid pDSVL-MkEPO. The liquid material taken from the culture after 65 hours of culture contained EPO monkeys at a concentration of 0.60 units / ml.

Liquid material from CHO pSVgHuEPO and CHO pDSVL-gHuEPO contained human EPO with immunological properties similar to that obtained with COS-1 cells transfected with plasmid pSVgHuEPO or pDSVL-gHuEPO. A liquid fraction of a 3 day old culture of CHO pSVgHuEPO contained 2.99 units / ml human EPO and a 5.5 day cultured CHO sample pDSVL-gHuEPO contained 18.2 units / ml human EPO as measured by RIA.

The amount of EPO produced by the above-mentioned cell strains can be increased by gene amplification to produce a new cell strain with higher productivity. The enzyme dihydrofolate reductase (DHFR), which is a product encoded by the DHFR gene, can be inhibited by methotrexate (MTX). Thus, cells grown in hypoxanthine-free and thymidine-free media can be inhibited in growth to kill MTX. Under appropriate conditions (e.g., minimum MTX concentration), cells may become resistant and able to grow in the presence of MTX. The resistance of these cells to MTX is due to the amplification of the number of their DHFR genes and thus to the higher production of the DHFR enzyme. Surviving cells can then be treated with increasing amounts of MTX to obtain cell strains that contain multiple DHFR genes. A gene carried by an expression vector (e.g., an EPO gene) concomitantly with the DHFR gene or the transformed DHFR gene usually also increases its copy number.

A practical example of this amplification is the case in which the CHO cell strain pDSVL-MkE was subjected to increasing MTX concentrations (0 nM, 30 nM and 100 nM). After hours of exposure to each concentration, the cultures were monitored by RIA and the liquid material could be shown to contain 0.60, 2.45 and 6.10 units per ml. The CHO pDSVL-gHuEPO cell strain was then treated with increasing MTX concentrations of 30 nM, 50 nM, 100 nM, 200 nM, 1 μΜ, 5 μΜ MTX. After three days of culture, the liquid material from the culture was treated with 100 nM MTX of human EPO at a concentration of 3089 ± 129 units / ml as determined by RIA. For 48 hours, 100 nM and 1 μΜ MTX treated samples contained 466 and 1352 units / ml human EPO as determined by an RIA (average of three determinations). A concentration of 1 x 10 ⁶ cells in 5 ml medium was used using 60 mm diameter plates. After 24 hours, the medium was removed and replaced with 5 ml serum-free medium (DMEM with high glucose concentration and 0.1 mM non-essential amino acids and L-glutamine). After 48 hours, the accumulation of EPO in serum free environment was monitored. The medium was collected for RIA analysis and cells were trypsinized and counted. Mean RIA values were 467 units / ml and 1352 units / ml, for cells grown at 100 nM and 1 μΜ MTX, the values were 2335 units / plate and 6750 units / plate. The average number of cells per plate was 1.94 x 10 ⁶ and 3.12 x 10 ⁶ . Thus, production efficiency was 1264 and 2167 units / 10 ⁶ cells per 48 hours.

The cells contained in the above cultures are genetically heterogeneous populations. Standard procedures were used to isolate genetically homogeneous clones with the highest production capacity. These procedures are outlined in the Characterization of Cell Lines used to Produce Biologics, Section A, Part 2, June 1, 1984, Office of Biologics Research Review, Center for Drugs and Biologics, U. S. Food and Drug Administration.

The productivity of the CHO-producing EPO cell lines can be improved by appropriately interfering with the cultivation of cultures. Cultivation of mammalian cells in culture usually requires the presence of serum in the culture medium. The method of producing mammalian cells in culture usually requires the presence of serum in the growth medium. A method for producing erythropoietin from CHO cells in a serum-free environment substantially facilitates the isolation of erythropoietin from the culture medium. Thus, this method ensures the economical recovery of erythropoietin in environments that do not contain large quantities of serum.

CHO pDSVL-gHuEPO cells grown under standard conditions are used to inoculate mines suitable for direct centrifugation in an environment consisting of a 50:50 mixture of DMEM with high glucose and Ham's F12 medium supplemented with 5% fetal serum, L-glutamine as well as penicillin, streptomycin, 0.05 mM non-essential amino acids and the appropriate concentration of methotrexate. Growing cells in suspension allows the use of a large volume of EPO-producing CHO cells. Suspended CHO cells are then used to inoculate larger 850 ml mines with 200 ml medium. The cells are allowed to grow until a continuous layer is formed in the form of a wall adhering layer for three days. The medium used in this phase is the same as that used to grow the cells in suspension. At the end of the three-day growth period, the serum-containing medium is removed and replaced with 100 ml of serum-free medium. It is a 50:50 mixture of DMEM with a high glucose content and Ham's F12 medium supplemented with 0.05 nM non-essential amino acids and glutamine. The flasks are returned to the incubation apparatus for 1 to 3 hours, the medium is removed again and replaced with 100 ml of fresh medium, serum-free. Incubation with serum-free media for 1 to 3 hours will reduce the concentration of contaminating serum proteins. The flasks are then returned to the incubator for seven days at which time erythropoietin accumulates in the culture medium, without serum. At the end of the seven-day period, the medium is removed and replaced with fresh medium, serum-free for the second production cycle. After seven days, the sample thus obtained may contain, for example, 3892 ± 409 units / ml human erythropoietin according to RIA data. Based on the determined cell density of 0.9 to 1.8 x 10 ⁵ cells per ml, each flask contains 0.75 to 1.5 x 10 ⁸ cells and thus EPO production in 100 ml culture was 750 to 1470 units / 10 ⁶ cells per ml. 48 hours.

The culture medium from the cultivation of the CHO pDSVLMkEPO cell strain in the presence of 10 nM MTX was subjected to RIA in vitro and the in vivo efficacy was also monitored. The environmental sample contained 41.2 ± 1.4 units / ml MkEPO when measured by RIA, 41.2 ± 0.066 units / ml when measured in vitro and 42.5 ± 5 units / ml when measured in vivo biological activity. Monitoring of the polypeptide chain of the products showed the presence of EPO-type substances, containing three residues of the signal sequence after the amino-terminal residue of alanine. It is not yet clear whether it is a misproduction of the polypeptide in the CHO cell membrane or a reflection of the variation in the amino-terminal termination structure of monkey and human EPO.

The CHO pDSVL-gHuEPO culture medium was subjected to three types of assay. After 5.5 days, the sample contained recombinant human EPO at 18.2 units / ml in an RIA assay, 15.8 ± 4.6 units / ml in vitro, and 16.8 ± 3.0 units / ml in an in vivo assay.

Growth medium of pDSVL-gHuEPO gene-loaded CHO cells after using 100 nM MTX was also subjected to all three assays. After growing for 3 days, the sample contained recombinant human EPO at 3089 ± 129 units / ml in an RIA assay, 2589 ± 71.5 units / ml in an in vitro assay, and 2040 ± 160 units / ml in an in vivo assay. The amino-terminal termination shown in Table VI could be demonstrated in the amino acid sequence assay.

The medium from culturing CHO cells transfected with plasmid pDSVL-MkE in the presence of 10 nM MTX was decanted and MTX was dialyzed for several days to obtain an EPO concentration of 221 ± 5.1 units / ml (EPO / CCM) to determine The in vivo efficacy of EPO / CCM to hematocrit level in normal Balc / C mice was performed as follows: CHO cell culture (CCM) and EPO-CCM medium was adjusted by addition of PBS. CCM was used for a control group of three mice and two concentrations were used for the experimental groups (2 mice per group), 4 units and 44 units per dose. Within 5 weeks, this dose was administered to mice 3 times intraperitoneally. After the eighth injection, the mean hematocrit was 50.4% for the control group, 55.1% for the 4-dose group, and 67.9% for the 44-dose group.

Mammalian cell expression products can be readily isolated in substantially pure form from the culture medium using high pressure liquid chromatography (C ₄ ) and an ethanol gradient, preferably at pH 7.

Preliminary experiments were performed to determine the properties of recombinant glycoprotein products from conditioned media after expression by COS-1 and CHO cells of the human EPO gene compared to human EPO isolates from urine using various types of analysis (SDS-PAGE). These studies show that the EPO produced by CHO cells has a slightly higher molecular weight than the product of expression by COS-1 cells, which had a molecule that is larger in size than the product obtained from urine. All products were partially heterogeneous. Recombinant expression products of COS-1 and CHO cells of approximately the same molecular weight but having a greater molecular weight than the asialoproduct from human urine were obtained after the neuraminidase enzyme was treated to remove sialic acid. Endoglycosidase treatment

F (EC 3.2.1) to the recombinant product from CHO cells and to the urine product (to completely remove carbohydrates from both products), a substantially homogeneous product was obtained in both cases with identical molecular weight.

Thus, it is clear that the glycoproteins of the invention correspond in structure to the naturally occurring erythropoietin and also possess one or more of its biological properties in the carbohydrate composition, which is slightly different from the naturally occurring erythropoietin.

Example 11

Attempts have been made to fully construct the nucleotide bases of the two structural genes, which, as shown in Table VI, encode human EPO while incorporating preferred codons for expression in E. coli and for expression in S. cerevisiae. Based on these assays, it is possible to construct genes that code for analogs of human EPO. The implementation was in accordance with the method described by Alton et al. (WO 83/04053). The genes were assembled as multiple pairs that could be divided into three groups. These groups were designed for amplification, and it was possible to construct a suitable expression vector by multiple fragment binding.

The following Tables VIII to XIV show the constructs of the gene thus obtained which encode human EPO as a translation product without a signal or preliminary sequence but with an initial methionine residue at the -1 position. In addition, codons that are preferential for E. coli are incorporated into the gene to a large extent, hence this gene was called ECEPO.

Table VIII

ECEPO Oligonucleotides Region 1

First

Second

Third

and

5th

6th

7th

8th

9th

10th

11th

12th

AATTCTAGAAACCATGAGGGTAATAAAATA

CCATTATTTTATTACCCTCATGGTTTCTAG

ATGGCTCCGCCGCGTCTGATCTGCGAC

CTCGAGTCGCAGATCAGACGCGGCGGAG

TCGAGAGTTCTGGAACGTTACCTGCTG

CTTCCAGCAGGTAACGTTCCAGAACT

GAAGCTAAAGAAGCTGAAAACATC

GTGGTGATGTTTTCAGCTTCTTTAG

ACCACTGGTTGTGCTGAACACTGTTC

CAAAGAACAGTGTTCAGCACAACCA

TTTGAACGAAAACATTACGGTACCG

GATCCGGTACCGTAATGTTTTCGTT

Table IX ECEPO section 1

XbaI

Eco RII, 3

AATTCTAG AAACCATGAG GGTAATAAAA TAflTGGCTCC GCCGCGTCTG

GATC TTTGGTACTC CCATTATTTT ATTÄ ^ GAGG CGGCGCAGAC 4

ATCTGCGAck CGAGAGTTCT GGAACGTTAC CTGCTGfcAAG

TGAAAACÄTC IACCACTGGTT GTGCTGAACA CTGTTc (rTTG ACTTTTGTAG TGGTGÄCCAA CACGACTTGT GACAAGAÄÄČ]

CTAAAGAAGC

GATTľCTTCG

AACGAAAACA

TTGCTTTTGT

KpnI BamHI TTACGGTACC G AATGCCATGG CCTAG

Table X

Oligonucleotides ECEPO region 2

1. AATTCGGTACCAGACACCAAGGT

2. GTTAACCTTGGTGTCTGGTACCG

3. TAACTTCTACGCTTGGAAACGTAT

4. TTCCATACGTTTCCAAGCGTAGAA

5. GGAAGTTGGTCAACAAGCAGTTGAAGT

6. CCAAACTJCAACTGCTTGTTGACCAAC

7. 'TTGGCAGGGTCTGGCACTGCTGAGCG

8. GCCTCGCTCAGCAGTGCCAGACCCTG

9. - AGGCTGTACTGCGTGGCCAGGCA

10. GCAGTGCCTGGCCACGCAGTACA

11. CTGCTGGTAAACTCCTCTCAGCCGT

12. TTCCCACGGCTGAGAGGAGTTTACCA

13. GGGAACCGCTGCAGCTGCATGTTGAC

14. GCTTTGTCAACATGCAGCTGCAGCGG

15. AAAGCAGTATCTGGCCTGAGATCTG

16. GATCCAGATCTCAGGCCAGATACT

Table XI ECEPO section 2

EcoRI KpnI 1, 3. pTATTCGGTACC AGACACCAAG GTfTAACTTCT ACGCTTGGAA ACGTATGGAA

GCCATGG TCTGTGGTTC CAČŤGjQAGA TGCGAACCTT TGCATACČŤ]

GTTGGTCAAC

CAACCAGTTG

AAGCAGTTGA AGTtTGGCAG GGTCTGGCAC TTCGTCAACT TCAAÄÍCtTG CCAGACCGTG

TGCTGAGCGfc

ACGACTCGCT

GGCTGTACTG

UČGjACATGAC '

CGTGGCCAGG

GCACCGGTCC

CAÍ3TGCTGGT accg

AAACTCCTCT

TTTGAGGAGA

CAGCCGTfcGG

GTCGGCACÚČ. 15 BglU BamHI

AACCGCTGCA GCTGCATGTT GACftAAGCAG TATCTGGCCT GAGATCTG TljGGCGACGT CGACGTACAA CTGTTTCGJC ATAGACCGGA CTCTAGACCTAC

Table XII ECEPO section 3

First

Second

Third

4th

5th

6th

7th

8th

9th

10th

11th

12th

13th

14th

15th

16th

GATCCAGATCTCTGACTACTCTGC i

ACGCAGCAGAGTAGTCAGAGATCTG

TGCGTGCTCTGGGTGCACAGAAAGAGG

GATAGCCTCTTTCTGTGCACCCAGAGC

CTATCTCTCCGCCGGATGCTGCATCT

CAGCAGATGCAGCATCCGGCGGAGA

GCTGCACCGCTGCGTACCATCACTG

ATCAGCAGTGATGGTACGCAGCGGTG

CTGATACCTTCCGCAAACTGTTTCG

ATACACGAAACAGTTTGCGGAAGGT

TGTATACTCTAACTTCCTGCGTGGTA

CAGTTTACCACGCAGGAAGTTAGAGT

AACTGAAACTGTATACTGGCGAAGC

GGCATGCTTCGCCAGTATACAGTTT

ATGCCGTACTGGTGACCGCTAATAG

TCGACTATTAGCGGTCACCAGTAC

- 67 Table XIII ECEPO section 3

BamHX BglII GA TCCAGATCTCTG

GTCTAGAGAC

ACTACTCTGC IIIGCGTGCTCT TGATGAGACG ACGCA ^ GAGA

GGGTGCACAG

CCCACGTGTC aaagaggI: ta tctctccgcc

TTTCTCCGAT ÁGjAGAGGCGG

GGATGCTGCA TC1bcTGCAC CGCTGCGTAC CATCACTC ^ T GATACCTTCC CCTACGACGT AGACGÄČfíTG GCGACGCATG GTAGTGACGA CT ^ TGGAAGG

8

II, 13 GCAAACTGTT TCG ^ GTATAC TCTAACTTCC TGCGTGGTAJA ACTGAAAČTG CGTTTGACAA AGCACATA |

12

Sali

TATACTGGCG AAGCfrTGCCG TACTGGTGAC CGCTAATAG ATATGACCGC TTCGTACGGjC ATGACCACTG GCGATTATC AGCT ^Ί 16

Table ΧΓ7 ECEPO gene - • XbaI CTAG AAACCATGAG GGTAATAAAA -1 1 MetAla TAATGGCTCC GCCGCGTCTG TTTGGTACTC CCATTATTTT ATTACCGAGG CGGCGCAGAC ATCTGCGACT CGAGAGTTCT GGAACGTTAC CTGCTGGAAG CTAAAGAAGC TAGACGCTGA GCTCTCAAGA CCTTGCAATG GACGACCTTC GATTTCTTCG TGAAAACATC ACCACTGGTT GTGCTGAACA CTGTTCTTTG AACGAAAACA ACTTTTGTAG TGGTGACCAA CACGACTTGT GACAAGAAAC TTGCTTTTGT TTACGGTACC AGACACCAAG GTTAACTTCT ACGCTTGGAA ACGTATGGAA AATGCCATGG TCTGTGGTTC CAATTGAAGA TGCGAACCTT TGCATACCTT GTTGGTCAAC AAGCAGTTGA AGTTTGGCAG GGTCTGGCAC TGCTGAGCGA CAACCAGTTG TTCGTCAACT TCAAACCGTC CCAGACCGTG ACGACTCGCľ GGCTGTACTG CGTGGCCAGG CACTGCTGGT AAAČTCCTCT CAGCCGTGGG CCGACATGAC GCACCGGTCC GTGACGACCA TTTGAGGAGA GTCGGCAGCC AACCGCTGCA GCTGCATGTT GACAAAGCAG TATCTGGCCT GAGATCTCTG TTGGCGACGT CGACGTACAA CTGTTTCGTC ATAGACCGGA CTCTAGAGAC ACTACTCTGC TGCGTGCTCT GGGTGCACAG AAAGAGGCTA TCTCTCCGCC TGATGAGACG ACGCACGAGA CCCACGTGTC TTTCTCCGAT AGAGAGGCGG GGATGCTGCA TCTGCTGCAC CGCTGCGTAC CATCACTGCT GATACCTTCC CCTACGACGT AGACGACGTG GCGACGCATG GTAGTGACGA CTATGGAAGG GCAAACTGTT TCGTGTATAC TCTAACTTCC TGCGTGGTAA ACTGAAACTG CGTTTGACAA AGCACATATG AGATTGAAGG ACGCACCATľ TGACTTTGAC TATACTGGCG AAGCATGCCG TACTGGTGAC Are you CGCTAATAG all ATATGACCGC TTCGTACGGC ATGACCACTG GCGATTATCA GCT

Table VIII shows the oligonucleotides used to construct region 1 of the ECEPO genes, which is the code for the amino-terminal residues of the human polypeptide. Oligonucleotides were assembled as pairs, i. 1 and 2, 3 and 4, etc. and these pairs were then coupled to form region 1 of the ECEPO gene, as shown in Table IX. Note that this region contains EcoRI and BamHI digestion sites, then downstream from the EcoRI treatment site is an XbaI site, and upstream of the BamHI site an KpnI site is located. Section I can easily be amplified using M13 phage as a sequence verification vector. Some difficulties have been associated with the isolation of this region as a fragment after treatment with XBal / KpnI enzymes from E. coli DNA, possibly due to methylation of the KpnI site in the host. For this reason, the DNA of the single-stranded phage was isolated and then changed in vitro to double-stranded DNA, then the desired double-stranded fragment was isolated.

The ECEPO genes, Sections 2 and 3, as shown in Tables XI and XIII, were constructed in a similar manner from the oligonucleotides listed in Tables X and XII. Each section was amplified in an M13 vector to verify the sequence and then isolated from DNA phage. As shown in Table XI, ECEPO region 2 was constructed with EcoRI and BamHI digestion sites and could be isolated as a KpnI / BglII-treated fragment. Similarly, ECEPO region 3 was prepared with BamHI and SalI-treated sites capable of reclosing at the end and could be isolated from phage DNA as a BglII / SalI fragment. The 3 regions thus obtained can be readily linked to a continuous DNA sequence as shown in Table XIV, which encodes the entire human EPO polypeptide with an amino terminal methionine (ATG) codon for translation initiation in E. coli. It should also be noted that upstream of the ATG start codon, there is a base pair that essentially repeats the ribosome binding sequence in the expression of the E. coli OMP-f gene.

Any suitable vector for expression can be used as a carrier vector for ECEPO. The vector selected for this purpose was the temperature-sensitive plasmid pCFM536, which is a derivative of the plasmid pCFM414 (ATCC 40076), which has been described in U.S. Patent Application Ser. No. 636,727, filed Aug. 6, 1984 (Charles F. Morris). The pCFM536 gene was then digested with XbaI and HindIII. The large fragment was isolated and used for binding to the ECEPO gene. Sections 1 (XbaI / KpnI), 2 (KpnI / BglII), and 3 (BglII / SalI) were preceded in the correct order in M13 and the EPO gene was then isolated as a single fragment (XbaI / HindIII). This fragment contained a portion of the binding chain from phage M13 mp9 between the SalI to HindIII sites. To verify expression of the plasmid P536 was used promoter, the lambda P _L, which itself may be subject to repressor gene C ₈ & ₇ as is the E. coli strain K12 ^ HTRP.

The above-mentioned ECEPO gene can then be modified in various ways to obtain codes for erythropoietin analogs, for example, [Asn ² , des-Pro ² to Ile ⁶ ] hEPO and [His 1 JhEPO], as mentioned above.

A. [Asn ² , des-Pro ² to Ile ⁶ ] hEPO

Plasmid 536, which carries the engineered ECEPO gene of Table XIV as an inserted region, terminated with the XbaI and HindIII sites, was digested with HindIII and XhoI. The other endonuclease cleaves said gene at a site 6 bp away from the codon encoding Asp ⁸ to Arg ¹⁰ . The XbaI / Xhola binding chain was then produced in the following sequence:

+1 2 7 8 9 XbaI Met Ala same time Cys Asp XhoI 5'-CTAG ATG GCT AAT TGC GAC-3 1 3 '- TAC CGA TTA ACG CTG AGCT-5 '

The XbaI / XhoI binding chain and the ECEPO XhoI / HindIII gene sequence fragment were inserted into a large fragment resulting from the digestion of plasmid pCFM526, a derivative of plasmid pCFM414 (ATCC 40076), using the XbaI and HindIII enzymes, as described in U.S. Pat. No. 636,727, filed Aug. 6, 1984 (Charles F. Morris) to obtain a DNA sequence that encodes the Met- ¹ form of the desired analog when expressed in E. coli.

B. [His ⁷ ] hEPO

Plasmid 536 was digested with HindIII and XhoI as in paragraph A. The XbaI / XhoI chain was constructed with the following sequence:

+1 2 3 4 5 6 7 8 9 XbaI Met Ala for for Arg Leu Ile His Asp Xhol 5'-CTAG ATG GCT CCG CCA CGT CTG ATC CAT GAC-3 ' 3 ’- TAC CGA GGC GGT GCA GAC TAG GTA CTG AGCT-5 '

A chain monovalent fragment ECEP (XhoI / HindIII, was then incorporated into a plasmid pCFM526 to obtain the DNA sequence carried by the plasmid coding for Met ^"1 form the desired analog when expressed in E.

The construction of the SCEPO gene, which incorporates codons that facilitate expression in yeast, is described in the following Tables XV to XXI. As in the case of the ECEPO gene, the entire construct involves the formation of three groups of oligonucleotides as shown in Tables XV, XVII and XIX below, which are base pairs which are then arranged into sections as shown in Tables XVI, XVIII and XX, it should be noted that the synthesis may be partially facilitated by the use of some suboptimal codons in both SCEPO and ECEPO construction, i. in the case of both genes, identical oligonucleotides 7 to 12 in region 1 are identical, and also in region 2, oligonucleotides 1 to 6 are identical in both genes.

Table XV

Oligonucleotides SCEPO region 1

1. AATTCAAGCTTGGATAAAAGAGCT

2. GTGGAGCTCTTTTATCCAAGCTTG

3. CCACCAAGATTGATCTGTGACTC

4. TCTCGAGTCACAGATCAATCTTG

5. GAGAGTTTTGGAAAGATACTTGTTG

6. CTTCCAACAAGTATCTTTCCAAAAC

7. GAAGCTAAAGAAGCTGAAAACATC

8. GTGGTGATGTTTTCAGCTTCTTTAG

9. ¹ ACCACTGGTTGTGCTGAACACTGTTC

10. CAAAGAACAGTGTTCAGCACAACCA

11. TTTGAACGAAAACATTACGGTACCG

12. GATCCGGTACCGTAATGTTTTCGTT i

Table XVI

SCEPO section 1

EcoRI HindIII 1 AATTCA AGCTTGGATA

GT TCGAACCTAT 2

AAAGAGCTfcc ACCAAGATTG ATCTGTGACT cfcAGAGTTTT TTTCTCGAGG TG ^ TTCTAAC TAGACACTGA GCTCJCAAAA, 7

GGAAAGATAC TTGTTGpAAG CTAAAGAAGC TGAAAACATC ftCCACTGGTT

CCTTTCTATG AACAACtľTTČ] GATTTCTTCG ACTTTTGTAG TGGT'tyCCAA _| 11 Kpnl BamHI

GTGCTGAACA CTGTTCjTTTG AACGAAAACA TTACGGTACC G

CACGACTTGT GACAAGAÄAl TTGCTTTTGT AATGCCATGG CCTAG

Table XVII

Oligonucleotides SCEPO region 2

1. AATTCGGTACCAGACACCAAGGT

2. GTTAACCTTGGTGTCTGGTACCG

3. TAACTTCTACGCTTGGAAACGTAT

4. TTCCATACGTTTCCAAGCGTAGAA

5. GGAAGTTGGTCAACAAGCAGTTGAAGT

6. CCAAACTTCAACTGCTTGTTGACCAAC

7. <TTGGCAAGGTTTGGCCTTGTTATCTG

8. GCTTCAGATAACAAGGCCAAACCTTG

9. AAGCTGTTTTGAGAGGTCAAGCCT

10. AACAAGGCTTGACCTCTCAAAACA

11. TGTTGGTTAACTCTTCTCAACCÄTGGG

12. TGGTTCCCATGGTTGAGAAGAGTTAACC

13. AACCATTGCAATTGCACGTCGAT

14. CTTTATCGACGTGCAATTGCAA

15. AAAGCCGTCTCTGGTTTGAGATCTG

16. GATCCAGATCTCAAACCAGAGACGG

- 75 TABLE XVIII SCEPO section 2

KpnI

EcoRI 1

TTÄTTCGGTffCC AGACACCAAG GCCATGG TCTGTGGTTC

GTkAACTTCT ACGCTTGGAA ACGTAThGAA GTTGGTCAAC AAGCTGTTGA CAAŤTGhAGA TGCGAACCTT TGCATACCT CAACCAGTTG TTCGACAACT. 6 agtJttggcaa

TCAAÄČC | JTT

GGTTTGGCCT

CCAAACCGGA

TGTTATCTGA AGCTGTTTTG AGAGGTCAAG ACAATAGACT TCGhCAAAAC TCTCCAGTTC cctItgttggt

GGAÄÄÄ / ^ CA, 13

TAACTCTTCT CAACCATGGG pACCATTGCA ATTGCACGTC ATTGAGAAGA GTTGGTACCC TTGGTjAACG 'TAACGTGCAG' 14

GAThAAGCCG

CTATŤŤČpGC

TCTCTGGTTT

AGAGACCAAA

BglII BamHI GAGATCTG CTCTAGACCTA G

Table XIX

Oliganucide SCEPO section 3

1. GATCCAGATCTTTGACTACTTTGTT

2. TCTCAACAAAGTAGTCAAAGATCTG

3. GAGAGCTTTGGGTGCTCAAAAGGAAG

4. ATGGCTTCCTTTTGAGCACCCAAAGC

5. CCATTTCCCCACCAGACGCTGCTT

6. GCAGAAGCAGCGTCTGGTGGGGAA

7. CTGCCGCTCCATTGAGAACCATC

8.! CAGTGATGGTTCTCAATGGAGCG

9. ACTGCTGATACCTTCAGAAAGTT

10. GAATAACTTTCTGAAGGTATCAG

11. ATTCAGAGTTTACTCCAACTTCT

12. CTCAAGAAGTTGGAGTAAACTCT

13. TGAGAGGTAAATTGAAGTTGTACAC

14. ACCGGTGTACAACTTCAATTTACCT

15. CGGTGAAGCCTGTAGAACTGGT

16. CTGTCACCAGTTCTACAGGCTTC

17. GACAGATAAGCCCGACTGATAA

18. GTTGTTATCAGTCGGGCTTAT

19. CAACAGTGTAGATGTAACAAAG

20. TCGACTTTGTTACATCTACACT

Tables XX SCEPO section 3

BamHI BqIII 1 ^

GATC CAGATCTTTG ACTACTTTGT TGAGAGCTTT GTCTAGAAAC TGATGAAACA ACTCT ^ GAAA

5

GGGTGCTCAA AAGGAAGfcCA TTTCCCCACC AGACGCTGCT CCCACGAGTT TTCCTTCGt tyAGGGGTGG TCTGCGACGA

6

TCTGCCGCTC

AÍÄCTľGCGAG

1

CATTGAGAAC CATCf \ CTGCT GATACCTTCA GTAACTCTTG GTAGTGÄČfcA CTATGGAAGT '10

GAAAGTThľT

CTTTCAATÄÄ

TCCAACTTCT IAGAGAGGTAA ATTGAAGTTG TACACfcGGTG AGGTTGAAGA ACCT |

19

AACTGGTfcAC AGATAAGCCC GACTGATAAp AACAGTGTAG TTGACCACIG fJjATTCGGG CTGACTATTG TTGpTACATC ii

CAGAGTTTAC

GÍTCTCAAATG

AAGCCTGTAG

TTCGGACATC

Sali

ATGTAACAAA G TACATTGTTT CAGCT

Table XXI

Gen SCEPO

-1 +1

Hindlll ArgAla

ÄSUTtggata aaagagctcc accaagattg atctgtgact cgagagtttt

ACCTAT TTTCTCGAGG TGGTTCTAAC TAGACACTGA GCTCTCAAAA

GGAAAGATAC TTGTTGGAAG CTAAAGAAGC TGAAAACATC ACCACTGGTT CCTTTCTATG AACAACCTTC GATTTCTTCG ACTTTTGTAG TGGTGACCAA

GTGCTGAACA CTGTTCTTTG AACGAAAACA TTACGGTACC AGACACCAAG CACGACTTGT GACAAGAAAC TTGCTTTTGT AATGCCATGG TCTGTGGTTC

GTTAACTTCT ACGCTTGGAA ACGTATGGAA GTTGGTCAAC AAGCTGTTGA CAATTGAAGA TGCGAACCTT TGCATACCTT CAACCAGTTG TTCGACAACT

AGTTTGGCAA GGTTTGGCCT AGTGTTTTG AGAGGTCAAG TCAAACCGTT CCAAACCGGA ACAATAGACT TCGACAAAAC TCTCCAGTTC

CCTTGTTGGT TAACTCTTCT CAACCATGGG AACCATTGCA ATTGCACGTC GGAACAACCA ATTGAGAAGA GTTGGTACCC TTGGTAACGT TAACGTGCAG

GATAAAGCCG TCTCTGGTTT GAGATCTTTG ACTACTTTGT TGAGAGCTTT CTATTTCGGC AGAGACCAAA CTCTAGAAAC TGATGAAACA ACTCTCGAAA

GGGTGCTCAA AAGGAAGCCA TTTCCCCACC AGACGCTGCT TCTGCCGCTC CCCACGAGTT TTCCTTCGGT AAAGGGGTGG TCTGCGACGA AGACGGCGAG

CATTGAGAAC CATCACTGCT GATACCTTCA GAAAGTTATT CAGAGTTTAC GTAACTCTTG GTAGTGACGA CTATGGAAGT CTTTCAATAA GTCTCAAATG

TCCAACTTCT TGAGAGGTAA ATTGAAGTTG TACACCGGTG AAGCCTGTAG AGGTTGAAGA ACTCTCCATT TAACTTCAAC ATGTGGCCAC TTCGGACATC

AACTGGTGAC AGATAAGCCC GACTGATAAC AACAGTGTAG TTGACCACTG TCTATTCGGG CTGACTATTG TTGTCACATC

Sali

ATGTAACAAA G

TACATTGTTT CAGCT

The entire region of SCEPO was expressed in M13 and regions 1, and 3 were isolated from phage as HindIII / KpnI, KpnI / BglII and BglII / SalI fragments.

The system for expression of the SCEPO gene, which is currently believed to be advantageous, is based on the secretion of S. cerevisiae α-factor as described in U.S. Pat. No. 487,753, filed April 22, 1983 (Grant A. Bitter), and published October 31, 1984 as European Patent Application no.

123 294. This system includes constructs in which the DNA encoding the yeast α-factor gene leader sequence is inserted just 5 ' of the coding region of the exogenous gene to be expressed. As a result, the translation product contains a leader or signal sequence that is removed by the endogenous yeast enzyme during secretion of the remainder of the product. Since the ATG codon, which is the codon for the α-factor translation translation initiation codon, is used in construction, it is not necessary to position this codon at the -1 position of the SCEPO gene. As shown in Table XXI, there is a binding chain upstream of the alanine (+1) code that allows direct insertion into the plasmid, including DNA, for the first 80 residues of the α-factor signal sequence, followed by the α-factor promoter. The specific construction for the expression of the SCEPO gene involves a four-step binding including the above fragments and a large fragment after digestion of the paC3 plasmid with HindIII / SalI. From the resulting plasmid paC3 / SCEPO, the α-factor promoter, the signal sequence and the SCEPO gene can be isolated by digestion with BamHI and binding to the plasmid pYE after

I digested with BamHI to give plasmid pYE / SCEPO.

Example 12

Expression of recombinant products using the engineered ECEPO and SCEPO genes by the expression system of Example 11 can be achieved as follows:

Plasmid p536 of Example 11 is transformed into AM7 cells

E. coli, which have already been transformed with the appropriate plasmid pMW1 with the _C857 gene. Cell cultures in LB medium (ampicillin 50 μg / lnl and kanamycin 5 μς ^ Ι, preferably with 10 mM magnesium sulfate) are maintained at 28 ° C and the cells are grown to a density of 0.1 (optical density), EPO expression is maintained. caused by raising the temperature to 42 ° C. EPO is produced in an amount of approximately 5 mg / liter.

Cells are harvested, disrupted and then treated with NP-40 lysozyme and wetting agent. The centrifuged pellet (24,000 g) is then solubilized with guanidine hydrochloride and further purified by reverse phase high pressure liquid chromatography (C? - Vydac), 0-80% ethanol, 50 mM ammonium acetate, pH 4.5. When determining the sequence of the protein, it can be shown that the purity of the product is greater than 95% and that the product obtained has 2 possible amino terminal ends, namely A-P-P-R or P-P-R in a relative amount of 3: 1. This observation of products that correspond to human hEPO and the same alanine-free product at position 1 indicates treatment with host cells that removes terminal methionine and, in some cases, initial alanine. The radioimmunological activity of the isolates was 150,000 to 160,000 units / mg, the in vitro activity was 30,000 to 62,000 units / mg, and the in vivo activity was 120 to 720 units / mg. (Comparison was made with material isolated from human urine with an efficiency of 70,000 units / mg). The dose-response curve for the recombinant product in vivo was significantly different from the same curve for EPO from human urine.

‘I

The plasmids for the EPO analogs generated in Parts A and B of Example 11 were individually transformed into E. coli AM7 cells that had already been transformed with pMW1 and the cells were cultured as described above. Purified isolates were examined by RIA in vitro. Both values for [Asn ² , des-Pro ² to Ile ⁶ ] hEPO expression products were approximately 11,000 units / mg and 6000 units / mg protein, while the same values for [His ⁷ ] hEPO were 41,000 units / mg and 14 000 units / mg protein, indicating that analogous products had a potency of 1/4 to 1/10 as full expression products.

In an expression system for use in S. cervisiae host cells, the plasmid pYE / SCEPO was transformed into two different strains, YSDP4 (genotype and pep4-3 suffer) and RK81 (αα pep4-3 suffer genotype). Transformed YSDP4 host cells were grown in SD (Cold Spring Harbor Laboratory, Cold Spring Harbor,

N.Y., p. 62 (1983)), supplemented with casein amino acids at a concentration of 0.5%, pH 6.5 at 30C. The medium was separated at an optical cell density of 36 and contained EPO at a concentration of 244 units / ml (97 µg / liter according to RIA). Transformed RK81 cells were grown to an optical density of 6.5 or 60, with an EPO concentration of 80-90 units / ml (34 µg / liter according to RIA). Preliminary analysis showed the heterogeneity of the products, presumably due to different protein glycosylation and relatively high mannose content.

The plasmids PaC3 and pYE in HB101 E. coli were deposited on September 27, 1984 in American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland under the numbers ATCC 39881 and ATCC 39882. Plasmids pCFM526 in AM7, pCFM536 in JM103 cells and pMW1 in JM103 cells were also deposited on November 21, 1984 under ATCC Nos. 33932, 33934 and 33933. Saccharomyces cerevisiae strains YSPD4 and RK81 were deposited on November 21, 1984 under ATCC Nos. 20734 and 20733.

It is clear that numerous exceptionally valuable products can be obtained by the process of the invention.

Polypeptides obtained by the method of the invention are useful materials, whether they are microbial expression products or synthetic products, the primary, secondary and tertiary structures of these products being first recognized by the implementation of the method of the invention.

As mentioned above, recombinant products and synthetic products share, to varying degrees, the in vitro biological activity of both EPO isolates from natural sources and expression products and can thus be used as substitutes for EPO isolates in blood cell culture media. To a certain extent, the polypeptides produced by the method of the invention also have in vivo efficacy similar to the natural EPO isolate and can therefore be used to treat erythropoietin in mammals including humans, for example where reticulocyte stimulation is desired, ferokinetic effects such as iron exchange between plasma and bone marrow where it is necessary to achieve a change in erythrocyte numbers, stimulation of hemoglobin C (Eschbach et al., supra) and an increase in hematocrit in mammals, as shown in Example 10. Thus, these are patients who usually require blood transfusion, for example in trauma, in the case of surgical diseases, in kidney diseases, including the use of artificial kidney, and in patients with various types of blood composition disorders such as hemophilia, blood cell disorders, physiological anemia, sickle cell anemia and the like. In this way, it is possible to reduce the need for transfusions, thereby utilizing EPO to reduce the materials by which infectious diseases can be transmitted. The products according to the invention with respect to production by recombinant methods are devoid of pyrogens, natural inhibitory substances and the like and are therefore very effective in comparison with natural products. They can also be used where oxygen transfer needs to be improved, for example when staying in a reduced amount environment and in circulatory diseases.

A preferred route of administration for these polypeptides is parenteral administration, the agents usually being administered with acceptable diluents, carriers, and / or excipients. Preliminary pharmacokinetic studies have shown a longer half-life in vivo for monkeys

EPO by intramuscular administration than by intravenous administration. Effective doses vary substantially from disease to disease, but are in the range of 0.1 to 100 µg / kg, i. about 7 to 7000 units / kg. Conventional diluents such as human serum albumin and standard carriers such as saline may be used.

Other adjuvants such as testosterones, germ cell stimulators, insulin-like growth factors, prostaglandin, serotonin, cyclic AMP, prolactin, and triiodothyronine may also be used in the compositions, as well as agents used in the treatment of aplastic anemias such as metenolene, stanozol and pandrolone (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.

(Supp. 8), 283-291 (1980) and Kurtz, FEBS Letters, 14a (1), 105-108 (1982). Compounds known to enhance the effect of erythropoietin or asialo-EPO, such as adrenergic agents, thyroid hormones, androgens or BPA, as described in Dunn, Current Concepts in Erythropoiesis, John Wiley, may also be used as adjuvants. 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), Fischer 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) and compounds which are referred to as liver factors for blood production, as described in Naughton et al., Acta Haemat. 69, 171-179 (1983) and the erythropropins described in Congote et al. In Abstract 364, Proceedings 7th International Congress of Endocrinology (Quebec City, Quebec, Jul 1-7, 1984), Congote, Biochem. Biophys.

Res. Comm., 115 (2), 447-483 (1983) and Congote, Anal. Biochem., 140, 428-43 (1984) and the erythrogenins described in Pu84 flashing Rothman et al., J. Surg. Oncol., 20, 105-108 (1982). Preliminary screening shows promising results in hypoxic polycythaemic mice pretreated with 5-α-dihydrotestosterone or nandrolone and then erythropoietin.

The diagnostic use of the polypeptides produced by the method of the invention is also very broad and involves the use of both labeled and unlabeled forms in various immunoassays, including RIA, ELISA, and the like, as well as a variety of in vitro and in vivo assays. These procedures are described in Dunn et al., Expt. Hematol., 11 (7), 590-600 (1983); Gibson et al., Pathology, 16, 155-156 (1984), Expt. Hematol., 11 (7), 649-660 (1983); Saito et al., Jap. J. Med., 23 (1) to 21 (1984), Natan et al., New Eng. J. Med., 308 (9).

520-522 (1983) and also in various articles referred to in these publications. Polypeptides of the invention, including synthetic peptides with sequences or residues of EPO, are also highly useful pure polyclonal antibody substances and monoclonal antibody banks specific for recognition of continuous and discontinuous EPO epitopes. Examples include preliminary analysis of the amino acid sequence of Table VI by Hopp et al., PNAS (USA), 78, pp. 3824-3828 (1981) and secondary structure analysis by Chou et al., Ann. Rev. Biochem, 47, p. 2.51 (1978) has shown that synthetic peptides whose structure corresponds to continuous sequences of natural peptides in regions 41-57, 116-118 and 144-166 inclusive each time are capable of eliciting a highly antigenic response and thereby producing monoclonal and polyclonal antibodies which react with both the synthetic peptide and the whole protein. These antibodies could be useful for the detection and purification of EPO and related products.

The following three synthetic peptides were prepared:

(1) hEPO 41-57, V-P-D-T-K-V-N-F-Y-W-K-R-M-E-V-G,

2) hEPO 116-128 K-E-A-I-S-P-D-A-A-S-A-A

3) hEPO 144-166; V-Y-S-N-F-L-R-G-K-L-K-L-Y-T-G-E-A-C-R-T-G-D-R.

A preliminary immunization study using these polypeptides showed a relatively weak positive response for hEPO 41-57, almost no response for hEPO 116-128, and a strong positive response to hEPO 144-166, measured by the ability of rabbit serum antibodies to precipitate EPO isolate from human urine, No. ¹²⁵ I. Preliminary in vivo studies of the three peptides did not show practically no activity, either individually or in admixture.

The amino acid sequence of mammalian EPO residues, as demonstrated in the examples, defines the primary structure of full EPO, but the specific sequence of monkey amino acid 165 amino acids in Table V and 166 human EPO residues in Table VI do not limit the range of useful polypeptides of the invention. According to the method of the invention, it is possible to obtain various allele forms of EPO which, according to the results of earlier investigations, exist, for example human gamainterferon, as can be compared with data suggesting an argenine residue at position no. 140 EPO or a glutamine residue at position 140 in Gray et al., Nature 295, pp. 503-508 (1982). However, it is always a complete sequence of human gamma-interferon. The allelic forms of full EPO may differ from each other and from the sequences in Tables V and VI by the sequence length or by the fact that some residues are omitted, replaced, segmented or added with subsequent changes in activity and changes in glycosilization. As mentioned above, one allele form of human EPO appears to contain the methionine residue at position 126. It is expected that there are still genome DNAs and cDNA sequences that encode allele polypeptides or simply contain different codons to designate the same polypeptide.

In addition to the naturally occurring allele forms of full length EPO, the invention also includes other EPO products, such as EPO polypeptide analogs and fragments thereof. By the method of published applications of Alton et al. (E.g. WO / 83/04053), it is possible to design and construct genes for microbial expression of polypeptides whose structure differs from full EPO in one or more residues by substitution, addition in the middle or at the end or omitted. It is also possible to make modifications to the monkey cDNA and EPO genes in the genome by well-known site-directed mutagenesis to obtain analogs and derivatives of EPO. These products may have some biological properties but differ in other EPO properties. Some products omit the amino acids [Asn ² , des-Pro ² to Ile ⁶ ] hEPO, [des-Thr ¹⁶³ to Arg ¹⁶⁶ ] hEPO, and delta 27-55hEPO, the latter having the entire exon deleted. Some derivatives are more stable against hydrolysis and thus have a longer effect, or one or more glycosylation sites are omitted, which may lead to greater efficacy in yeast production, or the cysteine residue may be replaced by, for example, histidine or serine as in [His ⁷ ] hEPO and can be more easily isolated or have phenylalanine-substituted tyrosine, such as [Phe ¹⁵ ] hEPO, [Phe ⁴⁹ ] hEPO, and [Phe ¹⁴⁵ ] hEPO, and may bind poorly or more readily to receptors in target cells. They may also be fragments containing only part of the sequence of full EPO and only part of the activity of the substance, for example, binding to receptors and not erythropoietic activity. Of particular importance are potential fragments of the human genome DNA sequence of Table VI, which are special domains of biological activity. It should be noted that in vivo ineffectiveness may not indicate therapeutic failure or lack of antagonism. This antagonism may be useful in the treatment of polycythaemia in excess of EPO as described in Adamson, Hosp. Practice, 18 (12), 49-57 (1983), and Hellmann et al., Clin. Lab. Haemat., 5, 335-34 (1983).

According to another aspect of the invention, it is possible to obtain cloned DNA sequences which encode human and simian EPO and which are valuable with respect to information that they report on the mammalian erythropoietin amino acid sequence that had previously not been obtained despite decades of analytical research on naturally occurring products. DNA sequences can also be used for the synthesis of erythropoietin by recombinant means. In addition, they can be used to construct viral and circular plasmids, host cells, both prokaryotic and eukaryotic after transfection or transformation, and yeast and mammalian cells in culture, making them novel and valuable methods of growing said cells capable of producing EPO and EPO-products. The DNA sequences are also useful as labeled samples in the isolation of EPO and the genome DNA, including the cDNA that encodes these products. While it is not possible to estimate to what extent the DNA sequences of the invention can be used in protein synthesis, for example in insects. These sequences can be generated by different types of mammalian cells, which can then be eukarytic hosts for the production of erythropoietin and the like, as described in Palmiter et al., Science, 222 (4625), 809-814 (1983).

Thus, it is to be understood that the examples set forth in the Examples section of the application are not intended to limit the scope of the invention since many modifications and variations are possible in principle, since the DNA sequences include cDNA and genomic DNA sequences. Thus, they may also code for EPO, as in Example 12, for fragments and analogs thereof, i. EPO products which may have some biological properties identical to the natural product, others not.

Thus, it is understood that the DNA sequences of the invention include all sequences allowing expression in prokaryotic or eukaryotic host cells, wherein the expression product is a polypeptide having at least a portion of the primary structure of EPO and one or more of its properties, such as:

(a) DNA sequences from Tables V and VI;

(b) DNA sequences which hybridize to the sequences in paragraph (a) or fragments thereof; and

(c) DNA sequences which hybridize with the sequences of (a) and (b) for reasons other than the degeneracy of the genetic code.

It should be noted that existing allele monkeys and human EPO gene sequences are likely to hybridize to sequences from Tables V and VI or fragments thereof. In addition, the SCEPO and ECEPO genes and cDNA or DNA genome sequences coding for different fragments and analogs of EPO can also hybridize to the above DNA sequences. This hybridization can be carried out under the conditions described above, including limiting conditions.

The above examples illustrate microbial expression of EPO and expression in mammalian cells, wherein the DNA is incorporated into a hybrid bacterial plasmid and virus vector, but a wide variety of similar systems could be used. These could be, for example, vectors of homogeneous origin introduced into a variety of cells of bacteria, yeast and mammals in culture, or systems in which vectors are not used, for example using calcium phosphate. Obviously, expression of, for example, monkey-derived DNA is carried out in a culture of monkey or human cells, and although in this case it is an expression of exogenous DNA, a high level of expression can be achieved even though not DNA of genomic origin. host. The expression systems of the invention further contemplate the formation of EPO and similar products in the cytoplasm or membrane with subsequent accumulation89, possibly also in the periplasmic space of the bacteria or in the culture supernatant of the above cultures or in less common systems, for example those using

P.aeruginosa according to Gray et al., Biotechnology,

2, pages 161-165 (1984).

The improved hybridization methods of the invention have been applied to DNA / DNA, but can also be applied to RNA / RNA and RNA / DNA. Mixed samples can allow for greatly improved hybridization with faster isolation of the polynucleotide. These may include, for example, improved colony transfer, the use of nylon-based filters, such as GeneScreen and GeneScreen Plus, which allow reuse, the use of new proteases according to Taub et al., Anal. Biochem., 126, p. 222-230 (1982), the use of very low concentrations of the order of 0.025 picomoles of a number of mixed samples, for example containing more than 32 different sequences, and carrying out hybridization and further processing under restrictive conditions, i. 4, preferably 2 ° C from the lowest calculated temperature for each sample. These improvements can deliver results that could not have been expected earlier. Thus, it is clear that the improvement achieved by the method of the invention is very important in view of the fact that the mixed samples containing four times the number of different sequences compared to what has been done hitherto could be successfully used to isolate a single of the gene sequence from a mixture containing 1,500,000 phage coatings. This result was achieved essentially in parallel with the published opinion of Anderson and others in the above article, that ... this method is impractical and inconvenient for the isolation of the mammalian protein gene in the absence of the corresponding RNA.

Claims (31)

PATENT CLAIMS 1. A DNA sequence for use in providing expression of a polypeptide product whose structure at least partially corresponds to the primary structure of erythropoietin to provide its biological properties of enhancing reticulocyte and red blood cell production in bone marrow cells and enhancing hemoglobin synthesis or iron uptake in prokaryotes. or eukaryotic host cells, wherein said DNA sequence is selected from the group consisting of (a) the DNA sequences set forth in Tables V and VI and their complementary strands; (b) DNA sequences hybridizing under stringent conditions to the DNA sequences defined in (a) or fragments thereof; and (c) DNA sequences that would hybridize to the DNA sequences defined in (a) or (b) if not> sW; degeneracy of the genetic code. The DNA sequence of claim 1 encoding human erythropoietin. The DNA sequence of claim 1 coding for monkey erythropoietin. DNA sequence according to claim 3 comprising a protein coding region shown in Table V. DNA sequence according to claim 1 or 2, which is a genomic DNA sequence. The DNA sequence of claim 5 encoding human erythropoietin. 7. The DNA sequence of claim 6 comprising the protein coding region shown in Table VI. The DNA sequence of claim 1 or 2, which is covalently linked to a detectable label. The DNA sequence of claim 8, wherein the detectable marker is radioactive. The DNA sequence of claim 8 or 9, which is single stranded. The DNA sequence of claim 1 encoding [Phe ¹⁵ ] hEPO, [Phe ⁴⁹ ] hEPO, [Phe ¹⁴⁵ ] hEPO, [His ⁷ ] hEPO, [Asn ² -des-Pro ² to Ile ⁶ ] hEPO, [des-Thr ¹⁶³ to Arg ¹⁶⁶ ] hEPO or [delta 27-55] hEPO. íd,, A prokaryotic or eukaryotic host cell transformed or transfected with a DNA sequence according to any one of claims 1, 2, 3, 6, 7, and 8 in a manner allowing said host cell to express said polypeptide product. id id A prokaryotic or eukaryotic host cell according to claim 13, which is capable of glycosylating said polypeptide. The prokaryotic or eukaryotic host cell of claim 13, which is a mammalian cell. - 'c-L The prokaryotic or eukaryotic host cell of claim 13, which is a COS cell. - 36 rpm icL The prokaryotic or eukaryotic host cell of claim 13, which is a CHO cell. A biologically functional circular plasmid or viral DNA vector comprising the DNA sequence of any one of claims 1, 2, 3, 5, 6, 7 or 11. ichi. ¹ , A prokaryotic or eukaryotic host cell stably transformed or transfected with the DNA vector of claim 17. 19. A recombinant polypeptide having all or part of the primary structural conformation of human or simian erythropoietin as set forth in Table VI or V, or any portion thereof, or any allelic variant or derivative thereof, exhibiting biological activity consisting in increasing reticulocyte and red blood cell production in bone marrow cells and a son. . . This polypeptide is a product of eukaryotic expression of an exogenous DNA sequence and has a molecular weight by SDS-PAGE higher than that of erythropoietin isolated from urine. The polypeptide of claim 19, which is a glycoprotein whose average carbohydrate composition differs from human erythropoietin isolated from urine. The polypeptide of claim 19 or 20, wherein the exogenous DNA sequence is a cDNA sequence. The polypeptide of claim 19 or 20, wherein the exogenous DNA sequence is a genomic DNA sequence. The polypeptide of claim 19 or 20, wherein the exogenous DNA sequence is part of an autonomously replicating circular DNA plasmid or viral vector. 24. The polypeptide of any one of aspects 19 to 23, which is covalently attached to a detectable label. The polypeptide of claim 24, wherein the detectable label is radioactive. A polypeptide product obtainable by expressing a DNA sequence according to any one of claims 1, 2, 3, 5, 6, and 7 in eucalypts of a host cell. 27. A method of producing a polypeptide having at least a portion of the primary structural conformation of erythropoietin to enable biological efficacy of increasing reticulocyte and red blood cell production in bone marrow cells and enhancing hemoglobin synthesis or iron uptake, characterized in that it is prokaryotic or eukaryotic the host cell transformed or transfected with the DNA sequence of any one of claims 1, 2, 3, 5, 6 and 7 in a manner allowing the host cell to express the polypeptide is cultured under suitable nutrient conditions and optionally the desired polypeptide expression product of the DNA sequence is isolated. 28. The method of claim 27, wherein the host cell of any one of claims 12 to 16 is cultured. A method according to claim 27 or 28 for the production of a polypeptide according to any one of claims 19 to 23 and 26, characterized in that it is carried out under conditions resulting in said poly peptides. 30. A pharmaceutical composition comprising a polypeptide obtainable by a process according to claim 27, 28 or 29 and a pharmaceutically acceptable diluent, excipient or carrier. A pharmaceutical composition according to claim 30, characterized in that it comprises a polypeptide according to any one of claims 19 to 23 and 26.