CZ443885A3 - Dna sequence used for assuring expression of polypeptide product - Google Patents

Description

A DNA strand for use in ensuring the expression of a polypeptide product

Field of technology

The invention relates to a DNA strand for use in expressing a polypeptide product whose structure at least partially corresponds to that of human or monkey erythropoietin. Vanález also relates to a process for the manufacture of these chains and to pharmaceutical compositions containing the expression products of these chains.

State of the art

A. Manipulation with genetic materials

Genetic materials can be broadly defined as those chemical structures that plan and carry out the production of cell components and viruses. Long-chain polymeric substances, deoxyribonucleic acid (DNA), carry the genetic material of all living cells and viruses, with the exception of some viruses whose genetic material carries ribonucleic acid (PNA). each consists of a purine (adenine or guanine) or pyrimidine (thymine or cytosine) bound to a phosphate-bound deoxyribose. The binding of nucleotides in linear polymeric form takes place via the nucleotide 5-phosphate on the one hand and via the 3-hydroxyl group on the other. Functional SNA occurs in the form of double permanent chains, which arise from single nucleotide chains (called deoxyoligonucleotides), the chains being joined by a hydrogen bond between purine and pyrimidine bases (thus a "complementary linkage between adenine (A) and thymine (T) or guanine (G) and cytosine (C)). Nucleotides are usually named according to the 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." Ribonucleic acid is a polynucleotide that contains adenine, guanine, cytosine and instead of thamine uracil (U) bound to ribose and phosphate group ·

Very briefly, the planning function of DNA is usually performed by a process in which a sequence of nucleotides (genes) is transcribed into relatively unstable mRNA. This mRNA serves as a template for the formation of structural, control and catalytic proteins from amino acids. The process of mRNA translation involves small RNA (tRNA) interactions, which are key to the transfer of individual amino acids throughout the mRNA strand, so that polypeptides with the correct amino acid sequence can be generated. This DNA-derived mRNA "message", which provides the tRNA interaction and orientation of any of the twenty amino acids, is the basis for expression based on triplets (codons), ie sequences of three consecutive nucleotides. Protein production is the culmination of the 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 placed in front of a gene in a DNA polymer and represent the site at which transcription into mBNA begins. The DNA control sequence, usually also located in front of the gene in a given DNA polymer, includes a protein that determines the frequency or rate of onset of transcription.

Thus, a promoter / control sequence or control system only is a sequence that is placed in front of a gene (or genes) in a functional DNA polymer and that determines whether a particular gene is transcribed and, where appropriate, expressed. DNA sequences that are located downstream of the gene or genes in polymeric DNA are a signal to terminate transcription into mRNA, and are therefore termination sequences.

The focus of microbiologists in the last decade has been on the industrial production of substances that are important from an industrial or pharmaceutical point of view using orgymes that did not initially include information in their genetic material regarding the desired product or (in the case of mammalian cells in tissue tissue) the chromosomal gene is usually not expressed on a larger scale. In this case, a gene that is specific for the structure of the desired buS polypeptide is isolated from an organism that contains the gene or produced chemically and then permanently incorporated into another organism, which is usually a normally reproducing unicellular organism, such as bacteria, yeast or mammalian cell in tissue culture. Once this occurs, the existing pxBxtxaRxfBxmssx cell mechanism in this transformed cell is transfected into the transformed cell to construct the desired product, with exogenous DNA being used as a template for mKNA transcription followed by translation to form a continuous sequence of amino acid residues.

A variety of publications are known in the art that relate to recombinant DNA methods for isolating, synthesizing, purifying, and propagating genetic material for use in transforming a selected host organism. For example, U.S. Patent No. 4,237,224 (Cohen et al.) Discloses the transformation of a single cell host by the action of hybrid viral or circular plasmid DNA that contains selected sequences of exogenous DNA. In carrying out the method included in said patent, the procedure is to first obtain a vector for transformation by enzymatic digestion of viral or circular plasmid DNA to form linear DNA strands. Using similar enzymes, selected exogenous or heterologous chains are usually 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 the exogenous DNA in the presence of binding enzymes that allow the fragments to rejoin to form hybrid vectors that contain a selected segment of exogenous DNA that is inserted into the viral or circular plasmid DNA.

Transformation of a suitable unicellular host with the hybrid vector thus obtained results in the production of multiple copies of exogenous DNA in the host cell population. More often, however, the whole transformation goal is to express exogenous DNA by host cells on a larger scale, so that isolatable amounts of commercially important protein fragments or polypeptides encoding exogenous DNI can be obtained. Similar procedures have also been described in other patents, such as U.S. Patent Nos. 4,264,731 (Shine), 4,273,875 (Manis), 4,293,652 (Cohen), and European Patent Specification No. 93,619, November 1983 «

The development of specific DNA sequences for incorporation into DNA vectors can be accomplished in a variety of ways, which depend to a large extent on the foreignness of the donor to the intended host and the size of the polypeptide molecule to be expressed in the host organism.

In great simplification, the basic three ways can be mentioned, namely

1) the method involved in isolating the double-stranded DNA sequence from the donor genome,

2) a method which chemically produces a DNA sequence which encodes a desired polypeptide a

3) double-stranded DNA synthesis in vitro using an enzyme called mfiNA reverse transcription, which was isolated from donor cells.

Methods for generating DNA that is complementary to a given mENA are generally described in \ As methods using cDNA. IN

DNA sequence production is often the method of choice when the entire amino acid residue sequence of the desired polypeptide is known. This procedure is described, for example, in U.S. Patent Application No. 483,451 (Alton), filed April 15, 1983 and published November 24, 1983 as WO83 / 04D53). This publication describes means that allow very good results to be obtained, such as: the possibility of introducing alternating codons that are usually found in genes, especially those which are highly expressed in the host organism (for example, preferential codons for the use of a certain host, such as yeast or E. coli), to ensure the absence of an untransmitted intron, ie within a stored sequence that is often and commonly present in mammalian genomic LNA and polypeptide leader sequences that are normally present but not readily cleaved from the resulting polypeptide by bacterial or yeast cells, as well as ensuring good incorporation; and finally the construction of genes that encode polypeptide fragments and for a of the desired polypeptides.

If the entire sequence of amino acid residues in the desired polypeptide is not known, the DMA sequence cannot be produced directly and the DNA sequence encoding the desired polypeptide by the cDNA method becomes the method of choice despite the potential disadvantages. In this way, expression vectors can be obtained which provide a high level of microbial expression, as mentioned above. One of the standard procedures for isolating the cDNA sequence of the desired end product is the preparation of cDNA plasmids, obtained by reverse transcription of mENA, which is found in large quantities in the donor's cells. For example, it is a whole group of cDNA samples derived from pituitary cells that use relatively large amounts of growth hormone as a final product. Where substantial portions of a sequence of amino acid residues of polypeptides are known, labeled single-stranded DNA sequences can be used that join to the sequences, presumably present in the target cDNA. These sequences are then used for DNA / DNA hybridization procedures, which are performed on cloned copies of cDNA denatured to a single-stranded form. These procedures have been described, for example, in U.S. Patent No. 4,394,443 (Weisman et al.) And throughout A number of publications, such as Wallace et al., Nuc. Acids. Res. 6, pp. 3543-3557 (1979), Reyes et al., P.N.S.S. (USA) 79, pp. 3270-3274 (1982) and Jaye et al., Nuc. Acids Res. 11, pp. 2325-2335 (1983). Also in U.S. Pat.

U.S. Patent No. 358,535 (Falkow et al.) Discloses DNA / DNA tyn hybridization in diagnosing, and European Patent Application Publication Nos. 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) pp. 55-58 and 174-176 describe the hybridization of colonies and plaques, and instructions for the transfer and hybridization of DNA and RNA can be found in the brochure New England Nuclear (Boston, Mass.) For membrane materials intended for piseax gene search. (Catalog number NEF-972).

Among the major innovations in recombinant cloning hybridization procedures are, for example, the use of labeled mixed samples of synthetic oligonucleotides, each of which is a potential complete complement to a specific DNA sequence in the hybridization sample, including a heterogeneous mixture of SNA or single-stranded DNA. These methods are particularly useful in screening cDNA clones derived from sources in which a very small amount of mBNA is available for the desired polypeptide. The purpose of the procedure is to select limited hybridization conditions so that, as far as possible, non-specific binding does not occur in the subsequent autoradiographic visualization of a specific cDNA clone when hybridizing the target DNA to this single sample, which is a complete complement of the sequence. These procedures have been described in various publications, such as Wallace et al., Nuc. Acids Res. 9, pp. 879-897 (1981), Suggs et al., P.N.A.S. (USA) 78, pp. 6613-6617 (1981), Choo et al., Nature 299, pp. 178-180 (1982), Kurachi et al., P.N.A.S. (USA) 79, pp. 6461-6464 (1982),. Ohkubo et al., P.N.A.S. (USA) 80, pp. 2196-2200 (1983) and Kornblihtt et al., P.N.A.S. (USA) 80, pp. 321S-3222 (1983). In general, mixed sample procedures, as described in Wallace et al. (1981), have been further developed by various workers to obtain satisfactory results in isolating DNA clones using a mixture of 32 strands of 16 bases. with a different sequence of baits along with a single strand of 11 baits to perform double-sided confirmation of the presence of the desired cDNA, as described in Singer-Sam and other PNAS (USA) 80, pp. 802-806 (1983).

The use of DNA isolated from genomes is a common feature of at least the three methods mentioned above for obtaining DNA sequences specific for use in recombinant methods. This is especially true for methods to ensure microbial expression of mammalian polypeptides, where the complexity of the mammalian genome DNA is a major problem. There are relatively suitable procedures for phage-borne mixtures of human and other mammalian genome DNA, as described, for example, in Lawn et al.

Cell 15, pp. 1157-1174 (1978), which describes a procedure for obtaining a human genome library called the Maniatis Library, Kam et al., P.N.A.S. (USA) 77, pp. 5172-5176 (1980), the publication relates to a human genomic library based on endonuclease digestion, and Blattner et al., Science 196, pp. 161-169 (1977), which describes the construction of a bovine genome library. On the other hand, very few successful hybridization procedures have been performed in the genome to obtain DNA from the genome in the absence of a well-known amino acid sequence or sequence of DNA. Examples include Piddes et al., J. Mol., And App. Genetics 1, pp. 3-18 (1981), which describes the successful isolation of the gene encoding the subunit and human pituitary glycoprotein (hormone) from the Maniatis Library "using a" full length "sample of 621 pairs based on a pre-isolated sequence Another example is Das et al., PN «AS (USA)

80, pp. 1531-1535 (1983), describe the isolation of clones of the human genome for the human HLA-DR using synthetic oligonucleotide ₃ 175 pores covet · Finally, Anderson et al in P.NoA.S. (USA) 80, pp. 6838-6842 (1983) describe the isolation of a clone from the genome for a pancreatic bovine trypsin inhibitor (BPTI) using a single sample of 86 bp, the sample was constructed according to the known amino acid sequence in BPTI. The authors report insufficient methods for isolating mRNA that would be suitable for obtaining cDNA collection due to apparently too low levels of mRNA in the salivary gland and lung tissue, which were the source of this acid, and describe genome monitoring using a mixture of labeled samples. that:

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

Thus, there is a continuing need to design improved methods for rapid and efficient isolation of cDNA clones in cases where insufficient information is available regarding the amino acid sequence of the polypeptide to be encoded and where enriched tissues are not available as source of mRNA for the construction of 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 in polypeptides encoded by the genes sought. .

Erythropoietin as a target polypeptide

Erythropoiesis, ie the formation of red blood cells, takes place continuously throughout cell life to replace cells that have been destroyed. This is a very precisely controlled physiological process that allows sufficient red blood cells to be available in the blood for good tissue oxygenation, but there must not be enough blood cells to restrict blood circulation. Red blood cells are made 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 carbohydrate component, but have the same potency, biological effect and molecular weight Form asialo is form a or β, from which the terminal carbohydrate group is removed. Erythropoietin is present in plasma at very low concentrations when the tissues are sufficiently aerated with full erythrocytes in good health. This normal low concentration is sufficient to stimulate the replacement of red blood cells, which normally undergo physiological breakdown during their aging.

The amount of erythropoietin in the circulation increases in the absence of oxygen when the oxygen transfer by the red blood cells is reduced. Hypoxia can be caused by the loss of large amounts of blood by bleeding, the destruction of red blood cells when exposed to radiation, decreased oxygen intake at high altitudes or due to prolonged unconsciousness or in the case of various forms of anemia. In response to hypoxic stress, erythropoietin increases red blood cell production by stimulating the conversion of primitive germ cells in the bone marrow to proerythroblasts, which gradually mature, synthesize hemoglobin and are released into the bloodstream as intestinal blood cells. If the number of red blood cells in the circulation is greater than is needed for normal tissue oxygen requirements, circulating erythropoietin levels decrease.

These ratios are described, for example, in Dough et al., Extp Hemato !, B (Supp. 8), 144-152 (1980); Tong et al., J. Biol. Chem. 256 (24), 12666-12672 (1981), Goldwasser, J. Cell Physiol. 110 (Suppl.) 133-135 (1982), Finch, Blood, 60 (6), 1241-1246 (1982), Sytowski et al., Expt. Hernatol, 8, (Supp.8), 52-64 (1980), Naughton, Ann. Clin. Lab. Sci. 13 (5), 432-438 (1983), Weise 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. Ϊ.Ϊ. Acad. Sci. 414, 66-72 (1983); Murpby et al.

Acta Haematologica Jqmnica 46 (7), 1380-1396 (1983), Dessypris et al., Brit. J. Haematol 56, 295-306 (1983); Emannouel et al., Am. J. Physiol 247 (l? T 2) F 168-76 (1984).

Because erythropoietin is an essential factor in red blood cell production, this hormone is potentially useful in the diagnosis and treatment of renal disorders that are characterized by low or defective red blood cell production. These facts have been described in Pennarthur-Das et al., Blood 63 (5), 1168-71 (1984) and Haddy, Am. J. Ped. Hematol./Oncol. 4, 191-196 (1982), the publications relate to erythropoietin as a possible drug for a disease in which red blood cells have a crescent-shaped shape and

Esbach et al., J. Clin. Invest. 74 (2), pp. 434-441, 1984), which describes a method of treatment of uremic sheep and the effect of infusion of erythropoietin-rich plasma, proposing doses of IC units of erythropoietin (E0O) / kg per day. for 15 to 40 days to correct the anemia that occurs in chronic renal failure. A similar problem is discussed by Krane, Henry Ford Hosp. Copper. J., 31 (3), 177-181 (1983).

Recently, it has been shown that if erythropoietin was available in sufficient quantities, 1,600,000 patients in the United States alone could treat anemia, as described in Morrison, Bioprocessing in Space overview, p. 557-571 in The World Biotech. Report 1984, Volume 2, USA (Online Publications, New York, N.Y., 1984). Recent studies have been the basis for estimating the efficacy of erythropoietin in various disease states, disorders and hematological irregularities, as described in Vedovato et al., Acta Haematol 71, 211-213 (1984) (β-thlassemia), Vichinsky et al., J Pediatrician. 105 (1), 15-21, 1984 (cystic fibrosis), Cotes et al., Brit. J. Obstet · Gyneacol. 90 (4), 304-311 (1983) (pregnancy, menstrual problems), Haga et al., Acta Pediatr. Scand. 72, 827-831, 1983 (early anemia at immaturity), Claus-Walker et al., Arch. Phys. Copper. Rehabil. 65, 370-374, 1984 (spine injury), Dunn et al., Eur. J. Appl. Physiol. 52, 178-182

1984 (spaceflight), Miller et al., Brit · J. Haematol. 52, 545-590, 1982 (acute blood loss), Udupa et al.,

J. Lab. Clin. Copper. 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 obtain erythropoietin in good plasma or urine yields have been relatively unsuccessful. It requires complex laboratory equipment and even in this case it is usually only possible to obtain small amounts of an impure and volatile extract that contains erythropoietin.

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

Early attempts to isolate erythropoietin from urine resulted in unstable, biologically ineffective preparations of the hormone. U.S. Pat. No. 3,865,801 discloses a method for stabilizing the biological activity of a crude substance which contains erythropoietin isolated from urine. 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. St. 252, No. 15 (August 10, 1977), pp. 5558-5564. mg of protein in a yield of 21%.

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

British Patent Specification No. 2,085,887 (Sugir moto et al., Issued May 6, 1983) discloses a process for the production of human hybrid lymphoblastoid cells using 3 to 420 units of etathropoietin per ml of cell suspension in culture after multiplication of mammalian host cells. At the highest production, the rate of erythropoietin production can be expressed as 40 to 4,000 units / 10 ⁶ cells / 48 hours in in vitro culture when transferred from living cells. 4,377,513 · A number of methods have been proposed for the isolation of erythropoietin from tissues, including the use of tumor cells, but the yields have been very low, as evidenced, for example, by Jelkman et al., Expt. Hernatol. 11 (7), 581-588 ( 1983), Tambourin et al., PNAS (USA) 80, 6269-6273 (1983), Katsuoka et al., Gann, 74, 534-0541 (1983), Hagiwara et al., Blood, 63 (4), 828-835 ( 19845 and Choppin et al., Blood 64 (2), 341-347 (1984).

Another possible way to isolate purified erythropoietin is by immunological procedures. Injection of human erythropoietin to animals, preferably animals or rabbits, induces serum antibodies to human erythropoietin. Human erythropoietin, injected into an animal, is recognized as a foreign substance, ie as an antigen for the animal's immune system, and triggers the production of antibodies against that antigen. When blood is drawn, the antibody remains in the blood serum. Both unpurified serum and purified antibodies, such as the serum immunoglobulin G fraction, can be used to detect and complex human erythropoietin, but these materials have the major disadvantage of running on polyclonal antibodies derived from different cells and therefore binding in addition to erythropoietin still on other components of crude extracts.

Also interesting are current attempts to develop continuous cell cultures capable of producing a homogeneous antibody that reacts specifically with a single antigen. This procedure was generally described in Chisholm,

High Technology Vol. 3, δ. 1, pp. 57-63 (1983). Attempts have also been made to use cell fusions and hybridizations to obtain anti-erythropoietin monoclonal antibodies and to use these antibodies to isolate and quantify human erythropoietin. An example is the successful development of mouse hybridoma cell lines that secrete monoclonal antibodies to human ethythropoietin, as described in Lee Huang, Abstract No. 1463 in the Fed. Why. 41, 520 (1982). Another example is the detailed description of the preparation and use of an anti-human erythropoietin monoclonal antibody according to Weiss et al., P.N.A.S. (USA) 79, 5465-5469 (1982) and also in Sasaki, Biomed. Biochemistry. Acta 42 (11/12), pp. 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 concerning the immunological activity of synthetic peptides that substantially mimic the amino acid sequence that exists in naturally occurring proteins, glycoproteins and nucleoproteins. Lower molecular weight proteins are involved in immunological responses that are similar in scope and duration to physiologically important proteins, such as viral antigens, polypeptide-type hormones, and the like. Immunological responses of these polypeptides include the induction of specific antibody production in animals, as described, for example, in Lerner et al., Cell, 23, 309-310, 1981; Rosa et al., Nature 294, 654-656, 1981. , Walter et al., PNAS (USA) 77, pp. 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 3-25 (1984), which discusses in particular the biological and immunological activity of synthetic peptides which have approximately the same secondary structures as peptide hormones, but may not have the same primary structure. However, the above studies mostly refer to amino acid sequences of proteins other than erythropoietin, for which no relevant information has yet been published regarding the amino acid sequence of this substance.

U.S. Patent Application No. 463,724, filed February 4, 1983 (J. Egrie), published August 22, 1984 as European Application No. 0 116 446, discloses a mouse hybridoma cell line (ATCC No. HB 8209) that produces highly a specific type of antibody that is simultaneously specifically reactive to a polypeptide consisting of the following amino acid sequence:

NH2- Ala-Pro-Pro-Arg-Leu-Ile-Cys-Asp-Ser-Arg-Val-LeuGlu-Arg-Tyr-Leu-Leu-Glu-Ala-Lys-COOH.

This sequence corresponds to the sequence expected for the first twenty amino acids of the complete human erythro25 poetin, isolated according to the method of Miyake et al., J. Biol. Chem. 252, 5558-5564 (1977), where amino acid sequence analysis was performed in the 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 described in Sue et al., Proc. Nati. Acad, Sci. USA 80, pp. 3651-3655 (1983), the publication relates to the production of polyclonal antibodies against a synthetic sequence of 26 amino acids and in Sytowski et al., J. Immunol. Methods 69, pp. 181-186 (1984).

The polyclonal and monoclonal antibodies described above are highly useful materials for use in immunology for the detection and quantification of erythropoietin and may 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 , sufficient for further analysis, clinical trials and therapeutic use of this substance, for example in chronic kidney disease, in which the tissue is insufficient to maintain sufficient erythrocyte production.

As a result, it can be assumed that the best prospects for the full characterization of mammalian erythropoietin and for obtaining large amounts of this substance for potential diagnostic and clinical use have the successful application of recombinant techniques to ensure microbial synthesis of said material in large quantities.

Great efforts have been made to isolate the DNA sequence that encodes human erythropoietin and similar substances in other mammals, but so far without success. This failure is probably due to the fact that a very small number of tissues are available as sources of this substance, especially in humans, especially mBNA-enriched materials, which could allow the construction of a series of cDNAs from which a sequence could be isolated in a conventional manner. which codes for erythropoietin. In addition, so little is known about the continuous amino acid sequence in erythropoietin that it is not possible to construct, for example, longer samples of polynucleotides that could be further used in DNA / DNA hybridization using cDNA and especially to obtain a certain genome assembly. For example, the above sequence of twenty amino acids, which was used to obtain a monoclonal antibody produced by ATCC No.

HB 8209 does not allow 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 encoding human erythropoietin makes up less than 0.00005% of the total DNA in the human genome.

Even the best results described so far, obtained using recombinant methods to obtain DNA sequences in which isolatable amounts of mammalian erythropoietin could be obtained in a microorganism, have been far from a real success. For example, in Farber et al., Exp. Hernato1 · 11, Suppl. 14, Abstract 101 (1983) describes the extraction of BHX mRNA from baboon kidney tissue after phenylhydrazine treatment followed by mRNA injection into Xenopus laevis oocytes, in which a transient mixture of translation products having, inter alia, some erythropoietin-like activity has been achieved. In Farber et al., Blood 62, No. 5, Supp. No. 1, Abstract 392 and on page 122a (1983) describe the translation of mRNA from human kidney into frog oocytes. The resulting mixture of translation products contained 220 units of translation product with active erythropoietin per mg of mRNA.

This level of translation of the exogenous erythropoietin mRNA code in vitro is very low (even compared to previously reported results of baboon mRNA translation), but the results appear to confirm that the human kidney is the site where erythropoietin is expressed, so it should be it may be possible to obtain from the kidney a set of cDNAs enriched in the desired gene (Farber, Clin. Res. 31 (4), 769A (1983))

Until U.S. Patent Application Nos. 561,024 and 582,185, only one report of human erythropoietin cDNA cloning and expression in E. coli was known. A large number of cDNA clones were inserted 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 in Lee-Huang, Proc. Nati. Acad. Sci. USA 81, pp. 2708-2712 (1984).

According to the invention, it is possible for the first time to obtain novel purified and isolated polypeptide products which contain part or all of the primary structure (i.e. amino acid residue sequence) and also have one or more biological properties (e.g. immunological properties and biological activity in vitro and in vivo). ) of naturally occurring erythropoietin, including its allelic variants. These polypeptides are also products of expression of a eukaryotic or rparyotic host (e.g., bacterial, yeast, and mammalian cells in culture) using exogenous DNA sequences obtained by genome or cDNA cloning or gene synthesis. Furthermore, microbial expression products in vertebrate cells, such as mammals and birds, are free of human protein and other impurities that may accompany erythropoietin in its natural environment in extracellular fluid, such as plasma or urine. Typical yeast cell products, such as Saccharomyces cerevisiae or prokaryotic cells, such as E. coli, are free of the presence of any mammalian protein. Depending on the host used, the polypeptides obtained by the method of the invention may be glycosylated by a carbohydrate of a mammal or eukaryotic organism or left in non-glycosylated form. The polypeptides obtained by the method of the invention may also contain a 1-amino acid residue of methionine.

The novel glycoproteins obtained by the process of the invention, including those whose primary structure follows the structure of a naturally occurring, e.g. human erythropoietin, sufficient to achieve one or more biological properties with a carbohydrate composition different from that of the naturally occurring occurring, for example, human erythropoietin.

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

The method according to the invention also provides synthetic polypeptides which correspond, in whole or in part, in structure to the contiguous sequence of amino acid residues of erythropoietin which has thus been elucidated for the first time. These sequences, which may share primary, secondary or tertiary structural properties with naturally occurring erythropoietin, may also have biological activity and / or immunological properties in common with the naturally occurring product, so that they may be used as biologically or immunologically active erythropoietin substitutes for medical and immunological purposes. Correspondingly, mono31 clonal and polyclonal antibodies can also be obtained which are immunoreactive with respect to the above-mentioned polypeptides and preferably also immunoreactive with naturally occurring erythropoietin.

Examples of flies obtained by the method of the invention include cloned DNA sequences of human or ape origin and polypeptide sequences obtained using these materials, which are the first confirmation of the erythropoietin structure of monkeys and humans.

The method of the invention also provides 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. listed vectors. In this way, according to the invention, valuable polypeptides can be obtained by culturing a transformed microbial host in culture under conditions that allow or facilitate the expression of exogenous, vector-incorporated DNA in large quantities followed by isolation of the desired polypeptides from growth medium, cell lysate or cell fractions. membranes ·

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

After illuminating the sequence of amino acid residues in erythropoietin, the complete and / or partial DNA sequence encoding erythropoietin can also be established, including the preferred properties of the sequence, e.g. for restriction endonuclease digestion and the addition of initial, terminal, or interleaved DNA sequences that may facilitate construction or facilitate expression of the vector. Thus, it is possible to obtain (and further develop by specific mutagenesis of cLNA and genome DNA) DNA sequences that encode microbial expression of erythropoietin polypeptide analogs or derivatives that differ from the naturally occurring form by the identity or location of one or more amino acid residues. thus, for example, analogues resulting from the omission of less than all erythropoietin (EPO) -specific residues and / or from substitutions in which one or more residues are ί (Wz

- 33 is replaced by other residues and / or resulting in the addition of one or more amino acid residues to the terminal or medial portion of the polypeptide, all of which may have some or all of the properties of naturally occurring forms.

The essence of the invention

The invention provides a DNA strand for use in expressing a polypeptide product whose structure at least partially corresponds to the primary structure of human or monkey erythropoietin of Table VI or Table V or any variant or derivative of these strands with erythropoietin biological activity, in particular production of reticulocytes and red blood cells in the bone marrow and an increase in hemoglobin synthesis or iron uptake in prokaryotic or eukaryotic cells, this DNA strand being selected from

a) DNA strands from Table VI or V or from complementary strands for these strands,

(b) DNA strands capable of hybridizing under specified conditions to the DNA strands of paragraph (a) or to fragments of such strands; and

(c) DNA strands that are capable of hybridizing to the strands of paragraphs (a) and (b) for reasons other than the degeneracy of the genetic code.

A specific representative of chain b) are the chains for human and monkey erythropoietin, including allelic variants and / or the chains for erythropoietin of other mammals. A typical strand c) may be a synthesized DNA strand that encodes EPO, EPO fragments or EPO analogs, which strands may contain codons that facilitate translation of the mRNA into a vertebrate host.

The invention also relates to polypeptides for which the above chains are coding chains.

The invention also provides a process for the manufacture of a polypeptide product whose structure at least partially corresponds to the primary structure of human or monkey erythropoietin of Table VI or V or any variant or derivative thereof with erythropoietin biological activity, in particular to increase reticulocyte and red blood cell production. bone marrow and an increase in hemoglobin synthesis or iron uptake, the method consists in culturing, under suitable conditions in a suitable nutrient medium, a prokaryotic or eukaryotic host cell which has been transformed or transfected with the above DNA strand and expressed the appropriate polypeptide, after which, the desired DNA expression polypeptide product is isolated in these cells.

34a

The invention also relates to a pharmaceutical composition having the action of erythropoietin, which comprises as its active ingredient a polypeptide obtained in the above-mentioned manner and in addition a carrier, diluent or excipient which is pharmaceutically acceptable.

These compositions are suitable for use where erythropoietin deficiency occurs, for example in various types of anemia, especially chronic renal failure.

Polypeptides that are the product of the above procedure can be covalently attached to a detectable material. For example, it may be radiolabeled with I isotope, which provides 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 can also be labeled, either radioactively or with biotin, for example, and then used in hybridization to determine

EPO fragments or EPO analogs, which sequences may contain codons that facilitate translation of the mBNA into a host other than vertebrate.

Also within the scope of the invention are polypeptides encoded by a portion of DNA complementary to the upper strand of Table VI, i.e., a complementary invert protein as described in Tramontano et al., Nucleic Acids Besearch 12, pp. 5049-5059 (1984). .

Also within the scope of the invention are pharmaceutical compositions comprising effective amounts of the polypeptides obtained by the method of the invention together with suitable diluents, excipients and / or carriers that allow treatment with erythropoietin, especially anemia, which occurs in chronic renal failure.

Polypeptides. which are the product of the invention can be labeled by covalent bonding with a detectable material, e.g., may be a radiolabel isotope of iodine ^y i, whereby it is possible to obtain a reagent useful for the detection and quantitative detection of erythropoietin in solid tissue and samples of liquids, e.g. urine or blood DNA obtained by the method of the invention can also be labeled, either radioactively or with biotin, 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 on their chromosomal map. Erythropoietin gene disorders at the DNA level or related gene disorders can also be screened in this way.

and

As will be discussed in more detail below, the invention represents a substantial improvement in the detection of a specific single-stranded polynucleotide of unknown sequence in a heterogeneous cell or viral sample, including multiple single-stranded polynucleotides, by:

(a) prepare a mixture of labeled single-stranded polynucleotide samples with uniformly varying and different bases, 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 on a solid substrate,

c) the substrate with the fixed sample is processed in such a way as to reduce the further mutual possibility of binding of the array of nucleotides, with the exception of hybridization to the polynucleotides of the ul sample, I

d) contacting the treated α-fixed substrate with a mixture of labeled samples under conditions that facilitate hybridization only between fully complementary polynucleotides; and

e) the specific polynucleotide is detected by detecting the presence of a hybridization reaction of the polynucleotide with a completely complementary sample from a mixture of labeled samples, as evidenced by the presence of a higher density of labeled material on the substrate at the site of the specific polynucleotide compared labeled sample on the substrate.

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

As mentioned above, this procedure allowed the identification of DNA clones encoding erythropoietin of simian origin from a group of sequences prepared from mRNA of anemic monkey kidney 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 human erythropoietin fractions in colony hybridization, thus showing seven positive cDNA clones out of the total number.

200,000 colonies. In addition, three positive clones could be rapidly isolated from 1,500,000 plaques of the human genome.

In this case, too, a mixture of 128 20-base polynucleotide samples was ingested together with a small set of 128 nucleotides of 17 bases according to amino acid analysis in x & zHysh another continuous sequence of human erythropoietin.

The above procedures represent the first use of mixed oligonucleotide samples in DNA / DNA hybridization to isolate mammalian genome clones, with a mixture of more than 32 oligonucleotide samples being isolated for the first time in isolating cDNA clones.

Further advantages of the method according to the invention will be apparent from the following detailed description of preferred embodiments of this method.

The method of the invention can isolate and characterize DNA sequences that encode part or. for the complete human and monkey erythropoietin polypeptide sequence. Furthermore, human and monkey DNA could be expressed in eukaryotic and prokaryotic cells to obtain isolatable amounts of polypeptides with biological (e.g., immunological) activity of naturally occurring EPO both in vivo and in vitro.

Monkey DNA was isolated from a group of cDNAs generated using mRNA from monkey kidney tissue with chemically induced anemia, the monkey's serum containing high levels of EPO compared to normal monkey gloom. Isolation of the desired clones (DNA containing DNA encoding erythropoietin was performed using DNA / DNA colony hybridization using a mixture of 128 20-base radiolabeled oligonucleotides with rapid evaluation of 200,000 colonies formed. The status of the oligonucleotide samples was determined by amino acid sequence information in enzymatic fragmentation and sequencing in a small portion of human EPO.

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

Positive clones and plaques were verified by detecting the sequence of the LNA clone using an auxiliary set of 16 sequences for a 20-base sample mixture, and selected clones were subjected to nucleotide sequence detection, which resulted in the primary structure of EPO-type polypeptides for which this

DNA code. The polypeptide sequence thus detected was highly homologous to the partial amino acid sequence determined by amino acid analysis of human EPO fragments.

The selected positive monkey cDNA clone and the selected positive human cDNA clone were inserted into a DNA transfer vector that was propagated in E. coli and then used to transfect mammalian cells in culture. These cells were then further cultured, and up to 3 OCO EPO units in 1 ml could be detected in the culture supernatant.

The invention will be illustrated by the following examples, which are specifically directed to procedures that were performed prior to the identification of cDNA clones encoding the EPO monkey and human genome clones, to procedures that were to identify and sequence, to develop a system for expression and for immunological verification of EPO expression in these systems.

Example 1 is directed to the determination of the amino acid sequence in human EPO fragments and to the construction of a mixture of radiolabeled samples based on the sequence determination results.

Example 2 focuses on procedures for identifying positive monkey cDNA clones and provides information for animal treatment and for preliminary radioimmunoassay (RIA) of animal sera.

Example 3 focuses on the preparation of a cDNA mixture and assembly, colony hybridization, positive colony screening and verification of positive clones, detection of the DNA sequence in the positive cDNA clone, and confirmation of the primary structure of the monkey EPO (amino acid sequence determination).

Example 4 focuses on procedures involving the identification of positive genome human genes and provides information on the source of the genomic library, plaque hybridization, and verification of positive clones.

Example 5 focuses on the detection of a DNA sequence in a positive clone of the genome and on obtaining information about the amino acid sequence in human EPO, including its comparison with the monkey EPO sequence.

Example 6 shows procedures for constructing a vector with inserted DNA encoding human EPO derived from a positive cDNA clone, using this vector to transfect COS-1 cells, and culturing these cells after transfection.

Example 7 focuses on the construction of a vector with inserted DNA encoding EPO obtained from a positive clone of the human genome, the use of this vector for the transfection of COS-1 cells and the cultivation of these cells after transfection.

AND

Example 8 relates to immunological procedures using the supernatant from cell cultures after transfection according to Examples 6 and 7.

Example 9 focuses on monitoring the in vivo and in vitro biological efficacy of EPO, obtained by microbial expression in Examples 6 and 7.

Example 10 relates to the development of mammalian expression systems for monkey EPO 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 products obtained by expression using these systems and their properties.

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

on

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

The invention will be illustrated by the following examples.

Example 1

A. Determination of the amino acid sequence in a fragment of human erythropoietin

Human EPO was isolated from urine and digested with trypsin to give and isolate 17 separate fragments at approximately 100 to 150 pmol.

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

Sample no.

T4a T4b T9 T13 T16

T18

T21 T25 T26a T26b

T27

T28 T30

T31

T33

T35 T38

Table I

The result of anal.vz in sequence

A-P-P-R

G-K-L-K

A-L-G-A-Q-K

V-L-E-R

A-V-S-G-L-R

L-F-R

K-L-F-R y-1-e-a-k 1-i-c-d-s-r

L-Y-T-G-E-A-C-R

T-I-T-A-D-T-F-R

E-A-I-S-P-P-O-A-A-M-A-A-P-L-R

E-A-E-X-I-T-T-G-X-A-E-H-X-S-L

N-E-X-I-T-V-P

V-Y-S-N-F-L-R

S-L-T-T-L-L-R

V-N-F-Y-A-W-K g-q-a-1-1-v-x-s-s-q-p-wE-P-L-Q-L-H-V-D-K

B. Composition and construction of a mixture of oligonucleotide samples

The amino acid sequences listed in Table I were used with respect to possible degeneracy of the genetic code to determine whether a mixed sample procedure could be used for DNA / DNA hybridization to cDNA and / or DNA from the genome library. that fragment T35 contained a sequence of inherited amino acid residues (Val-Asn-Phe-Tyr-Ala-Trp-Lys), which could be encoded by one of 128 possible DNA sequences containing 20 bp. Therefore, a first set of 128 sequences of 20 pairs of bases was compiled in a standard phosphoamidite manner according to Beaucage et al., Tetrahedron Letters 22, pp. 1859-1862 (1981) on a solid support according to the following

Table II. Table II Residue - Val - Asn - Phe - Tyr - Ala - Trp - Lys 3 * OAA TTG AAG ATG CGA AAC TT T AND AND AND T G G

C

G

Further analysis showed that there was a sequence of six amino acid residues (Gln-Pro-Trp-Glu-Pro-Leu) in fragment T38, based on which a mixture of 128 17-pore oligonucleotides could be prepared according to the following Table III.

Table III

Residue

Gin - Pro - Trp - Glu - Pro - Leu

GTT GGA ACC CTT

C T c

G

C

GGA GA - 5 'T A

G

C

Oligonucleotide samples were labeled at the 5- ^{terminus with 32} P-ATP, 7500-8000 Ci / mol (ION) using N-polynucleotide kinase (NEN).

Example 2

A. Monkey pretreatment and RIA analysis

Female Macaca fascicularis monkeys weighing 2.5 to 3 kg at 1.5 to 2 years of age were given a pH 7 solution containing 12.5 mg / kg phenylhydrazine hydrochloride subcutaneously on days 1, 3 and 5 · Before each injection the hematocrit was checked. On day 7 or whenever the hematocrit decreased to 25% of the original value, blood serum and kidneys were isolated after administration of 25 mg / kg ketamine hydrochloride. The material was immediately frozen in liquid nitrogen and stored at -70 ° C.

B. PIA 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 sample tubes were cooled in ice, ¹² .mu.l of erythropoietin was added, and the tubes were incubated at 0 DEG C. for at least 15 hours. Each tube contained 500 [mu] l of incubation mixture, consisting of 50 [mu] l of diluted immune serum, 10,000 [mu] l pulses per minute of erythropoietin, 5 [mu] l of trasyl and 0-250 [mu] l of EPO standard or unknown sample, the rest of the volume was made up using PBS containing 0.1% BSA. The antiserum used was from a second experimental blood sample from rabbits immunized with 1% pure human urinary erythropoietin. The final dilution of antiserum was adjusted during the experiment so that the amount of ¹² μl-EPO bound to the antibody was at most 10 to 20% of the possible input pulses, which is generally the final dilution of antiserum in the range of 1: 50,000 to 1: 100,000.

125

The I-erythropoietin-bound antibody was precipitated by adding 150 μl of Staph A. After 40 minutes of incubation, the samples were centrifuged and the pellets were washed twice with 0.75 ml of 10 mM Tris-HCl pH 8.2 containing 0.15 M sodium chloride. , 2 nuu 3ΙΧΓΑ and 0.05% Triton X-100. The number of pulses in the washed pellets was determined to determine the percentage of bound labeled ε-ythropoietin. Pulses obtained for serum before 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 monkey serum chewed in Part A and to healthy monkey serum. Normal serum contained approximately 36 mJ / ml, whereas serum from pretreated monkeys contained amounts ranging from 1000 to 1700 mJ / ml.

Example 3

A. Construction of monkey cDNA library

For kidneys and anemic monkey mRNA was isolated using guanidiniumthiokyanótu described by Chirgwin et al Biochemistry 18, pp. 5294 (1979) and poly (A) ⁺ MSNA was purified twice by passing a column packed with oligo (dT) -cellulozy as described by Maniatis et al., Molecular Cloning, and Laboratory Manual (Cold Springs Harbor Laboratory, Cold Springs Harbor, NY 1982), pp. 197-198. The cDNA library was constructed according to a modification of the general procedure described by Okayama et al. Biol. 2, pp. 161-170 (1982). The key steps in the preferred procedure are as follows:

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

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

3) first strand synthesis and oligo dT binding was performed according to the publication,

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

5) RNA strand replacement with DNA strand was performed in the presence of two helper binding strands, GATCTAAGACCGTCCCCCCCCC and ACGGTCTTTA in a three-fold molar excess, relative to the oligo dG-terminated vector.

B. Colline hybridization to find the sequence in the monkey cDNA assembly

The transformed E. coli microorganism was at a density of 9,000 colonies per 10 x 10 cm broth plates containing 50 micrograms of ampicillin. GeneScreen filters (New England Nuclear Catalog No. Nef972 were pre-wetted on a BHI-CAM plate (Bacto brain heart infusion 37 g / l, casino acids 2 g / l and agar 15 g / l containing 500 micrograms Chloramphenicol) and then used to remove colonies Colonies were grown in the same medium for 12 hours or more to obtain multiple copies of the plasmid, and the colonies (colonies were processed upward by placing the filters sequentially on two layers of Whatman 3 MM paper, saturated with the following solutions:

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

2) 0.5 M HaOH 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 for two hours at 80 ° C.

The filters were then digested with Proteinase K by a solution of 50 micrograms / ml of this enzyme in buffer K [0.1 M Tris-HCl pH 8.0, 0.15 M NaCl, 10 mM EDTA pH 8.3 and 0.2% SDS is added. 5 ml of the solution is added to each filter and the digestion is carried out at 55 ° C for 30 minutes, after which the solution is removed.

4 ml of prehybridization buffer (5x SSPE, 0.5% SDS, 100 micrograms / liter SS E coli DNA and 5x BFP) is then added to the filters. The buffer is allowed to operate at 55 ° C, usually 4 hours or more, and then the buffer is removed.

Hybridization was then performed as follows: To each filter was added 3 ml of hybridization buffer (5x SSPE, 0.5% SDS, 100 micrograms / liter yeast tBNA) containing 0.025 picomoles per ml of each of the 128 samples listed in Table II (this mixture referred to as EPV mixture) 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, and then washed two to three more times 6x SSC with 1% SDS at a hybridization temperature of 48 ° C.

Autoradiography of the filters was able to prove that out of the original number of 200,000 colonies, seven colonies are positive.

The sequence of one of the monkey cDNA clones (designated clone 83) was then analyzed by modifying the procedure described in Wallace et al., Gene 16, p.

21-26 (1981). The procedure was performed by linearizing the plasmid DNA of monkey clone 83 cDNA with EcoRI and then denaturing by heating in a boiling water bath. The sequence of deoxynucleotides was determined according to the method of Sanger et al., P.N.A.S. (USA) 74, pp. 5463-5467 (1977). A subset of the EPV mixture, consisting of 16 sequences, was used as a primer for this reaction.

C. Determination of monkey cDNA sequence for EPO

Nucleotide analysis of clone 83 was performed according to Messing, Methods in Enzymology 101, pp. 20-78 (1983). Table IV summarizes the restriction enzyme analysis of approximately 1600 base pairs of EcoRI / Kind III digested clone 83. The approximate location of the restriction endonuclease sites was determined by determining the number of bases from the 3 * end to the EcoRI site near the 5-terminus of this fragment. Nucleotide sequence analysis was performed by determining the sequence of individual fragments after treatment with individual restriction enzymes so that overlapping fragments could also be detected. Thus, for example, overlap in nucleotide sequence analysis was found for the restriction fragment designated C113 (Sau3A at about 11 / SmaI at about 324) and the reverse sequence for the fragment designated C73 (Alu I at about 424 / BstEII at about 203).

The following table ΒΓ shows the approximate site of action of each restriction endonuclease.

T bulka IV

ÍO ·; L r · ΐ. k ·: 'ri' <íir. '.

Mí í; t.o pro ;; and reindeer * ·· her nearby location

EcoRI 1 Sau3A 111 Sma I 180 BstEII 203 Sma I 324 K ^ nl 371 Rsa I 372 Alul 424 Pstl 426 Alul 430 Hpa I 466 Alul 546 Pstl 601 PvuII 604 Alul 605 Alul 782 Alul 788 Rsa I 792 Pstl 807 Alul 841 Alul 927 Ncol 946 Sau3A 1014 Alul 1072 Alul 1115 Alul 1223 Pstl 1301 Rsa I 1343 Alul 1384 HindHI 1449 Alul 1450 HindlII 1585

Determination of the 1342 base pair region (in the region from the Sau3A enzyme site near the 3-terminus to the EcoRI and HindIII site) and analysis of the reading direction allowed to obtain information on the amino acid sequence and the relevant DNA as given in In this table, the starting amino acid of complete EPO (as verified by correlation with the aforementioned sequence of 20 terminal amino acids) is indicated by the number +1. The presence of the methionine-specific ATG codon (numbered -27) upstream of the terminal alanine residue as the first residue for complete EPO suggests that EFO expression first occurs in the cytoplasm in the form of a precursor that contains a 27-residue leader region. amino acids that are cleaved before EPO enters the bloodstream. Potential glycosylation sites are indicated by asterisks in polypeptides. The determined molecular weight of the transferred region was determined to be 21,117 units and the molecular weight of 165 polypeptide residues constituting complete monkey EPO was determined to be 18,236 units.

O

> N cfl O C. c xS n * r-l O. . 1 n what Φ F-t f> ω C ω to AND

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Gly Cys Ser Glu Ser Cys Ser Leu Asn Glu Asn Ile Thr Val Pro GGC TGT TCC GAA AGC TGC AGC TTG AAT GAG AAT ATC ACC GTC CCA »1

- 51continued table

□ et O 1— □ ι- CO CJ a> cj · -) CD > -t et l- <CJ at 1— —L CJ * - <h- CD CD The CD 0. CJ _l CJ d CD ►m d * - * O m cr C CD > .CJ □ d M O CO 1— QJ o * —T d - <CD <—I d X O > CD CO 1— CD CJ CD CD CD CD H · d □ cd O Σ3 CJ Im CJ O Im Ι- C CD About CTd · —L d κ <di- a> cj Ο CD CD mm d KS A cd CD CD -Yeah co 1— - · CO d CD CJ —1 d CJ 4-J CD □ CD Im 1— OJ CJ CO CJ □ CJ ω t- <U 1- CD CJ —L 1— Ή CJ 0) t- X d _l CJ to ι- ι-t d d CD _J CJ ctcd CO CJ is o CO CJ xd About d Im a CD - * CJ * tn d rM CJ -i CD Im U et al and CD d d d CD CD CD tt. Cj tn CD □ CD CO CJ tn d □ CD CO 1— Xd 0) 1- —1 CJ > <d ID 1— rM CJ _J « _J CJ d CD _i d _l CJ d CD O. CD X (J The CD 0.1- CO CD <0 ►— Im O —L CD CD 1- <n d <- <CJ Ή CJ t— t- CD CD _J 1- d CD d CD d CD O CO CJ C CD O mM CD M-) CD About CTCD M CD J <-t CJ <-m et oo CO 1— 0) 1— "I have a CD." 0) CJ «I CD In CJ > CD X d HdU CO Ι- Im h- o. cd CO CJ tn cj □ 1— Ο o Xd Im a CD —1 CJ or d <D 1- -H CJ 1- Ι- 1- 1— ct CD X CJ _l CJ d CD Ο) CJ · —1 CJ C CD □ CD The CD The CD X Ι- CD 1— -i d a) i- OJ h- · —1 CJ Ο- Ι- > CD CD CJ _l CJ _l CJ d CD and CJ □ CC >> CJ C CD Im 1- Q.I- tn d —I CC * —1 CD r-M d X CJ cn d et cc CD CD CD CD CD CJ d cc cd · —T i- <-l d ctcd □ CD (M CJ o d CO 1- CO 1— Im a CD <D H- X o • m o > CD > CD d cj _J CJ d a. cj tn cr O CO H □ CD O O CJ with cj O □ CJ > .d V0 · —1 CJ ID 1— Os Im CJ · —1 1— CM 0) 1- _i <£ CC CD _l CJ 0- CJ l-t d —I _j cj Im CJ C CD <- <CJ □ CD ι-t CJ Im CJ X CJ > -t d CO I— Ή d ω cd 0) CJ I- d CD CJ > CD CD CD in d tn ►— About CJ C CD CO 1— <D CJ CTO a> cj tn cc and d -t CJ jz ί- Im a cd ř — t 1— and CD CD CJ d CD α. 1— d cj »-I d

Thr Ala Asp Thr Phe Cys Lys Leu Phe Arg Val Tyr Ser Asn Phe ACT GCT GAC ACT TTC TGC AAA CTC TTC CGA GTC I AC TCC AAT TTC continued table

O CJ O O Ι- CJ F- F- O O CTet ct ct ct CJ Ο Ι- (- F- ct et F O et ct ct et O Ο O et O O ct et CJ Ι- CJ CJ et O «X F- ct cc O Ο CJ et O ct CJ <t O CC cto et CJ et O et F- Ι- Ct Π- O F O CJ et CJ O et F- Ο O Ι- Ι- CC Ι- CJ ct O et F- F- et Ο Ο Ο Ι- O CJ <t «T O O Ct O O ΙΛ O CJ Ο O O CJ <t O et i- O CJ Ι- CJ CJ CJ O CC O ο Ct O I— Ο CJ ct O CJ et h— O ct CJ CJ O CJ Ct CJ O O O O ro o CJ CJ CJ Ι- O <t O et Ct ct r-t o et CJ et Ι- CJ O O O O O <t O CJ CJ O Ο F- CJ TO- ct O CJ et l · - et ct F- O CJ π- O 3 O ct CJ O ct ct F- O F- ι- ct - <CC CJ O AND- O O Ι- ct IN- ο O o o CJ F- ct O ct Ο 1— O O O t— et O CJ CJ CJ Ι- O et O > .O ct O et CJ ct O Ο O O F-. <- · o CJ O CJ CJ CJ CC et O O ct o o Ct O F- et ct O ct O O CJ O Ct O O CC O CJ F- O F CJ CJ Ct ct CJ CJ CJ et cr F- O _C o CJ ct O O et CJ O O 1— Ι- F- et CJ CJ CJ Ι- CJ et O ct O Ο AND- O ct Ο CJ O Ι- et O O F CJ CJ O O F- ct CJ Ο O et et >, <t CJ O O ct O F- ct O O O f- f- et Ι- CJ CJ Ι- «T O O et O CJ Ο F- CJ Ο CJ F- Ι- O Ι- 3 CJ CJ et CJ et CC F- cr Ο O Ο <U F— F- O Ct Ct ct F- O O O O _YEAH O O O Ι- ct 1— O F- ct O CJ et O Ο F- et et Ι- ’F— O t / J o O O i- F- F- ct O Ο F- et >, ct F- Ι- ο ct F- ct et O <t i- _ | et CJ Ο ct CJ O et CJ O ct ο CJ O O ct O O O Ι- ct O 3 CJ et O CC CJ ct CJ O Ο CC O UJ ►— CJ Ι- O Ι- O h— ct ct ct O _J CJ CJ Ο CJ Ο CJ O 'O O O O O t— Ι- <t O F- O O O· (Λ CJ ct O et Ο CJ ct <t O ct ct >. CC 0. C) Ct O ct ct O O O O O _j et About Ι- O O Ct CJ O O O O O ct O O ct CJ CC et et ct o >> ec in CT ct O IN- ct O CJ 1— O O ct m —4 o .CO U CJ O CJ O F- O O O O —I O O —4 Ct Ct CJ Ι- CJ F- Ι- O O Ι- O Ι- Ο ct Ct Ο <t O Ο O CTO 0.0 Ο O CJ O et O O F- O F O (Λ ct Ι- CJ ct CJ O O O ct Ι- et o ct CJ Ο 'ct •O ct Ι- F- ct et Ο O O O et Ο F- ct O F- 3 CJ > U CJ O Ι- CJ CJ t- ~ O O O 0) Ι- r-4 CJ Ι- CJ Ο O ct F- Ι- F- Ι- Ο CJ CJ CJ Ο O F- et CJ 1— Ο F- Ο

AGGATGGGGGCTGGCCTCTGGTTCTCGTGGGGTCCAAGCTT

HindlII

The polypeptide sequence listed in Table V can be readily analyzed for the presence of highly hydrophilic regions and / or analysis for secondary structural properties that are also potentially high, for example. immunogenic regions, for example, the method of Hopp et al., PNAS (USA) 78, pp. 3824-3828 (1981) and Kyte et al., J. Mol. Biol. 157, p.

105-132 (1982) and / or Chou et al., Biochem. 13, pp. 222-245 (1974) and Advances in Enzymology 47, pp. 45-47 (1978). Computer-assisted analysis can be performed using the PEP Reference Sectio 6.7 range, Intelligenetics Inc., 124 University Avenue, Palo Alto, California, USA (method according to Hoppe et al.).

Example 4

A. Human genome assembly

The human fetal liver genome assembly, carried by Cn4A phage, was obtained as described by Lawn et al., Cell 1z. 5s3-943 (1979), the assembly was then maintained for use in plaque hybridization.

Β. Plaque hybridization as a method for searching for sequences in the human genome

The phage phage sections were disrupted and the DNA was fixed on filters at 50 OCO plaques per filter as described in Woo, Methods in Enzymology 68, p.

389-395 (1979) except that GeneScreen Plus filters (New England Nuclear Catlog No. NEF-976) and NZYAM plates (NaCl 5g, MgGl 2 · 6 ^ 0 2g, NZ-Amin A 10 g, extract) were used. from yeast 5 g, casamino acids 2 g, maltose 2 g and agar 15 g per liter).

The air-dried filters were heated to 80 ° C for 1 hour and then digested with Proteinate K according to the method of Example 3, Part B. Preliminary hybridization was performed using 1 M NaCl, 1% SDS buffer for 4 hours at 55 ° C. C or slightly longer, after which the buffer was removed. Hybridization and post-hybridization treatment were performed in a manner similar to Example 3, Part B. Both a mixture of 128 20 bp samples, designated EPV, and a mixture of 128 17 bp samples from Table III, designated EPQ, were used. Hybridization was performed using the EPV mixture at 48 ° C and using the EPQ mixture at 46 ° C, i.e. 4 degrees below the lowest calculated Td value for the mixture components. Removal of the hybridized rehybridization sample was performed by boiling with 1x SSC, 0.1% SDS for two minutes. Autoradiography of the filters showed the presence of three positive clones (which reacted with both samples) from the group of the original 1,500,000 phages. These positive clones were then verified to encode EPO by LNA sequence determination and electron microscopy, where the structure of the monkey cDNA of Example 3 could be demonstrated. The presence of introns in the DNA genome sequence could also be demonstrated.

Example 5

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

AND

d O O O O and O X ω O O ω O P- »- O X O O O O et 0 O O -cr ω 0 O to- et O O ω C3 O CN · Η et O O O O et 0 O ct 1 χ ω CJ cZ et O O 0 O ω O (_) O et et 0 O P- -π C3 > - O O O O P- O <t (0 Ρ- O O O P- O 0 CC O > Ο ►— O et O O 0 O O O O O to- <t 0 et ω > Ο d , - p- O O 0 O O ω O α et O O P- O O ω ω Ι- C3 O ρ- O 0 O O Ο AT O ω and ω P— > - r ~ 4-> ω > - et O O O 0 0 p- CN QJ Ρ— ω O O O O 0 0 O 1 X et »- O O O O 0 , - O d <= c ω O et 0 Ο et O ►— et p— O 0 P- O Ο α ct O O O 0 O O et O ω O O O 0 O O Ο d ct et ρ- O 0 X O Ο O at O ω O ct 0 O ω O O O O et 0 0 O ο at O O O O 0 0 ct ω d ►— O and O ω 0 O ω d the O et O 0 0 O et O the O O O 0 0 Ι- Ct O Ο O O O P- 0 Ο ω d O O O p— O 0 O ω at O O et O O ct O ω ►— et et et Ι- O O O ο O O O O Ο Ι- O O ω d O O P— O Ο O P- ω d O O O O <í O O ω O O ct O et 0 O O ω O and O O θ ' CC O O cC (_) O O et et X O O 0 O O O O O 0 O O ω J— O O O et 0 ct p- ο > - O O O O 0 O 0 ct Ι- O O O O 0 O 0 Ο Ο ρ- p- O O cr O ω ř— d ω O O et 0 O 0 ω h- ρ- O O O CC O 0 ω C_) ω O O i- O ct ω ω O O O O Ο O 0 ct ρ- d CC P- O. O O 0 ρ- ω CJ O O et 0 O 0 ω ct O O et O 0 O 0 ρ- CC O and O ρ- ct O 0 ω ω d O O ω 0 O p— O ω O cc Ι- O 0 0 0 ρ- ω d O Ο O 0 0 0 ω ω O O O O 0 ct (- ρ- ω O O O O l · - O 0 ω ω ►— O O O 0 O p- ω ω h- O O O 0 O 0 ω ω O O ct CC 0 O 0 ω ω, O O et O 0 O 0 ω ω O P- O O ct O 0 ω ω O cr O et Ι- ct 0 ω ω ι- O O O Ο O 0 Ct ct Ο O O O O CC d ω ω H- O Ι- Ι- 0 CC 0 et ρ- J— O Ο Ο 0 p- 0 ω ω Ό O O Ι- and cc 0 ω ω 'O «X O Ο 0 O 0 Ρ— ω d O et O p— et 0 > - ω d ct et et 0 O 0 ω ο

GTGAGTACT.CGCGGGCTGGGCGCTCCCGGCGGCCGGGTTCCTGTTTGAGCGGGGATTTAGCGCCCCGGCT

Ο ο · 1

CJ □ <r (X | - 1— t— ►— CJ et CD —Í ct . > - <x J— CJ CJ <x ct CD co co 1— ι- «X what CJ O O d CJ what CJ Ι- O ο About ffl Ι- ω cj 1— et CJ Ο et (_> *Α * “H CJ C H- CJ WHAT et cr O Ο β o Q_ t- 1— ►— WHAT AND- O ο et «X Ι- 1— O ο ω t- C t- Ct what Ι- <r O ►— >> C0 (Λ et what ct Ο cr cr C3 cj * - cr et ex CJ CJ cr O ►— CJ <t 1— cr Ι- >> '(_) <—I i— CJ CJ 1— cr Ι- Ο —And what CD 1— AND- CJ et «X Ο ο co co > CO t— ct et ct O ο t— CJ CJ 1— O d Γ L. CO tn eí t— CJ <X 1— 1- ►— CM XZ O >> et CJ ct O 1— eX ο t- cr _i cr t— CJ CJ et O f * 1— what CJ O O υ.j h- CJ 1— (- 1- <X Cl d C CJ CJ CX «X cx O ο t— a: CJ <t CJ «X O d t- «X «X O O ο what Q.CJ t— et CJ cr O CD cr ΙΛ ex t— i- O Ι- O d O and CO t— ο O Ο O (J and- 1— what CJ Ι- ct t— o ct 1— et CJ Ο O ο H- (H CJ 1— ex 1— 1— i- cr cr 0. CJ 1— «X et <x ο d O 1— WHAT O O O cr what > -t CJ (- what et O O ο i- CD 1— t— what WHAT O «X ο Ο > CO t— what Ι- O cr cr Ι- 1— what Ο O O ο Ο O 1-, t— (- CJ WHAT 1— O ο Ι- <i r u 1- t— what «X 1— cr ο h- et CJ ct cr O 1— d what CJ what O ct 1— ο what O) CJ t— 1- O O ct ο what - <►— 1— ct O Ι- AND- Q O · - »<x CJ ct 1— Ο et d O «X WHAT et ct O Η- O C t- CJ cr et h— ct ο O ♦ w cr 1— what 1— Ct O d 1— ex cr CJ CJ el O O d O CJ ι- O O d et about what IA □ CO et Ο ct Ι- ο what Cr lA «—l et et Ι- O Ο ►— ►— about what CO CO «X Ο O ct (_) »- CJ O O H- CJ what C h- 4-> CO 1— et ct O d O tn <s. OJ I— cr O fr— O μ- O cr cr Σ ex CJ i- O Ι- d et CJ ο O Ο ο what □ co cno AND- what et O ο what oj »- Ci co t— CJ ct 1— d what _J 1- CX «X 1- cr O <x d et 1— CJ O O d CJ l-l CJ tn CO et <x et O CJ AND- <u CO >, cr CJ O O Ι- O i- IZ> <x _l ct 1— CJ <t Ο O ο CJ O O O O (_) tn CJ CL CO CJ what 1— Ι- O what >> co Who what CJ Ι- Ο O cr CJ 1— t— t— et what Ι- ct Ι- what WHAT Ι- Ο ct Ο ct W CJ o co cj CJ Ο cr O O O • H eí IA · —1 O what O O Ι- 1— O X CJ ex CO t— what O Ο

TTTGAGGCTGCAGTGAGCTGTGATCACACCACTGCACTCCAGCCTCAGTGACAGAGTGAGGCCCTGTCTCA. 65 _

»- what <0 k— □ co Π Π- X to- to Π- ο O - <o CD k— b) H— what O —I, ο d π- x co _l CJ _l CJ what O x co ο ι- what what Ι- ►— ο X □ X O O CJ □ co what Ο to Π- ο O i- —1 x C CJ CD k— X O rd O d · to- ο co co CL CJ _J CJ X X X CO ο WHAT to- what what Η- WHAT <_> d CO □ co d k— X O d X ο t— O ω o · —1 X -C CJ X what CD O d t— <_> CZ) k— co co k— X π- X what π- ο O Ι- ω O Η— O Ο O □ co CL CO d CJ π- what I o ►— X X r- ~ <U k— d co -C. CJ ω Ι- r-k O d O O _l o Η- ί- i- x H— Ο X co ο i- O to- X Η * α to- □ co about what □ CJ Ι- O to co > - what O ω k— li CJ <D k- Ο what rd o d to- X _l o Q_ CJ _l CJ O . what X co Η to- what O to- Η— to- X to o c co d CJ what what Q-k- Ή d WHAT O rd o r-l X (D CO to- to- ω x £ ο i- O X co CO CJ (Z) X Ι- O X co Η— α O Ο to- ►— Π- to- □ co d CJ CTCJ what π- or X 0 d Ι- O <D k— CD CJ d CO Ι- ι- d o CJ Ο X _1 o cn k- X CJ Ο ο CL O d O what π- O £, ►— Ct O >, <_> d k— — K— ι- O About H- Ý1 d to- to- rd CO CD CJ (D H- ο π- d O 0 d X O co co cz> n- _J CJ X ι- CL O Ο O O O ο * ' ο WHAT X c co C CJ >> CJ X O O d O Η- to- O —1 x ♦ ωχ n CO O to- CN <D O d to- Ι- co O X X co co what to- cn π- AND Η- X Ο O X 1 Ο to- O CL CO r-l CJ O d Ι- what Ι- ω o ►— to- O d co CO k— Ο DO X Ο —1 H- ο O and- k- H- > co —1 tZ) X what O kd X _ι ο Ι- X O O ο Ο O rd c_) □ CO rl CJ X π- to o ο x X to k— CD k— to k— π- ω rd O d ct O > co _l Ι- > co ω to- x co ο O Ι- X O Η- <£ Ο □ X Ο co to CJ what X □ X »Υ d X what n X CO CD k— n CJ X —Ι X * · d O what co co _Yeah X co what what co co ι Ί Η- to- X what ¹ d to- and —I X CO CJ ω x m c co X what ω co • ' <£ X X what k— rl CJ >> x »—4 O >> x d O O > co X co _J X -1 O O X -Η _i x t 1 d t— O π- d > - what O (0 CJ c co CLk— CO O ω C3 «X X NO —1 CJ r — 1 X ω x rl CJ X d O O X co CO CJ X co X co what d to- O what what d to- X c what > O rd CO > .x X X d X O rl X r-l CO what i— —L co X O d O O CO CJ co co > co ω co Ι- what d to- what Ο what O to- what c co co co ω π- about what what Π- d X what - X d a —K X CD t— Π- Ι- d O X co o X CJ X CJ _Yeah Ι- Ο d to- what Ο O d to- Ι- > -co □ co about what what i— Ι- Ι- O X Ο r-l co <D k— (D Π- n O Ο Ο d O what co co _J CJ _J CJ X co what to- d Ι- Ι- what to- d Ο · Ο what r-k CJ r-l CJ c co About CTCO X to- ct X what m co k- co n- rl X n d CO X 1— d O what > co > co CO CJ —I X o what what

Table 71 - continued o

a o

Li o > -CD ex O O CJ ο Ο Ο α a> o —1 CD . Ο O «X CD α CD α α IZ) t— CD O «X Ι- O μ- μ- α CD CD Ο Ο t— α μ— ο α μ- in o t-ι tí ο «X CD O μ- ο α ο >> <x X O Ο O μ- O α μ- Ο α t- t- t- β Ι- « α: CD Ο ο μ- ο Ο CD CD α Ο μ- α α • -I CJ cn cd μ- CD CD CD Ο μ— ο ο CO I— C-. CD μ- > - <X α Ο ο ο α > O «I« X ο «X ex O α α α CD Ο CJ μ- α CD CD < μ- σ> α (Λ CJ t— O O CD CD μ- CD ι- In the CD > .CD μ- O μ- CD μ— Ο Ο α α cj About μ- α μ— «X CD μ— μ- CD μ- CJ CD CD α Ι- Ο Ι- ο O) u Ο CO CJ ex t— ex O Ο Ο Ο ο JC h- Ό —l CJ «X CJ CD μ- Ο α CD α D. h- - <x o ο CJ Ι- O Ο α μ- α CJ CD Ο α α ο Ο ο □ CJ □ CD ex <x μ- α ο CD ο μ- <l) H- - <«X O CJ O α α Ο ο Ο _j α CD CD i- CJ ex μ- Ο μ- ο μ- ο CD <X t- Ο Ο ο μ- w α > - .CD O O O μ- α Ο ο α >. « -i CD O CD CD ο CJ α α μ- _J α CD CD μ- (X ex CD CJ μ- ο ο O CJ CD α Ο μ- α μ- COCJ μ. α O Ι- <X ο μ- Ο ο ο P o SW CJ ex Ο CD CD α α μ- μ- c £ CJ 1— α O CD ex α Ο α μ- μ- O α O Ο μ- CD ο μ- <u o In CJ α O O α μ- α ο μ- r μ- > ^ <X O i- μ— CD μ- Ο ο ο Ο. t- 1—> - O ο CD α α Ο ο α μ- μ- μ- ο α μ- ο α K 1— □ CD α ο ο μ— α ο ο ο .c cj CD Ι- μ- CD «X μ- α ο α ο 1— «X Ο CJ α CD «X α CJ α ο μ- O CD CD ο Ο CD α ο DUTY ω cd CD CD CD CD μ- μ- α ο ΙΛ α CD ex ex α ο α ο ο α CD _l <X CD CD CD α • CJ Ο ο ο i- O ex Ο ο CD ο μ- CO h- D CD ο t— O CD α α μ— α · —1 O a> t- O CD O α ο ο CD ο CD _J CJ ex O CD ο Ο α Ο Ι- O O CD CD Ο CD μ- Ο Cí> - on CD O O CD α Ο Ο »- μ- jC O >> <x to- O CD Ο μ- Ο ο Ο 1- α _J α CD < <X α ο α ο ο μ- «X O ο α ο ο ο 0) o >> <x O CD μ- ο ο μ- μ- μ- —H μ- r-4 CD μ— Ι- Ο ο μ- α ο ο l-l <t CD CD CD Ο μ- CD ο ο α μ- CD O α Ο Ο ο ο Ο M α O O CD ex μ— O CD α α ο Ο r: o ιΛ M CD O O α α CD μ- ο Ο μ- <X —Ι «X CJ O «X CD α μ- Ο CD Ο O i- CD ο Ο μ- μ- cna □ CJ «X O α μ— ο α α ο CDs oj μ- O. CD CD α μ- Ο ο ο ο «I o Ο CJ o μ- O O ο Ο Ο ο ο O CD α α Ο the * ο □ o ω cj vO C3) <X O α α Ο μ— CD ο 0? AND- XZ h- Μ) μ · cd O O ο α μ- Ο X _l o α. μ- '-I ex ex O O ο α μ— α μ- CD CD Ο μ- ο α o <t ε μ- CLU O TO· CD Ο CD μ- ο ti O ω α (Λ «X O CD Ο Ο μ- ο X CL O <χ ex «X CD O α α μ- ο μ- α

ACACAATATGAC

In Table VI, the initial continuous DNA sequence is the 620-base upper sequence, which appears to be the untranslated sequence immediately preceding the transferred portion of the human EPO gene. The sequence appears to contain the 5-terminus of the gene and then the transferred region of DNA, which encodes the first four amino acids (-27 to -24) of the leader or preliminary sequence. The four pairs in the sequence before the sequence, which is the code for the beginning of the leading sequence, have not been unambiguously determined and are therefore marked as X. Then

639 is followed by an intron of two base pairs (439 base pairs from this sequence were identified, the remaining 200 are designated I.S.) and then a codon for glutamine, which was designated position -23 in the transferred polypeptide. The exon sequence immediately following encodes amino acid residues from the bacon residue (designated residue +1 of the full-length human EPO amino acid sequence) to the codon for theonine 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 to 55 and then a third intron of 612 pounds bases. The following exon codes for residues 56 to 115 of human EPO, followed by a fourth intron of 134 bases, as indicated. It is followed by the exon, which codes for residues 116 to 166, and the stop codon (TGA). Table VI shows a sequence of 568 base pairs in which the untranslated 3-region of the human EPO gene appears to be located, 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 complete human EPO, including 166 fully specified amino acid residues (determined molecular weight 18,399). As shown in the table, the diss sequence DNA encodes a 27-residue signal sequence along with 5- and 3-sequence DNAs that may be important for the promoter / operon function of the human gene operon. Sites for potential glycosylation of whole human EPO polypeptides are indicated by asterisks in the table. It should be noted that the specific amino acid sequence listed in Table VI is likely to be the sequence of the naturally occurring allelic form of human erythropoietin. Support for this claim can be found in the results of systematic studies of urine-isolated sequences of human erythropoietin, which have shown that a substantial number of erythropoietin molecules contain methionine at position 126, in contrast to the sherina shown in the table.

Table VII shows the extent of sequence homology between human and monkey erythropoietin. In the upper continuous line, the table uses the letters used to indicate the sequence of human polypeptides after translation, starting at position -27, the lower continuous line listing the sequence for monkey erythropoietin, starting again at the position indicated at position -27. Asterisks are used to illuminate the homology of individual sequences. It should be noted that human and monkey EPO sequences show an additional lysine (K) residue in human erythropoietin 116. A comparison of Table VI shows that this residue lies at the junction of the genome sequence (mRNA). The presence of a lysine residue in the human polypeptide sequence was further verified by detecting the human cLNA sequence in a clone obtained with mRNA isolated from C03-1 cells. transformed with human genome DNA, as described in Example 7 below.

Example 6

The expression system chosen for the initial attempts to perform microbial synthesis of isolatable amounts of erythropoietin, which is the code of the monkey

the relevant cDNA and which is given in Example 3 was a system in which mammalian cells (i.e. COS-1 cells, ATCC No. CRL-1650) were used as hosts. These cells were transfected with a vector that allowed both autonomous replication in E. coli as a host due to the presence of DNA derived from plasmid pBR322) and replication in mammalian cells (due to the presence of SV40-derived DNA).

In more detail, the expression vector was constructed as follows. Plane clone 83, in Example 3, was amplified in Ξ. coli with DNA of approximately 1.4 kilobases, which encodes monkey erythropoietin, was isolated by digestion with EcoRI and HindIII. Separately, a fragment of pBR322 of approximately 4.0 kilobai was isolated using 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 enzymes. This fragment contained EcoRI-digestible cleavage, followed by SstI, SmaI, BamHI and XbaI digestion pD sites and SalI-digestible cleavage after SalI digestion. All three of the above fragments were ligated to give an intermediate plasmid (pERS) of approximately 5.4 kilobases in which the DNA for erythropoietin was flanked on one side by a group of sites for various restriction enzymes. Plasmid pERS was then digested with HindIII and SalI to obtain DNA for EPO and the EcoRI to SalI binding strand (M13mp10). The 1.4 bp fragment was ligated to a 4.0 kilobase fragment of plasmid pBR322 after digestion with BamHI and SalI and to another binding strand obtained by digestion of M13mp10 with RF enzymes HindIII and BamHI of approximately 30 bp. This second binding chain from M13 had a re-binding site at one end after the HimdlII enzyme, followed by sites at the PstI restriction enzyme,

SalI, XbaI, followed by termination with BamHI digestion with the possibility of resuspension. The binding product was again a useful plasmid (pBR-EPO), which contained DNA for j

erythropoietin, which was bounded on both sides by 1 chain with a number of sites for the action of various restriction enzymes.

The vector used to express erythropoietin DNA in COS-1 cells (pDSVL1) was designed to allow selection and autonomous replication in E. coli.

These properties of said vector are ensured by the origin of replication and the DNA sequence, which encodes the ampicillin resistance, which is located between nucleotides 2448 and 4362 in the sequence of plasmid pBR322. This sequence was structurally modified by the addition of an auxiliary binding strand to create a site for the action of the restriction enzyme HindIII immediately adjacent to nucleotide 2448 prior to incorporation into the vector. Among the useful properties of the chosen vector, it is necessary to mention in particular the ability of autonomous replication in COS-1 cells and the presence of a virus promoter sequence functional in mammalian cells. These properties are provided by the DNA sequence for the origin of replication and the viral promoter sequence, which is 342 bp in length and extends from position 5171 of the sequence to position 270 of the SV40 genome. There is a single restriction enzyme site (BamHI) in the vector, and a commercially available binding sequence (Collaborative Research) is bound in close proximity to the viral promoter. The vector also incorporates a sequence of 237 bass pairs (nucleotide position 2> 53 to 277C HV4C) containing: u <, A viral lysing signal (usually indicated by the transcription terminator. This fragment was placed in the correct orientation with respect to the vector). Another mammalian gene is present in the vector at a position that is not critical for the potential transcription of the gene, which is a single BamHI site, between the viral promoter and the termination sequence (this gene was not a gene of approximately 2500 feathers is based on mouse dihydrofolate reductase (DHKF), isolated from plasmid pMG-1 as described in Gasser et al., PNAS (USA).

79, pp. 6522-6526 (1982)). Main operational components »

of plasmid pDSVL1 are at nucleotides 2448 to 4362 of plasmid pBR322 together with nucleotides 5171 to 270 (342 bp) and 2553 to 2770 (237 bore) of SV40 DNA.

Following the above procedures, as described, for example, in Maniatis et al., DNA encoding erythropoietin was isolated from plasmid pBR-EPO as a fragment after BamHI digestion 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 the plasmid pDSVL-MkE is shown. Vectors with EPO genes in the wrong orientation were maintained as negative controls for transfection experiments in which the expression level of EPO in a host transformed with vectors containing erythropoietin DNA in the correct orientation was determined.

Example 7

A. Initial EPO expression system, COS-1 cells

The system that was chosen for the initial experiments with microbial synthesis of isolatable amounts of human erythropoietin, which encodes the human genome DNA, also included expression in mammalian cells as host cells (i.e., COS-1 cells, ATCC No. CSL-1650).

The human EPO gene was first subjected to initial cloning in a vector capable of autonomous replication in both E. coli hosts (due to the presence of DNA from plasmid pBR322) and in the mammalian cell line COS-1 (due to the presence of SV40-derived DNA ). This erythropoietin gene vector was then introduced into COS-1 cells. The EPO-type polypeptide material was then produced by the cells after transfection and secreted into the medium in which the cells were grown.

Thus, the expression vector was constructed as follows: DNA isolated from the lambda clone> hE13 containing the EPO gene of the human genome was digested with the restriction enzymes Bamtil and HindIII, and a 5.6 kbase fragment known to contain the complete erythropoietin gene. This fragment was ligated to the bacterial plasmid pUC8 (Bethesda 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 to express EPO DNA in COS-1 cells (p3V4SEt) was constructed previously. It is a plasmid that contains a sequence of DNA that allows selection and autonomous replication in E. coli cells. These properties are provided by DNA sequences for origin of replication and ampicillin resistance, which are present in the region extending from nucleotide at position 2448 to nucleotide at position 4362 of bacterial plasmid pBR322, which sequence was then structurally modified by adding a binding site with a site of action. of the restriction enzyme HindIII immediately near position 2448. Plasmid pSV4SEt is also capable of autonomous replication in COS-1 cells. This property is provided by a 342 bp fragment containing the origin of replication from the SV4O virus (nucleotides 5171 to 270. This fragment was then modified by the addition of a gcoRI restriction site in the immediate vicinity of nucleotide 270 and a binding chain with a site for the action of SalL in the immediate vicinity of nucleotide 5171.

A 1061 bp fragment from SV40 virus (nucleotides 1711 to 2772 with a binding chain with a SalI enzyme site in the immediate vicinity of nucleotide 2772) is also included in this vector. there is a single BamHI restriction site in this fragment. Overall, plasmid pSV4SEt contains a single BamHI and HindIII site, thus allowing the incorporation of the human erythropoietin gene, sequences allowing replication and selection in E. coli, and sequences allowing replication in COS-1 cells.

In order to insert the pro-human erythropoietin gene into plasmid p3V43Et, plasmid pUCS-HuE was digested with BamHI and HindIII, and a 5.6 kbase fragment of human erythropoietin DNA was isolated.

Plasmid pSV43Et was also digested with BamHI and HindIII, and a major fragment of 2513 base pairs was isolated that contained all the necessary functions. These fragments were mixed and ligated together to form the resulting vector, pSVgHuEPO ”, as shown in Figure 3. This vector was propagated in E. coli and the DNA of this vector was isolated. Restriction enzyme analysis was then used to confirm the incorporation of the EDO gene.

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

B. Second pfco EPO expression system using COS-1 cells

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

In the previous system, EPO expression in CCS-1 cells was achieved using its own promoter, located in the restriction fragment region after treatment with 5.6 kbaza BamHI and HindIII. In the following construction, the EPO gene was altered so that it was expressed using the 3V40 virus promoter.

The cloned 5.6 kbp EPO human genome restriction fragment after digestion with BamHI to HindIII was modified as follows. Plasmid pUC8-HuE, described above, was digested with BamHI and BstEII. The restriction enzyme BstEII cleaves a 5.6 kbp EPO fragment at a position located 44 pairs 5 * to the beginning of the ATG code for the preliminary peptide and approximately 680 pairs 3 * to the HindIII cleavage site. A fragment of approximately 4900 bp was isolated. A binding fragment with ends capable of binding at SalI and BstEII digestion sites with a BamHI cleavage site within the fragment was also synthesized and purified.

Both fragments were mixed and ligated into the plaanid pBR322, which was digested with the restriction enzymes SalI and BamHI to give the intermediate plasmid pBRgHE. The EPO gene of the human genome was then isolated as a 4900 bp fragment after digestion with BamHI, the fragment contained the complete structural gene with a single ATG at 44 bp 3 'to the BamHI site near the end of the coding region with an amino group at the end.

These fragments were isolated and inserted as a fragment capable of binding after BamHI digestion into the plasmid pLSVL1 described in Example 6 as an expression vector digested with BamHI. The resulting plasmid pSVLgHuEPO, which is shown in Figure 4, was used to express EPO-type polypeptide material in COS-1 cells according to the method of Examples 6 and 7A.

Example 8

The culture medium in which COS-1 cells were grown (six cultures after transfection according to Example 6) was analyzed by radioimmunoassay according to Example 2.

Part B. The individual samples contained 250, 125, 50 and 25 .mu.l of the indicated medium. Culture supernatants that contained vectors with incorrect EPO gene orientation were i

clearly negative. For samples of two supernatants from COS-1 cell cultures after transfection with vectors (H and L) that contained EPO DNA in the correct orientation, the percent inhibition ranged from 125 _χ . _{Ερο vé2 guided ns antibody} " _ro2 .

between 72 and 88%, so all values were at the top of the standard curve. The exact concentration of EPO in the culture could not be easily determined accurately. From a sample containing 250 .mu.l of supernatant, the EPO content was estimated to be at least 300 units / ml.

The supernatant obtained from the culture of Example 6 and the five- and seven-day-old cultures of Example 7A were examined by RIA to compare the potency of recombinant monkey and human EPO and naturally occurring EPO as a standard, the results are shown graphically in Figure 1. . As expected, recombinant simian EPO competed with the anti-human EPO antibody, although the inhibition of hiss was not complete under the experimental conditions. However, the maximum percent inhibition values for recombinant human EPO were close to those for standard human EPO. Because the dose and response curves run parallel, it is possible to judge the immunological identity of the 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 amount of human EPO detected in the sample after culturing for five days was 392 units per ml and 567 units per ml in a sample that was cultured 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 prepared according to Examples 6 and 7 was tested in vitro for EPO efficacy as described in Goldwasser et al., Endocrinology 97, No. 2, pp. 315-323 (1975). The values determined for monkey EPO in the culture medium of the examined cultures ranged from 3.2 to 4.3 units / ml. The culture medium containing human EPO was also effective in this monitoring, and in addition, the effectiveness of these media could be neutralized by an anti-EPO antibody. Sultax The monkey EPO recombinant culture medium of Example 6 was also examined for in vivo biological activity as described in Ses et al., Nature, No. 191. pp. 1065-1067 (1961) and according to Hammond et al.,

Ann. N.Y. Acad, Sci. 149, pp. 516-527 (1968) and an efficiency of 0.94 to 1.24 units / ml has been found.

Example 10

In the previous examples, recombinant human or simian EPO material was obtained using vectors 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 useful amounts of EPO can be obtained from COS-1 cells 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 able to produce the appropriate material after transfection. Next, a ^{Chinese hamster ovary DHFR 5} DHFR 5 ovary cell expression and selection-capable labeling system, DHFR, will be described. Related expression systems are described in U.S. Patent Nos. 4,399,216 and European Patent Nos. 117,057, 117,059 and 117,060, issued August 29, 1984.

CHO DHFR ^- [(DuX-B11) CHO K1J cells have been described by Urlaub et al. Hat. Acad. Sci. ISA, 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-kkE (Example 6) or pDSVL-gHuEPO (Example 7B) were transfected with carrier DNA into CHO DHFR * cells grown in hypoxanthine, thymidine and glycine medium on 60 mm plates. Plasmid pSVgHuEPO (Example 7A) was mixed with plasmid pkG2 containing the mouse dihydrofolate reductase gene cloned into 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 contain a second plasmid). After three days, the cells were dispersed by trypsinization on several 100 mm plates in a hypoxanthine and thymidine-free medium. Only those cells that have been permanently transformed with the DHFR gene and thus the EPO gene survive in this environment. After 7 to 21 days, colonies of surviving cells can be observed. After transformation by trypsinization, these transformant colonies can be grown continuously on beXz hypoxanthine and thymidine media to obtain new cell lines (e.g., CHO, pDSVL-MkEPO,

CHO pSVgHuEPO, CHO-pDSVL-gHuEPO).

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

The liquid material from CHO pSVgHuEPO and CHO pDSVLgHuEPO contained human EPO with immunological properties similar to that obtained using COS-1 cells after transfection with plasmid pSVgHuEPO or pLSVL-gHuEPO. EPO and 5.5 days cultured CHO sample pDSVL-gHuEPO contained 1b, 2 units / ml human EPO, measured by RIA.

The amount of EPO produced by the above cell lines can be increased by amplifying the gene to produce a new cell line with higher productivity. The enzyme dihyrofolate reductase (DHFR), which is the product encoded by the DHFR gene, can be inhibited by methotrexate (MTX). Thus, cells grown in a hypoxanthine- and thymidine-free environment can be inhibited in growth to kill MTX.

Under appropriate conditions (e.g., minimal MTX concentrations), 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 higher production of the DHFR enzyme. Surviving cells can then be treated with increasing amounts of MTX to obtain cell lines that contain multiple DHFR genes. A gene "carried" by an expression vector (e.g., an EPO gene) concurrently with a DHFR gene or transformed with a DHFR gene usually also increases its copy number.

A practical example of this amplification is the case in which the CHO pDSVL-MkE cell line was subjected to increasing concentrations of MTX (0 nM, 30 nM and 100 nM). After 65 hours of treatment, 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 cell line pDSVL-gHuEPO was then treated with increasing concentrations of MTX 30 nM, 50 nM, 100 nM, 200 nltt, 11 yUM, 5 yUM MTX. After three days of cultivation, the liquid material from the culture was treated with 100 nM MTX human EPO at a concentration of 3,089 ± 129 units / ml as determined by RIA. Samples grown for 48 hours after treatment with 100 nM and 1 μM MTX contained human EPO at 466 and 1352 units / ml as determined by RIA (average of three determinations). A 1 x 10 cell configuration in 5 ml medium using 60 mm plates was used to perform these procedures. After 24 hours, the medium was removed and replaced with 5 ml of serum-free medium (high glucose 0.1 mm non-essential amino acids and L-lutamine). Accumulation of EPO in a serum-free environment was monitored after 48 hours. The medium was removed for RIA analysis and the cells were trypsinized and counted. The mean RIA values were 47 units / ml and 1352 units / ml, for cells grown at 100 nM and 1 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, the production efficiency was 1264 and 2167 units / 10 cells in 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 proauction capacity. These procedures are described in Points to Consider in the Characterization of Cell Lines in Production Biologics, Section A, Part 2, June 1, 1984, Office of Biologics Research Review, Center for Drugs and Biologics, U.S. Pat. Food and Drug Administration.

The productivity of CHO-producing EPO cell lines can be improved by appropriate culture intervention. Growing 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. The method of producing erythropoietin from CHO cells in a serum-free medium will significantly facilitate the isolation of erythropoietin from the culture medium. Thus, this method guarantees the economical recovery of erythropoietin in environments which do not contain serum in large quantities.

CHO pDSVL-gHuEPO cells grown under standard conditions are used to inoculate flasks suitable for direct centrifugation in a 50:50 mixture of high glucose DMEM and Ham's F12 supplemented with 5% fetal serum. -glu% tamin and also Penicillin, streptomycin, 0.05 mM non-essential amino acids and the corresponding concentration of methotrexate. Growing cells in suspension allows the use of a large volume of EPO-producing CHO cells. The CHO cells grown in suspension were then used to inoculate larger 850 ml flasks containing 200 ml of medium. The cells are allowed to grow until a continuous layer forms as a wall-adhering layer for three days. The medium used in this phase is the same as the medium 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 high glucose DMEM medium and Ham's F12 medium supplemented with 0.05 nM non-essential amino acids and glutamine.

The flasks are returned to the incubator for 1 to 3 hours, the medium is removed again and replaced with 100 ml of fresh serum-free medium. Incubation with serum-free medium for 1 to 3 hours will reduce the concentration of contaminating serum proteins. The flasks are then returned to the incubator for seven days, during which time erythropoietin accumulates in the serum-free medium. At the end of the seven - day period, the medium is removed and replaced with fresh serum - free medium for the second production cycle. After seven days, the sample thus obtained may contain, for example, 3892 + 409 units / ml human erythropoietin according to KIA data. Due to the determined cell density of 0.9 5 to 1.8 x 10 6 cells per ml, each flask contains 0.75 to 1.5

Thus, 0 x 10 cells and EPO production in 100 ml of culture was 750 to 1470 units / 10 cells in 48 hours.

The culture medium from the cultivation of the CHO cell line pLSVL-to EPO in the presence of 10 MTX was subjected to PIA in vitro and in vivo efficacy was also monitored. The medium sample contained 41.2 ± 1.4 units / ml IzkEPO when measuring RIA, 4, 2 ± 0.064 units / ml when measuring in vitro potency and 42.5 ± 5 units / ml when measuring in vivo biological potency. Monitoring of the polypeptide chain of the products showed the presence of EPO-type substances, which contained substances containing three residues of the signal sequence behind the amino-terminal residue of alanine. It is not yet clear whether this is due to improper production of the polypeptide in the CHO cell membrane or a reflection of the differences in the structure of the amino-terminal end of monkey and human EPO.

The CHO pDSVL-gHuEPO culture medium was subjected to three types of tests. Pattern after cultivation

5.5 days contained recombinant human EPO in the medium at a concentration of 18.2 units / ml in the RIA assay, 15.8 ± 4.6 units / ml in the in vitro assay and 16.8 ± 3.0 units / ml in the in vitro assay. vivo.

The CHO pDSVL-gHuEPO cell culture medium with the amplified gene after using 100 nM MTX was also subjected to all three assays. After culturing for 3 days, the sample contained recombinant human EPO at a concentration of 3,089 ± 129 units / ml in the RIA assay, 2589 ± 71.5 units / ml in the in vitro assay, and 2,040 ± 160 units / ml in the in vivo assay. The amino-terminal termination shown in Table VI could be demonstrated by amino acid sequence determination.

The medium from the cultivation of CHO cells after transfection with the plasmid pDSVL-MkE in the presence of 10 nM MTX was fused and MTX was dialyzed for several days to obtain a medium with an EPO concentration of 221 ± 5.1 units / ml (EPO / CCM). In order to determine the in vivo EPO / CCM efficacy at hematocrit levels in normal Balb / C mice, the following experiment was performed: CHO cell culture medium (CCM) and EPO-CCJVi were treated by the 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), namely 4 units and 44 units in one dose. This dose was administered intraperitoneally to mice 3 times a week for 5 weeks. After the eighth injection, the mean hematocrit was 50.4% for the control group, 55.1% for the 4-group group and 67.9% for the 44-unit group *

Mammalian cell expression products can be readily isolated in substantially pure form from the culture medium using high pressure liquid chromatography (CH 2) and an ethanol gradient, preferably at pH 7.

Preliminary experiments were performed to determine the properties of recombinant glycoprotein products from conditioned medium after expression by COS-1 and CHO cells of the human EPO gene compared to human EPO isolates from urine using different types of analysis. (SDS-PAGE)

These studies show that EPO produced by CHO cells has a slightly higher molecular weight than the expression product of COS-1 cells, which had a molecule slightly larger than the product obtained from urine. All products were somewhat heterogeneous. After treatment with the neuraminidase enzyme to remove sialic acid, recombinant ex91 products were obtained in COS-1 and CHO cells of approximately the same molecular weight, but having a higher molecular weight than the asialoproduct from human urine. By the action of Endoglycosidase F (EC 3.2.1) on the recombinant product from CHO cells and on the product from urine (to completely remove carbohydrates from both products) a substantially homogeneous product was obtained in both cases with identical molecular weight.

the purified human urine product and the recombinant CHO cell product obtained by the method of the present invention were subjected to carbohydrate analysis by the method of Ledeen et al., Iviethods in Enzymology, 83 (Part D) 139191 (1982) after modification using hydrolysis according to Nesser et al. Anal. Biochem. 142, 58-67 (1984). The experimentally determined values, expressed as the molar ratio of carbohydrates in the product for the substance isolated from urine, were as follows: hexoses 1.73, N-acetylglucosamine 1, N-acetylneuraminic acid 0.93, fucose O, N-acetylgalactosamine O. Corresponding values for recombinant *

product (from CHO pDSVL-gHuEPO cells after three days of cultivation in the presence of 100 nid kTX) were as follows: hexoses 15.09, acetyl,

N-acetylglucosamine 1, neuranic acid 0.958, fucose O and N-acetylgalactosamine 0. These values are consistent with analytical data.

So it is clear that the products made? According to the process of the invention, they correspond in structure to naturally occurring erythropoietin and also have one or more of its biological properties with a composition of hydrates which is somewhat different from naturally occurring erythropoietin.

Example 11

Experiments were performed with the complete construction of nucleotion-based 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, genes that encode human EPO analogs can be constructed. The embodiment followed the method described by Alton et al. (7/0 83/04053). The genes were assembled as multiple pairs, which could be divided into three groups. These groups were intended for amplification and could be used to assemble a multiple expression vector by multiple fragment binding.

The following Tables VIII to XIV show the construction of the gene thus obtained, which codes for human EPO as a translation product without a signal or preliminary sequence, but with an initial methionine residue in position -1. In addition, codons that are preferential for E. coli are substantially incorporated into the gene, which is why the gene has been termed ECEPO.

and

TABLE Fairies

ECEPO OLIGONUCLEOTIDES SECTION 1

1. AATTCTAGAAACCATGAGGGTAATAAAATA

2. CCATTATTTTATTACCCTCATGGTTTCTAG

3. ATGGCTCCGCCGCGTCTGATCTGCGAC

AND·. ¹ CTČGAGTCGCAGATCAGACGCGGCGGAG ', 5. TCGAGAGTTCTGGAACGTTACCTGCTG

6. CTTCCAGCAGGTAACGTTCCAGAACT

7. GAAGCTAAAGAAGCTGAAAACATC

8. · GTGGTGATGTTTTCAGCTTCTTTAG

9. ACCACTGGTTGTGCTGAACACTGTTC

10. CAAAGAACAGTGTTCAGCACAACCA

11. TTTGAACGAAAACATTACGGTACCG

12. GATCCGGTACCGTAATGTTTTCGTT

TABLE IX

ECEPO SECTION 1

Xbal

EcoRI 1, 3

AATTCTAG AAACCATGAG GGTAATAAAA TAJATGGCTCC GCCGCGTCTG

GATC TTTGGTACTC CCATTATTTT ATTAČČÍGAGG CGGCGCAGAC 2 A

ATCTGCGACT CGAGAGTTCT GGAACGTTAC CTGCTGGAAG CTAAAGAAGC TAGACGCTGA 'GČTČfrCAAGA CCTTGCAATG GACGACCTT ^ GATTTCTTCG

TGAAAACATC ACCACTGGTT ACTTTTGTAG TGGTG ^ CCAA

GTGCTGAACA

CACGACTTGT ctgttc (tttg

GACAAGAAAC

AACGAAAACA

TTGCTTTTGT

BamρηI BamHl TTACGGTACC G AATGCCATGG CCTAG

TABLE XII

ECEPO SECTION 3

1. GATCCAGATCTCTGACTACTCTGC

2. ACGCAGCAGAGTAGTCAGAGATCTG

3. TGCGTGCTCTGGGTGCACAGAAAGAGG

J

4. GATAGCCTCTTTCTGTGCACCCAGAGC

5. CTATCTCTCCGCCGGATGCTGCATCT

6. CAGCAGATGCAGCATCCGGCGGAGA

7. GCTGCACCGCTGCGTACCATCACTG

8. ATCAGCAGTGATGGTACGCAGCGGTG

9. CTGATACCTTCCGCAAACTGTTTCG

10. ATACACGAAACAGTTTGCGGAAGGT

11. TGTATACTCTAACTTCCTGCGTGGTA

12. CAGTTTACCACGCAGGAAGTTAGAGT

13. AACTGAAACTGTATACTGGCGAAGC

14. · GGCATGCTTCGCCAGTATACAGTTT

15. ATGCCGTACTGGTGACCGCTAATAG

16. TCGACTATTAGCGGTCACCAGTAC

TABLE XIII

ECEPO SECTION 3

BamHI BglII GA TCCAGATCTCTGGTCTAGAGAC.

ACTACTCTGC JrGCGTGCTCT ·. TGATGAGACG ACGCAjCGAGA

GGGTGCACAG

CCCACGTGTC

AAAGAGGfcTA TCTCTCCGCC TTTCTCCGAT AGjAGAGGCGG

GGATGCTGCA

CCTACGACGT

TCTbcTGCAC

AGACGÁČjGTG

CGCTGCGTAC

GCGACGCATG catcactgIct GATACCTTCC GTAGTGACGA CT ^ TGGAAGG

GCAAACTGTT

CGTTTGACAA

TCGtTGTATAC

AGCACÁTAjTG tctaacttcc TGCGTGGTAÍA AGATTGAAGG ACGCACCATT

ACTGAAACTG

TGA ^ íTTGAC

Hall

TATACTGGCG AAGC <br> ATGCCG TACTGGTGAC CGCTAATAG ATATGACCGC TTCGTACGdC ATGACCACTG GCGATTATC AGCT

16

Table VIII shows the oligonucleotides used to construct section 1 of the ECEPO genes, which code for the amino-terminal residues of the human polypeptide. The oligonucleotides were assembled as a pair, i.e.

and 2, 3 and 4, etc. ·) and these pairs were then ligated to form region 1 of the ECEPO gene, as shown in Table IX. It should be noted that this region contains sites after cleavage by the enzymes BcoRI and BamHI, r

then downwards from the EcoRI termination site there is an XbaI site and upwards from the BamHI site there is a KpaI site. Section I can be easily 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 XbaI / KpnI enzymes from H1A, generated in E. coli, probably due to methylation of the KpnI-treated site in the host. For this reason, a single-stranded phage INA was isolated and then converted to a double-stranded IMA in vitro, after which the desired double-stranded fragment was isolated.

- 101

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 the M13 vector to verify the sequence and then isolated from ZHUA-phage. As shown in Table XI, ECEPO region 2 was constructed with EcoRI and BamHI feedback sites and could be isolated as a KpnI / Bgl II-treated fragment. Similarly, ECEPO section 3 was prepared with 1 / BamHI and SalI-treated sites capable of end-linking and could be isolated from DNA phage as a Bgl II / Sal I fragment. as shown in Table XIV, which codes for the entire human EPO polypeptide with an amino-terminal methionine (ATG) codon for translation initiation in E. coli · It should also be noted that there is a group of pairs which essentially repeats the ribosome binding sequence in E. coli CBdP-f gene expression.

Any suitable expression vector can be used as a carrier vector for ECEPO. Vector,

102 selected for this purpose was the temperature-sensitive plasmid pCIM536, a derivative of plasmid pCIM414 (AICC 40076), which was described in U.S. Patent Application No. 636,727, filed August 6, 1984 (Charles F. Morris). The ρΟΈΜ53β gene was then digested with XbaI and HindIII. The large fragment was isolated and used to bind to the BCEPO gene. Sections 1 (XbaI / KpnI), 2 (KpnI / Bgl II) and 3 (Bgl II / Sal I) were pre-arranged in the correct order in M13 and the EPO gene was then isolated as a single fragment (XbaI / HindIII). This fragment contained part of the binding chain from phage M13 mp9 between the Sal I to Hind III enzyme sites. _{The lambda P L} promoter was used to verify the expression of the resulting plasmid p536, which may itself be subordinate to the repressor gene Cj 357 as found in the E. coli strain K12AHtrp.

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

- 103 A. [Asn ² , des-Pro ² to Ile ⁶ ] heP0

Plasmid 536, which carries the engineered BCEPO gene from Table XIV as an insert, terminated by XbaI and HindIII sites, was digested with Hind III and XhoI enzymes. which codes for Asp ⁸ to Arg ¹ ^.

The XbaI / Xho Ia binding chain was then produced as follows:

Xbal +1 Met Ala 2 Asn 7 Cys 8 9 Asp XHol 5 * -CTAG AIG GCT AAT TGC GAC-3 * 3 * - TAC CGA TTA ACG CTG AGCT-5 * Binding string XbaI / XhoI and sequence fragment

The BCEPO XhoI / HindIII gene is incorporated into a large fragment that results from the cleavage of plasmid pC5M526, a derivative of plasmid pC! M414 (ATCC 40076), by XbaI and HindIII enzymes, as described in U.S. Pat. no. 636,727, filed August 6, 1984 (Charles F. Morris) to give INA sequence coding for Met ^"1 form the desired analog when expressed in B. coli.

- 104 B. [His ⁷ ] hEP0

Plasmid 536 was digested with Hind III and Xho I enzymes as in paragraph A. The XbaI / XhoI binding chain was constructed as follows:

+1 2 3 4 5 6 7

Xbal Met Ala Pro Pro Arg Leu IleeHis

5 * -CTAG - ATG GCT CCG CCA CGT CTG ATC CAT 3 * - TAC CGA GGC GGT GCA GAC TAG GTA

9

Asp Xhol

GAC-3 *

CTG AGCT-5 *

The binding strand and ECEPO fragment (XhoI / HindIII) were then inserted into plasmid pCIM526 to obtain the HiA sequence carried by the plasmid, which encodes the Met ¹ form of the desired analog when expressed in E. coli.

The construction of the SCEPO gene, which has xkx-incorporated codons that facilitate expression in yeast, is described in the following Tables XV to XXI. As in the case of the ECEPO gene, the whole construct involves the formation of three groups of oligonucleotides, as shown in the following Tables XV, XVII and XIX, gde pairs, which are then arranged in the sections shown in Tables XVI, XVIII.

105 and XX, it should be noted that the synthesis can be partially facilitated by using some suboptimal codes in the construction of both SCEPO and ECEPO, ie in the case of both genes the identical oligonucleotides 7 to 12 in section 1 and also in section 2 are identical in both genes oligonucleotides 1 to 6.

- / r <rΤΛ BULKA XV

9I ^^ CUiOTim_S_CEPO ÚSEK

1. AATTCAAGCTTGGATAAAAGAGCT

2. GTGGAGCTCTTTTATCCAAGCTTG

3. CCACCAAGATTGATCTGTGACTC

4. TCTCGAGTCACAGATCAATCTTG

5. GAGAGTTTTGGAAAGATACTTGTTG ’’ 6. CTTCCAACAAGTATCTTTCCAAAAC '

7. GAAGCTAAAGAAGCTGAAAACATC

8. GTGGTGATGTTTTCAGCTTCTTTAG

9. ACCACTGGTTGTGCTGAACACTGTTC

10. CAAAGAACAGTGTTCAGCACAACCA

11. TTTGAACGAAAACATTACGGTACCG

12. GATCCGGTACCGTAATGTTTTCGTT

TABLE XVI SCEPO SECTION 1

EcoRI HindlII 1

AATTCA AGCTTGGATA GT TCGAACCTAT

AAAGAGCTfcc ACCAAGATTG ATCTGTGACT cfcAGAGTTTT TTTCTCGAGG TGpTTCTAAC TAGACACTGA GCTČŤ] CAAAA

GGAAAGATAC TTGTTGpAAG CTAAAGAAGC TGAAAACATC ^ CCACTGGTT CCTTTCTATG AACAACCTŤČ] GATTTCTTCG ACTTTTGTAG TGGTČSACCAA 'θ

11 KpnI BamHI

GTGCTGAACA CTGTTCPTTG AACGAAAACA TTACGGTACC G CACGACTTGT GACAAGAÁÁČ] TTGCTTTTGT AATGCCATGG CCTAG '12 / ol · TABLE OTI

OLIGONUCLBOTIDES SCEPO USEK 2

1. AATTCGGTACCAGACACCAAGGT

2. GTTAACCTTGGTGTCTGGTACCG

3. TAACTTCTACGCTTGGAAACGTAT

4. TTCCATACGTTTCCAAGCGTAGAA

5.. GGAAGTTGGTCAACAAGCAGTTGAAGT

6. CCAAACTTCAACTGCTTGTTGACCAAC

7. TTGGCAAGGTTTGGCCTTGTTATCTG

8. GCTTCAGATAACAAGGCCAAACCTTG

9. AAGCTGTTTTGAGAGGTCAAGCCT

IQ. AACAAGGCTTGACCTCTCAAAACA

11. 'TGTTGGTTAACTCTTCTCAACCATGGG ·

12. TGGTTCCCATGGTTGAGAAGAGTTAACC

13. AACCATTGCAATTGCACGTCGAT

14. CTTTATCGACGTGCAATTGCAA

15. AAAGCCGTCTCTGGTTTGAGATCTG

16. GATCCAGATCTCAAACCAGAGACGG

TABLE XVIII SCEPO SECTION 2

KpnI

EcoRI 1

TTETTCGGTtfCC AGACACCAAG GCCATGG TCTGTGGTTC gtItaacttct caaTTg ^ aga acgcttggaa tgcgaacctt acgtatHgaa tgcatacčTť]

GTTGGTCAAC

CAACCAGTTG

AAGCTGTTGA

TTCGACAACT

AGTfTTGGCAA GG ”TTGGCCT TCAAAČ ^ TT CCAAACCGGA

TGTTATCTGp AGCTGTTTTG AGAGGTCAAG ACAATAGACT TCGhCAAAAC TCTCCAGTTC ’10 li 12

CCTjrGTTGGT TAACTCTTCT CAACCATGGG hACCATTGCA ATTGCACGTC GGAto ^ CA ATTGAGAAGA GTTGGTACCC TTGGTjAACGT TAACGTGCAG

BglII BamHI

GAThAAGCCG TCTCTGGTTT GAGATCTG CTATTTČpGC AGAGACCAAA CTCTAGACCTA G fCN TABLE XIX

OLIGONUCLEOTILY SCEPO ÚHEK 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

- f / PΤΛ BULKA XX t

SCEPO USEK 3) j

BamHI BqlII _1

GATC CAGATCTTTG ACTACTTTGT TGAGAGCTTT GTCTAGAAAC TGATGAAACA ACTCTjSGAAA

5

GGGTGCTCAA AAGGAAGfcCA TTTCCCCACC AGACGCTGCT TCTGCCGCTC cccacgagtt ttccttcggt aJaaggggtgg tctgcgacga aEaFggcgag

6 ⁷ 2 II

CATTGAGAAC CATCfrCTGCT GATACCTTCA GAAAGTTfaTT CAGAGTTTAC GTÁACTCTTG GTAGTGÁČpA CTATGGAAGT CTTTCAATAA G | TCTCAAATG

12

TCCAACTTCT frGAGAGCTAA ATTGAAGTTG TACACfcGGTG AAGCCTGTAG

AGGTTGAAGA AČTČJTCCATT TAACTTCAAC ATGTGGČČAJC TTCGGACATC

I ⁷ 12

AACTGGTbAC AGATAAGCCC GACTGATAAfc AACAGTGTAG

TTGACCACtG TTJtATTCGGG CTGACTATTG TTGfTCACATC

Hall

ATGTAACAAA G

TACATTGTTT CAGCT 20

- // 7TABLE; c <l GELL / CUPO

HlndlII

SočTtggata

ACCTAT

GGAAAGATAC

CCTTTCTATG

GTGCTGAACA

CACGACTTGT

GTTAACTTCT

CAATTGAAGA

AGTTTGGCAA

TCAAACCGTT

CCTTGTTGGT

GGAACAACCA

GATAAAGCCG

CTATTTCGGC

GGGTGCTCAA

CCCACGAGTT

CATTGAGAAC

GTAACTCTTG

TCCAACTTCT

AGGTTGAAGA

AACTGGTGAC

TTGACCACTG

ATGTAACAAA

TACATTGTTT

-1 +1 ArgAla

AAAGAGCTCC ACCAAGATTG ATCTGTGACT CGAGAGTTTT TTTCTCGAGG TGGTTCTAAC TAGACACTGA GCTCTCAAAA TTGTTGGAAG CTAAAGAAGC TGAAAACATC ACCACTGGTT AACAACCTTC GATTTCTTCG ACTTTTGTAG TGGTGACCAA CTGTTCTTTG AACGAAAACA TTACGGTACC AGACACCAAG GACAAGAAAC TTGCTTTTGT AATGCCATGG TCTGTGGTTC ACGCTTGGAA ACGTATGGAA GTTGGTCAAC AAGCTGTTGA TGCGAACCTT TGCATACCTT CAACCAGTTG TTCGACAACT GGTTTGGCCT TGTTATCTGA AGCTGTTTTG AGAGGTCAAG CCAAACCGGA ACAATAGACT • TCGACAAAAC TCTCCAGTTC TAACTCTTCT CAACCATGGG AACCATTGCA ATTGCACGTC ATTGAGAAGA GTTGGTACCC TTGGTAACGT TAACGTGCAG TCTCTGGTTT GAGATCTTTG ACTACTTTGT TGAGAGCTTT AGAGACCAAA CTCTAGAAAC TGATGAAACA ACTCTCGAAA AAGGAAGCCA TTTCCCCACC AGACGCTGCT TCTGCCGCTC TTCCTTCGGT AAAGGGGTGG TCTGCGACGA AGACGGCGAG CATCACTGCT gataccttca ' GAAAGTTATT CAGAGTTTAC GTAGTGACGA CTATGGAAGT CTTTCAATAA GTCTCAAATG TGAGAGGTAA ATTGAAGTTG TACACCGGTG AAGCCTGTAG ACTCTCCATT TAACTTCAAC ATGTGGCCAC TTCGGACATC AGATAAGCCC GACTGATAAC AACAGTGTAG TCTATTCGGC Hall G CAGCT CTGACTATTG TTGTCACATC

112 The entire U3ek SCEPO was expressed in M13 and sections 1, 2 and 3 were isolatable from phage as HindIII / KpnI, KpnI / Bgl II and Bgl II / / Sal I fragments.

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

October 31, 1984, as European Patent Application No. 0 123 294. This system includes constructs in which the INA, which codes for the leader sequence for the yeast α-factor gene, is placed in a position just 5 * behind the coding region of the exogenous gene to which expression is to occur. As a result, the translated product contains a leader or signal sequence that is removed by the endogenous yeast enzyme during secretion of the remainder of the product. Because the ATG codon, which is the codon for the start of α-factor translation, is used in the construction, it is not necessary to place this codon in position -1 of the SCEPO gene. As can be seen from Table XXI, the alanine (+1) code is preceded by a binding chain

- 113 which allows direct insertion into a plasmid including INA for the first 80 residues of the α-factor signal sequence with the following α-factor promoter. The specific construct for SCEPO gene expression involves a four-step linkage including the above fragments and a large fragment after digestion of plasmid pocC3 with HindIII / Sal I. The α-factor promoter, signal sequence and SCEPO gene can be isolated from the resulting plasmid pKC3 / SCEP0 BamHI and binding to plasmid pYE after digestion with BamHI to give plasmid pYE / SCEPO.

Example 12

E x over recombinant products using the engineered ECEPO and SCEPO genes for the expression system of Example 11 can be achieved as follows:

Plasmid p536 from Example 11 was transformed into E. coli AM7 cells that had already been transformed with the appropriate plasmid pMW1 with the Cj 357 gene.

Cell cultures of LB medium (Ampicillin 5β / μl and CACAMYCIN 5 μg / ml, preferably 10 mM magnesium sulfate) were maintained at 28 ° C and the cells were grown to a density of 0.1 (optical density ), EPO expression is induced by raising the temperature to 42 ° C. EPO is produced at approximately 5 mg / liter.

The cells are isolated, disrupted, and then treated with lysozyme and HP-40 wetting agent. The pellet after centrifugation (24,000 g) was then solubilized with guanidine hydrochloride and further purified by reverse phase high performance liquid chromatography (CH 2 -Edac), 0 to 80% ethanol, 50 mM ammonium acetate, pH 4.5. By determining the protein sequence, it can be shown that the purity of the product is higher than 95% and the product obtained has 2 possible ends at the amino-terminal end, A-P-P-R or P-P-R in a relative amount of 3: 1. This observation of products corresponding to human hEPO and the same alanine-free product at position 1 indicates host cell processing 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

- 115 30,000 to 62,000 units / mg and in vivo efficacy 120 to 720 units / mg. (The comparison was made with material sued from human urine with an efficacy of 70,000 units / mg). The dose-response curve for the recombinant product in vivo differed significantly from the same curve for human urinary EPO.

The EPO analog plasmids 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 grown as described above. Purified isolates were also examined by SSi BIA in vitro. Bbe values for the expression products [Asn ² , des-Pro ² to Ile ^ jhEPO were approximately 11,000 units / mg and 6,000 units / mg protein, while the same values for [His ^ lhEPO were 41,000 units / mg and 14,000 units / mg protein, indicating that the analogous products had an efficiency of 1/4 to 1/10 than the complete expression products.

In an expression system for use in S. cervisiae host cells, pYE / SCEPO was transformed into two different strains, namely

- 116 YSDP4 (genotype apep4-3 suffers) and RK81 (genotype aa pep4-3 suffers 1). Transformed YSDP4 host cells were grown in SD medium (Methods in Yeast Genetics,

Cold Spring Harbor Laboratory, Cold Spring Harbor,

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

Plasmids PaC3 and pYE in HB101 E. coli were deposited on September 27, 1984 in the American Type Culture Collection.

12301 Parklawn Drive, Rockville, Maryland under ATCC Nos. 39881 and ATCC 39882. Plasmids pCiM526 in AM7, pCIM536 in JM103 cells and pMW1 in JM103 cells were also deposited on November 21, 1984 under ATCC Nos. 33932, 33934 and 33933 · Saccharomyces kmSP4 RK81 was deposited on November 21, 1984 under ATCC numbers 20734 and 20733.

£

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

The polypeptides obtained by the process of the invention are useful materials, and whether they are microbial expression products or synthetic products, the primary, secondary and tertiary structure of these products was first recognized by the process of the invention.

As already stated, recombinant products and synthetic products are ⁱⁿ different stages of the biological activity in vitro for both isolates of EPO from natural sources, and the expression products and may therefore be used as substitutes for isolates of EPO in the medium for the blood cells. To some extent, the polypeptides produced by the method of the invention also have in vivo activity similar to natural EPO isolates and can therefore be used for treatment with erythropoietin in mammals, including humans, for example where reticulocyte stimulation is required. , for example, iron exchange between plasma and bone marrow, where it is necessary to achieve a change in the number of erythrocytes, stimulation of hemoglobin C production (Eschbach et al., supra) and

118 increase in hematocrit, in mammals, as shown in Example 10. These are therefore patients who usually require blood transfusions, for example in the case of injuries, in the case of surgical diseases, in kidney diseases, including the use of an artificial kidney, and in patients with various types of disorders in blood composition, such as hemophilia, blood cell disorders, physiological anemias, sickle cell disease, etc. In this way, it is possible to reduce the need for transfusions and thus use EPO to reduce the materials through which infectious diseases can be transmitted. The products of the present invention are free of pyrogens, natural inhibitory substances and the like with respect to production by recombinant methods, and are therefore very effective in comparison with natural products. They can also be used where it is necessary to improve oxygen transfer, for example when in an environment with a reduced amount of oxygen and in the case of circulatory diseases.

The preferred route of administration of these polypeptides is parenteral administration, usually with acceptable diluents, carriers and / or excipients. Preliminary pharmacokinetic studies show a longer in vivo half-life for monkey EPO when administered intramuscularly than when administered intravenously. Effective doses vary considerably from one disease to another, but are in the range of 0.1 to 100 .mu.g / kg, i.e. about 7 to 7,000 units / kg. Conventional diluents, such as human serum albumin, and standard carriers, such as saline, may be used.

Other excipients, such as testosterones, germ cell stimulators, insulin-like growth factors, prostaglandin, serotonin, cyclic AβMP, prolactin and triiodothyronine, may also be used in the compositions, as may agents used in the treatment of aplastic anemias, such as methenolene, stanozol. and nandrolone (Resegotti et al., Panminerva Medica 23, 243-248 (1981), MeGonigle et al., Kidney Int. 25 (2), 437-444 (1984)),

Pavlovic-Kantera et al., Expt. Hematol. 8 (Supp. 8) 283-291 (1980) and Kurtz, FEBS Letters, 14a (1), 105-108 (1982)). It is also possible to use as excipients compounds which are known to increase the action of erythropoietin or asialo-EPO, for example adrenergic substances, thyroid hormones, androgens.

-> rí2o or BPA, as described in Dunn publications,

Current Concepts in Erythropoiesis, John Wiley and Sons (Chichester, England, 1983), Weiland et al., Blut, 44 (3), 173-175 (1982), Kalmanti, Kidney Int., 22, 383-391 (1982), Shahidi, New. Eng.

J. Med., 289, 72-80 (1973), Fischer et al., Steroids, 30 (6), 833-845 (1977), Urabe et al., J. Exp. Med.

149, 1314-1325 (1979) and Billat et al., Expt, Hernato1, 10 (1), 133-140 (1982) and compounds which are termed as "hepatic factors for blood formation" as described in Naughton et al., Acta Haemat., 69, 171-179 (1983) and erythrotropins ", described in Congote et al., Abstract 364,

Proceedings of the 7th International Congress of Ifadocrino- j logs (Quebec City, Quebec, July 1-7, 1984),

Congote, Biochem. Biophys.Res.Comm, 115 (2),

447-483 (1983) and Congote, Anal. Biochem., 140, 428-433 (1984) and the erythrogenins described in Rothman et al., J. Stujg. Oncol, 20, 105-108 (1982). !

Preliminary monitoring indicates hypoxic polycythemic mice pretreated with 5-α-dihydrotestosterone or nandrolone and then erythropoietin with promising results. h

The diagnostic use of the polypeptides produced by the method of the invention is also very broad and includes the use of labeled and unlabeled forms in a variety of immunoassays, including RIA,

ELISA, etc., 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 Crystal, Hematol, 11 (7), 649-660 (1983), Saito et al. , Jap.J.Med., 23 (1), 16-21 (1984), Nathan et al.,

New Ihg. J. Med., 308 (9), 520-522 (1983) and also in the various articles to which these publications refer. The polypeptides of the invention, including synthetic peptides with EPO sequences or residues, are also highly useful pure substances for polyclonal antibodies and monoclonal antibody banks specific for recognizing continuous and discontinuous EPO epitopes. Examples are the preliminary amino acid sequence analysis of Table VI of Hopp et al., PNAS (USA), 78, pages 3824-3828 (1981) and the secondary structure analysis of Chou et al., Ann. Rev. Biochem, 47, pp. 251 (1978)

122 showed that synthetic peptides whose structure corresponds to continuous sequences of natural peptides in the regions 41 to 57, 116 to 118 and 144 to 166, inclusive, are able to elicit a highly antigenic response and thus the formation of monoclonal and polyclonal antibodies that react with the synthetic peptide. 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-A-W-K-R-M-B-V-G,

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

Preliminary immunization studies using these polypeptides showed a relatively weak positive response to hEPO 41-57, almost no response to hEPO 116 to 128, and a strong positive response to hEPO 144 to 166, as measured by the ability of rabbit serum antibodies to EPO isolate from human urine, labeled 125 ¹ . Preliminary in vivo studies of all three peptides

- 123 showed almost no efficacy either individually or in a mixture.

The sequence of mammalian EPO residues, as demonstrated in the examples, defines the primary structure of complete EPO, however, the specific sequence of 165 amino acids of monkey EPO in Table V & 166 of human EPO residues in Table VI does not limit the scope of useful polypeptides of the invention. According to the method of the invention, it is possible to obtain various allelic forms of EPO which, according to the results of previous research, exist, for example human gamma-interferon, as compared with data suggesting an argenin residue at position 140 EPO or a glutamine residue at position 140 in the publication. Gray et al., Nature, 295, pages 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 sequence length or by omitting, replacing, segmenting or adding certain residues with consequent changes in effect and changes in glycosylation. As already mentioned, one allelic form of human EPO appears to contain a methionine residue at position 126. It can be expected that

124 there are still IRA genomes and cERA sequences that encode allelic polypeptides or simply contain different codons to designate the same polypeptide.

In addition to naturally occurring allelic forms of whole EPO, the invention also encompasses other EPO- * products, such as EPO polypeptide analogs and fragments thereof. According to the method of the published applications of Altona et al. at the end or by omission. It is also possible to modify genes by mutogenesis to obtain EPO analogs and derivatives. 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 ⁸ ] hEPO, [des-Thr ¹ ^ _to ž Arg ¹ ^] .. hEPO and delta27-55hEPO, the latter having the entire exon deleted. Some derivatives are more stable against hydrolysis and thus longer, or one or more glycosylation sites are deleted, which may lead to greater efficiency in yeast production, or the cysteine residue may be replaced by, for example, flutidine or serine, as in Chis ^ lhEPO analog and may be more easily recovered or are tyrosine replaced with phenylalanine, such as [Phe lhEPO ^^, ^ CPhe lhEPO and [Phe ¹ ^] hEPO and may be harder or easier to bind to receptors in target cells. They may also be fragments containing only part of the full EPO sequence and only part of the activity of this substance, for example receptor binding and not erythropoietic activity. Of particular interest are the potential fragments of the INA sequence of the human genome of Table VI, which are specific domains of biological activity. It should be noted that in vivo ineffectiveness does not necessarily mean treatment failure or lack of antagonism. This antagonism may be useful in the treatment of polycythemia in excess EPO, as described in Adamson, Hosp. Practice, 18 (12),

49-57 (1983), and Hellmann et al., Clin. Lab. Haemat.,

5, 335-342 (1983).

According to another method of the invention, cloned INA sequences encoding pro can be obtained

- 126 human and monkey EPOs and which are valuable for the information they provide on the mammalian erythropoietin amino acid sequence, which previously could not be obtained despite decades of analytical research on naturally occurring products. DNA sequences can also be used for recombinant synthesis of erythropoietin. methods for growing said cells which are capable of producing EPO and EPO-products. DNA sequences are also useful as labeled samples in isolating the EPO and DNA genomes, including the cDNA that encodes these products. It is not yet possible to estimate the extent to which the INA sequences of the invention can be used in protein synthesis, for example in insects. These sequences can give rise to various mammalian cell types, which can be eukaryotic hosts for the production of erythropoietin and similar products, 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 preamble of the application are not intended to limit the scope of the invention, as many modifications and variations are possible in principle, since LNA sequences include cINA and LNA genome sequences. Thus, they may code for EPO, as in Example 12, as well as for its fragments and analogs, i.e. EPO-products, which may have the same biological properties as the natural product, but not others.

Thus, it will be appreciated that the ΕΝΑ sequences of the invention include all sequences allowing expression in prokaryotic or eukaryotic host cells, the expression of which is a polypeptide having at least a portion of the primary structure of EPO and one or more of its properties, i.e.:

a) LNA sequences from Tables V and VI,

(b) LNA sequences that hybridize to the sequences in paragraph (a) or fragments thereof; and

(c) INA sequences that hybridize to the sequences in paragraph

(a) and (b) for reasons other than due to the degeneracy of the genetic code.

It should be noted that the existing allelic monkey and human EPO gene sequences are likely to hybridize to the sequences in Tables V and VI or fragments thereof. In addition, the SCEPO and ECEPO genes and the cIRA or INA genome sequences, which encode various EPO fragments and analogs, can also hybridize to the above INA sequences. This hybridization can be performed under the conditions described above, including pricing conditions.

The above examples illustrate the microbial expression of EPO and expression in mammalian cells, with INA being incorporated into a hybrid vector of a bacterial plasmid and a virus, but a wide variety of similar systems could be used. These could be, for example, vectors of homogeneous origin introduced into a variety of bacterial, yeast and mammalian cells in culture, or systems in which no vectors are used, for example using calcium phosphate. It will be appreciated that expression of, for example, monkey INA is performed in monkey or human cell culture, and although exogenous INA is expressed in this case, high levels of expression can be achieved even though it is not INA that originates in the host genome. The expression systems according to the invention further consider the formation of EPO and similar products in the cytoplasm or membrane with subsequent accumulation, possibly also

129 in the periplasmic space of 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, pp. 161-165 (1984).

The improved hybridization methods of the invention have been applied to H1A / IKA, but can also be applied to RNA / RNA and ENA / H1A. Mixed samples can allow great improvement in hybridization with faster polynucleotide isolation. 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 performing hybridization and further processing under restrictive conditions, i.e. 4, preferably 2 ° C from the lowest calculated temperature for each sample. These improvements may deliver results that were not previously expected. It is thus clear that the improvement achieved by the process according to the invention is very substantial since

- 130 pooled samples, which contain four times the number of different sequences, have been successfully used to isolate a single gene sequence from a mixture that contained 1,500,000 phage plaques compared to what has been done so far. This result was achieved essentially in parallel with the published opinion of Anderson and others in the above article, namely that ... this method is impractical and impractical for isolating a gene for mammalian proteins in the absence of adequate RNA.

PATENTS

Claims (35)

Claims o o czx o oc r < (* 1. A DNA strand for use in providing expression of a polypeptide product whose structure at least partially corresponds to the primary structure of human or monkey erythropoietin from Table VI or Table V, or is any variant or derivative thereof with biological activity of erythropoietin, in particular by increasing the production of reticulocytes and red blood cells in the bone marrow and increasing the synthesis of hemoglobin or iron uptake in prokaryotic or eukaryotic host cells, the DNA strand being selected from (a) DNA strands from Table V or VI or from complementary strands for these strands; (b) DNA strands capable of hybridising under specified conditions to DNA strands of (a) or fragments thereof; and (c) DNA strands capable of hybridising to the strands of (a) and (b) for reasons other than for the degeneracy of the genetic code. The DNA strand of claim 1, which encodes human erythropoietin. The cDNA strand of claim 1 or 2. 4. The DNA strand of claim 3, which is a coding strand for monkey erythropoietin. 5. The DNA strand according to claim 4, comprising a protein coding region with the strand shown in Table V. II 6. A DNA strand according to claim 1 or 2. The DNA strand of the genome of claim 6, which is coding for human erythropoietin. 8. The DNA strand of claim 7, which comprises a coding region for a protein whose strand is shown in Table VI. The DNA strand of claim 1 or 2, which is covalently bound to a detectable label. The DNA strand of claim 9, which is a strand covalently bound to a radiolabelled substance. The DNA strand of any one of claims 9 or 10, wherein the strand is a single strand. 12. The DNA strand of claim 1 which is encoded _at ~ 15 zq for the (Phe) hEPO, (Phe) hEPO, (Phe ¹⁴⁵ ) hEPO, (His ⁷ ) hEP0, / Asn ² -des-Pro ^2- Ile ⁶ / hEPO, / des-Thr ¹⁶³ to Arg ¹⁶⁶ / hEP0 or (delta 27-55 / hEPO. A prokaryotic or eukaryotic host cell which has been transformed or transfected using a DNA strand according to any one of claims 1, 2, 3, 5, 7 or 8, and express the polypeptide of interest as a product. The prokaryotic or eukaryotic cell of claim 13, which is capable of glycosylating said polypeptide. 15. A prokaryotic or eukaryotic cell according to III of claim 14, which is a mammalian cell after transfection or transformation. The host cell of claim 14, which is a COS cell after transformation or transfection has been performed. The host cell of claim 14, which is a CHO cell after transformation or transfection has taken place. A biologically functional circular plasmid or vector of viral origin containing DNA comprising a strand according to any one of claims 1, 2, 3, 6, 7, 8 or 12. A prokaryotic or eukaryotic host cell that has been transformed or transfected with the biologically functional DNA vector of claim 18. 20. A polypeptide with part or all of the chain corresponding to the primary structure of human or monkey erythropoietin of Table VI or V, or any allelic variant or derivative having a biological effect in the bone marrow to increase reticulocyte and red blood cell production and increase hemoglobin synthesis or iron uptake wherein the polypeptide is the product of expression of an exogenous DNA strand in prokaryotic or eukaryotic cells. The polypeptide of claim 20, which is the product of expression of an exogenous DNA strand in eukaryotic cells. The glycoprotein type polypeptide of claim 20, having an average carbohydrate composition that is different from that of human erythropoietin that has been isolated from urine. IV A polypeptide according to any one of claims 20, 21 or 22, therein? the exogenous DNA strand is the cDNA strand. The polypeptide of any one of claims 20, 21 or 22, wherein the exogenous DNA strand is a DNA strand λ genome. A polypeptide according to any one of claims 20, 21 ¹ or 22, wherein the exogenous DNA strand is stored in DNA of a circular plasmid or viral origin vector capable of autonomous replication. A polypeptide according to any one of claims 20 to 25, which is covalently bound to a detectable label. The polypeptide of claim 26, which is covalently bound to a detectable radiolabelled substance. The polypeptide of claim 20, which is the DNA chain product of claim 1 or claim 2 in prokaryotic or eukaryotic cells. A method of producing a polypeptide product whose structure at least partially corresponds to the primary structure of human or monkey erythropoietin from Table VI or from Table V * or any variant or derivative thereof having biological activity of erythropoietin, in particular with an effect on increasing reticulocyte production and red blood cells in the bone marrow and increase in hemoglobin synthesis or iron uptake, characterized in that a prokaryotic or eukaryotic host cell that has been transformed or transfected with the DNA strand of claim 1 is grown under appropriate conditions in a suitable culture medium. of claim 2, and expressing the respective polypeptides, whereupon optionally the desired polypeptide product of the expression of the DNA strand in said cells is isolated. 30. The method of claim 29, wherein the host cell of claims 13 to 17 is grown under suitable conditions. An erythropoietin pharmaceutical composition comprising, as an active ingredient, a polypeptide obtained by the method of claims 29 or 30 and a pharmaceutically acceptable carrier, diluent or excipient. 33. A pharmaceutical composition according to claim 32, wherein the active ingredient is a polypeptide of claims 20 to 28. 34. An antibody having immunological reactivity against erythropoietin and a synthetic polypeptide whose structure essentially corresponds to the primary structure of a continuous chain of amino acid residues in naturally occurring urinary erythropoietin, with the exception that the polypeptide comprises the whole a chain of amino acid residues A-P-P-R-L-I-C-D-S-R-V-L-E-R-Y-L-L-E-A-K-E-A-E-N-I-I. The antibody of claim 34, which is a monoclonal antibody. The antibody of claim 34, which is a polyclonal antibody. . VI The antibody of claim 34, which is immunoreactive against erythropoietin and a synthetic polypeptide whose chain is selected from any of the following chains: V-P-D-T-V-N-F-Y-A-W-R-M-E-V-G, K-E-A-I-S-P-D-D-A-S-A-A a In-Y-S-N-F-L-R-G-K-L-K-L-Y-T-G-E-A-C-R-T-G-D-R.