DD251786A5 - Process for the preparation of a polypeptide - Google Patents

Abstract

A method of producing a polypeptide having some or all of the primary structural design and biological function (s) of the naturally occurring erythropoietin, characterized by (a) breeding - under appropriate conditions - procaryotic or eucaryotic host cells, reshaped or transferred with a biologically active , circular plasmid, or a viral DNA vector, including purified and isolated DNA sequence coding for the procaryotic or eucaryotic host expression of a polypeptide having some or all of the primary structural design and one or more biological properties of erythropoietin and (b) the Isolation of the desired polypeptide products of the expression of the DNA sequences in said vector.

Description

Field of application of the invention

The invention relates to a method for producing a polypeptide. In general, the invention relates to the manipulation of genetic materials, in particular to recombinant methods that enable the production of polypeptides that have partial or total primary structural organization and / or one or more of the biological properties of naturally occurring erythropoietin.

Characteristic of the known technical solutions

A. Manipulation of genetic materials

Genetic materials can be broadly defined as those chemical substances that program and direct the production of cellular components and viruses, and direct the response of cells and viruses. A long-chain polymeric substance known as deoxyribonucleic acid (DNA) contains the genetic material of all living cells and viruses, except for certain viruses programmed by ribonucleic acids (RNA); The repeating units in DNA polymers are four different nucleotides, one of which is either a purine (adenine or guanine) or a pyrimidine (thymine or cytosine) found on a deoxyribose sugar to which a phosphate group is attached. Addition of nucleotides in linear form occurs via the fusion of 5'-phosphates of one nucleotide to the 3'-hydroxy group of another. Functional DNA occurs in the form of stable double-stranded assemblies of single-stranded nucleotides (known as deoxyoligonucleotides) resulting from hydrogen bonding between purine and pyrimidine bases (eg, "complementary" assemblages found between either adenine [A] and thymine [T By convention, the nucleotides are designated by the names of their constitutional purine or pyrimidine bases, and the complementary aggregations of the nucleotides in the double-stranded DNA (eg, - A-T and GC) are referred to as " Base pairs ". Ribonucleic acid is a polynucleotide consisting of adenine, guanine, cytosine and uracil (U) instead of thymine attached to ribose and a phosphate group.

In summary, the programming function of DNA is generally accomplished by a process in which specific DNA nucleotide sequences (genes) are "transcribed" into relatively unstable messenger RNA (mRNA) polymers, conversely, which serves as a template for the formation of structural amino acid regulatory and catalytic proteins. This mRNA "translation" process involves the operation of small RNA strands (tRNA) that transport and align single amino acids along the mRNA strand to allow the formation of polypeptides in proper amino acid sequence. The mRNA "message", derived from the DNA and forming the basis for tRNA support, and the orientation of any given one of the twenty amino acids for polypeptide "expression", is in the form of "lodons" - consecutive moieties In this sense, the formation of a protein is the last form of "expression" of the programmed genetic message present in the nucleotide sequence of a gene. "Promoter" DNA sequences usually "precede" a gene in a DNA polymer and provide a site for initiation of titering in mRNA. "Regulator" DNA sequences, also usually upstream of the (e.g., precursor) gene in a given DNA polymer, bind proteins that determine the frequency (or rate) of transcription initiation.

Commonly referred to as a "promoter / regulator" or "control" DNA sequence, these sequences, which precede a selected gene (or set of genes) in a functional DNA polymer, work together to determine whether transcription (and eventually expression) of a gene takes place. DNA sequences "following" a gene in a DNA polymer and providing a signal for cessation of transcription into mRNA are referred to as transcriptional terminator sequences.

The focus of microbiological processes in the last decade has been the attempt to produce industrially and pharmaceutically significant substances using organisms which either initially did not contain genetically encoded information regarding the desired product in their DNA or (in the case of human cells in culture) usually no chromosomal gene with corresponding Express content. Simplified, a gene that specifies the structure of a desired polypeptide product is either isolated from a "donor" organism or chemically synthesized and then stably introduced into another organism, preferably a self-replicating, multicellular organism such as bacteria , Yeast or mammalian cells in culture.

Once this has been done, the existing gene expression mechanism in the "transformed" or "transferred" microwell host cells is used to construct the desired product, using the exogenous DNA as a template for the transcription of mRNA, which is then transformed into a continuous sequence of amino acid residues is translated.

From the prior art there is a numerous patent and literature publication on "recombinant DNA" technology for isolating, synthesizing, purifying, and amplifying genetic materials for use in the transformation of selected host organisms, for example, U.S. Patent No. 4,237,224 (Cohen et al. ) the transformation of unicellular host organisms with "hybrid" viral or circular plasmid DNA, including selected exogenous DNA sequences. Among the procedures of the Cohen patent is the preparation of a transformation vector by enzymatic cutting of viral or circular plasmid DNA to form linear DNA strands. Selected foreign ("exogenous" or "heterologous") DNA strands, which usually include sequences encoding the desired product, are prepared in linear form through the use of similar enzymes. The linear viral or plasmid DNA is incubated with the foreign DNA in the presence of ligating enzymes capable of effecting a recovery process, and "hybrid" vectors, including the selected exogenous DNA segment, are spliced into the viral or circular DNA plasmid.

Transformation of compatible unicellular host organisms with the hybrid vector results in the production of replicated copies of the exogenous DNA in the host cell population. In many cases, the desired result is simply the amplification of the foreign DNA, and the recovered "product" is the DNA. It has recently been reported that the ultimate goal of the transformation is the expression by the host cells of the exogenous DNA in the form of a large-scale synthesis of isolatable amounts of commercially significant, coding therefor protein or polypeptide fragments by the foreign DNA, see also, for example, US Patent 4264731 (Shine), 4273875 (Manis), 4293652 (Cohen) and EP-OS 093619, published on 9 November 1983.

The development of specific DNA sequences for splicing into DNA vectors is accomplished via a variety of techniques, largely depending on the degree of "foreignness" of the "donor" to the intended host and the size of the polypeptide to be expressed in the host. To the risk of over-simplification, it can be stated that three alternative principal methods can be used: (1) the "isolation" of the double-stranded DNA sequence from the genomic DNA of the donor; (2) the chemical production of a DNA sequence and (3) the in vitro synthesis of a double-stranded DNA sequence by enzymatic "reverse transcription" of mRNA isolated from donor cells. The latter methods, which include the formation of a DNA "complement" of mRNA, are commonly referred to as "cDNA" methods.

The production of DNA sequences has recently been termed the method of choice when the entire sequence of amino acid residues of the desired polypeptide product is known. DNA production process of the co-pending, co-pending U.S. Patent Application No. 483451 to Alton et al. (filed on April 15, 1983 and corresponding to PCT US 83/00605, published on November 24, 1983 as WO-83/04053 see, for example, further means for achieving such highly desirable results as: providing alternating codons recently disclosed in U.S. Pat Genes have been found that are highly expressed in the host organisms selected for expression (eg, providing yeast or Eccoli "preferred" codons) Avoiding the presence of untranslated "intron" sequences (usually in human genomic DNA). Sequences and mRNA transcodes thereof) that are not readily processed by procaryotic host cells; avoiding the expression of unwanted "leade" polypeptide sequences, which are commonly encoded by genomic DNA and cDNA sequences, but not recently of the polypeptide of interest be cleaved by bacterial or yeast host cells, providing a slight insertion of the DNA into appropriate expression vectors in association with desired promoter / regulator and terminator sequences; and providing a lightweight construction of genes encoding polypeptide fragments and analogs of the desired polypeptides.

If the entire sequence of amino acid residues of the desired polypeptides is not known, direct production of the DNA sequences is not possible, and the isolation of DNA sequences encoding the polypeptide by a cDNA method becomes the method of choice, regardless the potential decreases in scope of the set of expression vectors capable of realizing a high level of microwave expression reported above. Standard methods for isolating cDNA sequences of interest include the preparation of plasmid-derived cDNA "libraries" derived from reverse transcription of mRNA rich in donor cells selected to be responsible for a high level of expression of genes (e.g. Libraries of cDNA derived from pituitary cells expressing relatively large amounts of growth hormone products.

Where substantial amounts of the amino acid sequences of the polypeptide are known, labeled single-stranded DNA probing sequences can be used which duplicate a sequence presumably present in the "target" cDNA in DNA / DNA hybridization procedures performed on cloned heads of the cDNA which have been degraded into a single-stranded form (see in general the disclosure and discussion of prior art in Weissman et al., U.S. Patent 4,344,443, and the recent demonstration of the use of long oligonucleotide hybridization probes described by Wallace et al. in Nuc. Acids Res. 6, pp. 3543-3557 [1979] and Reyes et al., PNAS [USA], 79, pp. 3270-3274 (1982) and Jaye et al., Nuc. Acids Res. 11, p See also U.S. Patent No. 4,358,535 [Falkow et al.], Which relates to DNA / DNA hybridization methods for effecting diagnosis; Published European Patent Application Nos. Nos. 4,325,23335 [1983]. 0070685 and 0070687 concerning the light-emitting labels on single-stranded polynucleotide probes; Davis et al. "A Manual for Genetic Engineering, Advanced Bacterial Genetics", Coldspring Harbor Laboratory, Cold Spring

Harbor, N.Y. [1980], pages 55-58 and 174-176, for colony and plaque hybridization techniques; and New England Nuclear (Boston, Mass.) brochures for "Gene Screen" hybridization transfer membrane materials with instructions for the transfer and hybridization of DNA and RNA, Catalog No. NEF-972].

Among the most significant recent advances in hybridization techniques for screening recombinant clones is the use of labeled mixed synthetic oligonucleotide probes, each of which is potentially the full complement of a specific DNA sequence in the hybridization probe, including a heterogeneous mixture of single-stranded DNA's or RNA's. These procedures have been confirmed to be particularly useful in the detection of cDNA clones derived from such sources as to allow extremely low levels of mRNA sequences for the polypeptides of interest. In summary, the use of harsh hybridization conditions directed to the avoidance of non-specific binding may e.g. For example, autoradiographic visualization of a specific cDNA clone in the case of hybridization of the target DNA to this single probe within the mixture which is its complete counterpart. See generally, Wallace et al., Nucl. Acids Res. 9, pp. 879-897 (1981); Suggs et al., P.N.A.S. (USA), 78 p.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, p.2196-2200 (1983); and Kornbluth et al., P.N.A.S. (USA) 80, p.3218-3222 (1983). Generally, the mixed probe methods of Wallace et al. (1981), p. o. by various investigators expanded to the point where reliable results were presumably obtained in cDNA clone isolation using a 32-part mixed pool of 16-base-long (16-mer) oligonucleotide probes of uniform, distinct DNA sequences with a single 11-mer to effect a two-site "positive" confirmation of the presence of cDN A of interest. See Singer-Sam et al., P.N.A.S. (USA), 80, p.802-806 (1983).

The use of genomic DNA isolates is the last common of the three above-mentioned methods for the development of specific DNA sequences for use in recombinant methods. This is particularly true in the field of recombinant methods directed to securing microwell expression of mammalian polypeptides and, in principle, does justice to the complexity of mammalian genomic DNA. Thus, while providing reliable procedures for the development of phagogenic libraries of human and other mammalian genomic DNA (see, e.g., Lawn et al., Cell 15, pp. 1157-1174 (1978) for methods for generating a human gene library, Kam et al., PNAS [USA] 77, pp. 5172-5176 (1980) for a human gene library based on alternative restriction endonuclease fragmentation method; and Blattner et al., Science, 196, p -169 [1977], which describes the construction of a bovine gene library), there are relatively unsuccessful attempts to use hybridization techniques to isolate genomic DNA in the absence of extensive prior knowledge of amino acid or DNA sequences.

As an example, Fiddes et al., J. Mol. And Appl. Genetics, 1, pp. 3-18 (1981) reported the successful isolation of a gene encoding the aipha subunit of the human pituitary glycoprotein hormone from the Maniatis library by using a "full-length" probe, a full 621 base pair Fragments of a previously isolated alpha subunit cDNA sequence.

As another example, Das et al., P.N.A.S. (USA), 80, pp. 1531-1535 (1983) for the isolation of human genomic clones for human HLA-DR using a 175 base pair synthetic oligonucleotide. Finally, Anderson et al., P.N.A.S. (USA), 80, pp. 6838-6842 (1983) for the isolation of a genomic clone for a bovine pancreatic trypsin inhibitor (BPTI) using a single probe of 86 base pairs in length and constructed according to the known amino acid sequence of BPTI. The authors observe a determination of low prospects for the isolation of mRNA suitable for the synthesis of a cDNA library, because of apparently low levels of mRNA in initially "targeted" parotid gland and lung tissue, and then direct the chances of success to probing a gene mRNA. Library using a mixture of labeled probes with the statement:

"More generally, mixed sequence oligodeoxynucleotide probes were used to isolate protein sequences of unknown sequence from cDNA libraries. Such probes are usually mixtures of 8-32 oligonucleotides, 14-17 nucleotides in length, representing each possible codon combination for a small stretch (5-6 residues) of the amino acid sequence. Under severe hybridization conditions that penalize non-correct base pair probes, these probes are able to detect the location of specific gene sequences in low to moderate complexity clone libraries. Nevertheless, because of their short length and heterogeneity, mixing probes often do not have the specificity required for probing sequences as a complex as a mammalian genome. This makes such a method unsuitable for isolation of mammalian protein genes if the corresponding mRNAs are not available. "(End of Time).

Thus, there continues to be a need for improved methods that provide rapid and efficient isolation of cDNA clones in cases where little is known about the amino acid sequence of the polypeptide encoding it and where "enriched" tissue material of mRNA is not readily available for use Such improved methods would be particularly useful if they were to be useful for the isolation of mammalian genomic clones where only dispersed information is available regarding the amino acid sequences of the polypeptides therefor by the genes of interest

 B. Erythropoietin as a polypeptide of interest

Erythropoiesis, the production of red blood cells, occurs continuously during human life to compensate for cell degeneration. The erythrocyte formation is a very well-controlled physiological mechanism that allows a sufficient number of red blood cells in the blood to supply the tissue with oxygen, but not so many that the cells would hinder circulation. The formation of red blood cells takes place in the spinal cord and is controlled by the hormone erythropoietin.

Erythropoietin, an acidic glycoprotein of about 34,000 daltons in molecular weight, can occur in three forms: α, β and asialo. The α and β forms differ slightly in the carbohydrate components but have the same efficacy, biological activity and molecular mass. The asialo form is an a- or / 3-form in which the terminal carbohydrate (sialic acid) is removed. Erythropoietin is present in very low concentrations in the plasma when the body is in a healthy state, whereby the tissue receives sufficient oxygen over the existing number of erythrocytes. This normal low concentration is sufficient to stimulate the replacement of the red blood cells, which are usually cast by aging.

The amount of erythropoietin in the circulation increases under the conditions of tissue oxygen deficiency (hypoxia) when oxygen transport through the blood cells in the bloodstream is reduced. Hypoxia can be caused by loss of large quantities of blood due to hemorrhage, destruction of red blood cells by overdose of radiation, reduction of high levels of oxygen intake or prolonged unconsciousness, or by various forms of anemia. In response to tissue hypoxic stress, erythropoietin increases red blood cell production by stimulating the conversion of simple precursor cells in the spinal cord into pro-erythroblasts that later mature, synthesize hemoglobin, and are released into the bloodstream as red blood cells. If the number of red blood cells in the circulation is higher than required for the normal oxygen requirements of the tissue, the erythropoietin is reduced in the circulation. See, in general, Testa et al., Exp. Hematol., 8 (Suppl.8), 144-152 (1980); Tong et al., J. Biol. ex., 256 (24), 12666-12672 (1981); Gold Water, J. Cell. Physiol., 110 (Supp.1), 133-135 (1982); Finch, Blood, 60 (6), 1241-1246 (1982); Sytowski et al., Expt. Hematol., 8 (Supp.8), 52-64 (1980); Naughton, Ann. Clin. Lab. Sci., 13 (5), 432-138 (1983); Weiss et al., Am. J. vet. Res., 44 (10), 1832-1835 (1983); Lappin et al., Esp. Hematol. 11 (7), 661-666 (1983); Bacin et al., Ann. N.Y.Acad. Sci., 414, 66-72 (1983); Murphy et al., Acta Haematologica Japonica, 46 (7), 1380-1396 (1983); Dessypris et al., Brit. J. Haematol., 56, 295-306 (1984); and Emmanoul et al., Am. J. Physiol., 247 (1 pt 2), F 168-76 (1984).

Since erythropoietin is essential in the process of red blood cell production, the hormone has a potentially useful application in both the diagnosis and treatment of blood disorders characterized by low or defective red blood cell production. See, in general, Pennathur-Das et al., Blood 63 (5), 1168-1171 (1984) and Haddy, at Jour. Ped. Hematol./Oncol. 4,191-196 (1982), which relates to erythroproietin for possible sickle cell anemia therapies, and Eschbach et al., J. Clin. Invest., 74 (2), pp. 434-441 (1984) describing a therapeutic regimen for uremic sheep based on in vivo responses to erythropoietin-rich plasma infusions, and a proposed dose of 10 units EPO / kg per day for 15 to 40 days as a corrective of anemia associated with chronic renal failure. She also cranes, Henry Ford Hosp., Med., 31 (3) 177-181 (1983).

It has recently been estimated that the availability of erythropoietin in amounts would allow the anemia treatment of 1,600,000 people in the United States alone. See, for example, For example, Morrison "Bioprocessing in Space - an Overview", pp. 557-571 in The World Biotech Report 1984, Vol 2: USA (Online Publications, New York, NY, 1984). Recent studies have provided a basis for planning the effectiveness of Erythropoietin therapy in a variety of disease states, diseases and conditions of hematological irregularity:

Vedovato, et al., Acta. Haematol. 71, 211-213 (1984) (beta-thalassemia); Vichinsky, et al., J. Pediatr., 105 (1), 15-21 (1984) (cystic fibrosis); Cotes, et al., Brit. J. Obstet. Gyneacol., 90 (4), 304-311 (1983) (pregnancy, menstrual cramps); Hage, et al., Acta. Pediatr. Scand., 72, 827-831 (1983) (early anemia of prematurity); Claus-Walker, et al., Arch. Phys. Med. Rehabil., 65, 373-374 (1984) (spinal cord injury) Dunn, et al., Eur. J. Appl. Physiol., 52, 178-182 (1984) (spaceflight); Miller, et al., Brit. J. Haematol., 52, 455-590 (acute blood loss); Udupa, et al., J. Lab. Clin. Med., 103 (4L 574-580 and 581-588 (1984); and Lipschitz, et al., Blood, 63 (3), 502-509 (1983) (aging); and Dainiak, et al., Cancer. 51 (6), 1101-1106 (1983) and Schwartz, et al., Otolaryngol., 109, 269-272 (1983) (various neoplastic disease states accompanied by abnormal erythropoiesia).

Previous attempts to recover erythropoietin in good yield from plasma or urine have been relatively unsuccessful. There are complicated and cumbersome laboratory procedures required, which generally resulted in the recovery of only very small amounts of impure and unstable erythropoietin containing extracts.

Initial attempts to isolate erythropoietin from urine have produced unstable, biologically inactive preparations of the hormone. US 3865801 describes a method for stabilizing the biological activity of a crude substance containing urine-derived erythropoietin. The resulting crude preparation containing erythropoietin apparently retains 90% of the erythropoietin activity and is stable.

Another purification method of human erythropoietin from urine of patients with aplastic anemia is described by Miyake et al., In J. Biol. Chem., Vol. 252, No. 15 (August 10, 1977) pp. 5558-5564. The seven-step procedure involves ion exchange chromatography, ethanol precipitation, gel filtration, and adsorption chromatography, yielding a pure erythropoietin preparation with a potency of 70,400 units / mg protein in 21% yield. U.S. Patent No. 4,379,840 (Takezawa et al.) Describes methods for making "an erythropoietin product" from urine samples of healthy humans with weakly basic ion exchangers and states that the resulting low molecular weight product "has no inhibitory effects against erythropoietin."

GB 2,085,887 (Sugitomo et al.), Published May 6, 1982, describes a method for producing hybrid human lymphoblastoid cells and reports production levels ranging from 3 to 420 units of erythropoietin per ml of cell suspension (distributed in the cultures after mammalian host propagation containing up to 10 ⁷ cells per ml, the best results reported to reach them were that the production rate of erythropoietin was 40 to 4000 units / 10 ⁶ cells / 48 hours in in vitro culture following cell transfer from in vivo propagation systems. (See also U.S. Patent No. 4,377,513.) Numerous proposals have been made for the isolation of tissue erythropoietin, including neoplastic cells, but the yields have been See, for example, Jelkman, et al., Ex. Hematol., 11 (7), 581-588 (1983), Tambourin, et al., PNAS (USA), 80, 6269-6273 (1983). Katsucka, et al., Gann, 74, 534-541 (1983); Hagiwara, et al., Blood, 63 (4), 828-835 (1984); and Choppin, et al., Blood 64 (2), 341-347 (1984). Other isolation methods used to obtain purified erythropoietin include immunological procedures. An anti-erythropoietin-directed polyclonal serum-derived antibody was developed by injecting an animal, preferably a rat or rabbit, with human erythropoietin. The injected human erythropoietin is recognized by the animal's immune system as a foreign substance and triggers the production of antibodies to the antigen. Different cells that respond to stimulation by the antigenic substance produce and release antibodies that are slightly different from those of other reacting cells. The antibody activity is retained in the animal's serum when the blood is extracted. While unpurified semm or antibody preparations, purified as a semm immunoglobulin G fraction, can then be used in assays to determine and complex with human erythropoietin, the materials have a significant drawback. This serum antibody, composed of all the different antibodies produced by single cells, is polyclonal in nature and will complex with components in crude extracts, unlike erythropoietin alone. Of interest to the background of the present invention are also recently known

Advances in the field of developing continuous cell cultures capable of producing a single type of antibody that is specifically immunologically reactive with a single antigenic determinant of a selected antigen. See, generally, Chisholm, High Technology, Vol.3, No. 1, 57-63 (1983).

Attempts have been made to use cell fusion and hybridization techniques to employ "monoclonal" antibodies in the isolation and quantitation of human erythropoietin By way of example, a report appeared on the successful development of mouse-mouse hybridoma cell lines containing human monoclonal antibodies Prodr, 41, 520 (1982) As another example, a detailed description of the preparation and use of monoclonal anti-erythropoietin antibodies by Weiss et al. See also Sasaki, Biomed., Biochim, Acta., 42 (11/12), 5202-5206 (1983), Yanagawa, et al., Blood 64 (2)., PNAS (USA) 79, 5465-5469 (1982). , 357-364 (1984), Yanagawa, et al., J. Biol. Chem., 259 (5), 2707-2710 (1984), and U.S. Patent 4,665,624. Background of the invention are also of interest to reports the immunological activity of synthetic peptides in natural occurring proteins, glycoproteins and nucleoproteins substantially double existing amino acid sequences. In particular, relatively low molecular weight polypeptides have been shown to participate in immune responses that are similar in duration and extent to the immune responses of physiologically important proteins, such as viral antibodies, polypeptide hormones, and the like. Immune responses of such polypeptides also include provoking the formation of specific antibodies in immunologically active animals. See, e.g. Lerner et al. Cell, 23, 309-310 (1981); Ross, et al., Nature, 294, 654-656 (1981); Walter, et al., P.N.A.S. (USA), 77, 5597-5200 (1980); Lerner, et al., P.N.A.S. (USA), 78, 3403-3407 (1981); Walter, et al., P.N.A.S. (US), 78, 4882-886 (1981); Wong, et al., P.N.A.S. (USA), 78, 7412-7416 (1981); Green, et al., Cell, 28, 477-487 (1982); Nigg, et al., P.N.A.S. (USA), 79, 5322-5326 (1982); Baron, et al., Cell, 28, 395-404 (1982); Dreesman, et al., Nature, 295, 158-160 (1982); and Lerner, Scientific American, 248, no. 2,66-74 (1983). See also Kaiser, et al., Science, 223, pp. 249-255 (1984) for biological and immunological activities of synthetic peptides that approximately share the secondary structures of the peptide hormones but not their primary structural arrangement. The above studies, of course, relate to amino acid sequences of proteins other than erythropoietin, a substance for which no essential amino acid sequence has been previously described. In co-owned U.S. Patent Application No. 467244, filed on February 4, 1983 by J. Egrie, published August 22, 1984, EP-OS 0116446 discloses a mouse-mouse hybridoma cell line (ATCC HB 8209 ), which produces a highly specific, monoclonal, anti-erythropoietin antibody that is also specifically immunoreactive with a polypeptide consisting of the following sequence of amino acids: NH 1 -Ala-Pro-Pro-Arg-Leu-Ile-Cys Asp-Ser-Arg-Val-Leu-Glu-Arg-Tyr-Leu-Leu-Glu-Ala-Lys-COOH.

The polypeptide sequence is one attributable to the first twenty amino acid residues of mature human erythropoietin prepared by the method of Miyake et al., J. Biol. Chem. 252, 5558-5564 (1977) and for which amino acid analysis was performed by gas-phase sequencer (Applied Biosystems, Inc.) according to the procedure of Hewick, M. et al., J. Biol. Chem., 256, 6990- 7997 (1981). See also Sue et al., Proc. Nat. Acad. Be. (USA), 80, pp. 3651-3655 (1983) regarding the development of polyclonal antibodies to a synthetic 26-mer based on a different amino acid sequence and Sytowski et al., J. Immunol. Methods, 69, p. 181-186 (1984).

While polygonal and monoclonal antibodies, as described above, lead to very useful products for use in immunoassays for the determination and quantification of erythropoietin and are used in the affinity purification of erythropoietin, it is unlikely that these materials are readily available for large scale isolation of larger quantities of erythropoietin can be used from mammalian material sufficient for further analysis, clinical investigation, and potentially far-reaching therapeutic use of the substance in the treatment of, for example, chronic kidney disease where diseased tissue is unable to sustain the production of erythropoietin. It is therefore believed in the art that the best prospects for fully labeling mammalian erythropoietin and providing it with large amounts for potential diagnostic and clinical use is to successfully use the recombinant techniques to allow for large scale microbial synthesis of the compound.

While it appears that substantial progress has been made in attempting to isolate DNA sequences encoding human and other mammalian species erythropoietin, it does not appear to have been so successful. This is principally due to the lack of tissue material, particularly human tissue material, which is mRNA-enriched and would allow to construct a cDNA library from which an erythropoietin-encoding DNA sequence could be isolated by standard techniques. In addition, the sequence of amino acid residues of erythropoietin is so little known that it is not possible, for example, to construct long polynucleotide probes capable of reliable use in DNA / DNA hybridization screening of cDNA and in particular genomic DNA libraries to become.

By way of illustration, the 20 amino acid sequence used to produce the above-mentioned monoclonal antibody produced by ATCC No. HB8209, no construction of a unambiguous 60 base pair oligonucleotide probe as described by Anderson et al., Ibid Wise. It is estimated that humanity for erythropoietin appears as a "single copy gene" within the human genome, and in any event, the genetic material encoded for human erythropoietin is likely to be less than 0.00005% of the total human genomic DNA, which would be present in a gene library.

To date, the most successful of the literature-based attempts at recombinant-related methods to provide DNA sequences for use in microwell expression of isolatable ³ levels of mammalian erythropoietin have been far ahead of the target. As an example, Farber et al., Exp. Hematol. 11, Supp. 1.4, Abstract 101 (1983) on the extraction of mRNA from kidney tissue from phenylhydrazine-treated baboons and the injection of mRNA into Xenopus ovary eggs with the rather volatile result of the in vitro production of a mixture of "translation products", including biological properties Recently, Farber et al., Blood, 62, No. 5, Suppl. No. 1, Abstract 392 on page 122a (1983) reported the in vitro translation of human mRNA frog egg cell mRNA It was estimated that the resulting translation product mixture contained on the order of 220 (milliunits) (mU) of a translation product with the activity of erythropoietin per microgram of injected mRNA, while those levels were confirmed to be rather low by the in vitro translation of exogenous mRNA ( compared to the previously reported levels of baboon mRNA

Translation into the product sought), it was believed that the results would confirm the human kidney as the site of erythropoietin expression and allow the construction of an enriched human kidney cDNA library from which the desired gene could be isolated (see also Farber, Clin. Res., 31 [4], 769A [1983]). Since filing US Patent Application Nos.561024 and 582185, a single report on cloning and expression has appeared, claiming to have obtained human erythropoietin cDNA in E. coli. A number of cDNA clones were inserted into E. coli plasmids and β-lactamase fusion products were found to react immunoreactively with a monoclonal antibody to an unspecified "epitope" of human erythropoietin. See Lee-Huang, Proc.Nat.Acad.Sci. (USA), 81, p.2708-2712 (1984).

Object of the invention

The object of the invention is to provide a novel purified and isolated polypeptide having some or all of the primary structural organization and one or more biological properties of naturally occurring erythropoietin.

Explanation of the essence of the invention

The invention has for its object to find new peptides with the desired properties and methods for their Herstellungsu ng for the manipulation of genetic materials.

The invention provides primarily novel purified and isolated polypeptide products that contain some or all of the primary structural organization (ie, the contiguous sequence of amino acid residues) and one or more biological properties (eg, immunological properties and in vivo and in vitro biological activity) of naturally occurring erythropoietin, including allelic variants thereof. These polypeptides are also characterized solely by being the product of procaroytic or eurocardial host expression . By bacterial, yeast and mammalian cells in culture) of exogenous DNA sequences obtained by genomic or cDNA cloning or by gene synthesis. Products of microbial expression in vertebrate (e.g., mammalian and avian) cells may be further characterized as being free from association with human or other contaminants found in their natural mammalian cell environment or in extracellular fluids such as plasma or human Urine may be associated with erythropoietin. The products of typical yeast (B. saccaromyces cerevisiae) or procaryotic (e.g., E. coli) host cells are free of association with any mammalian proteins. Depending on the host used, the polypeptides of the invention may be glycosylated with mammalian or other eucaryotic carbohydrates or remain unglycosylated. The polypeptides of the invention may also have an initial methionine amino acid residue (at position-1).

Novel glycoprotein products of the invention include those which have a primary structural arrangement with sufficient duplicability from a naturally occurring (eg, human) erythropoietin to yield one or more of the biological properties of that compound and have an average carbohydrates composition derived from naturally occurring (eg, human) erythropoietin. The vertebrate animals (eg, COS-1 and CHO) cells provided by the present invention are among the first ever available cells that can be propagated continuously in vitro and that are capable of growing in their growth medium in excess of 100E (preferably in excess of 500E and most preferably in excess of 1000 to 5000E) to produce erythropoietin per 10 ⁶ cells in 48 hours, as determined by radioimmunoassay. Further, the present invention provides synthetic polypeptides, in whole or in part duplicatively, of contiguous sequences of erythropoietin amino acid residues, which are described herein for the first time. These sequences may possess biological activity and / or immunological properties as a result of involvement of primary, secondary or tertiary structural or dispositional characteristics with naturally occurring erythropoietin, as well as the naturally occurring product, so as to be used as biologically active or immunological substitutes for erythropoietin in therapeutic and immunological procedures can be. Accordingly, provided by standard methods, monoclonal or polyclonal antibodies that are immunoreactive with such polypeptides, preferably also immunoreactive with naturally occurring erythropoietin.

The present invention illustrates monkey or human cloned DNA sequences and polypeptide sequences appropriately derived therefrom which are the primary structural arrangement of erythropoietins of their corresponding monkey or human origin.

The invention also relates to novel biologically functional, viral and cyclic plasmid DNA vectors containing the DNA sequences of the invention as well as microbial (e.g., bacterial, yeast and mammalian) host organisms that transform or transfer by intervention of such vectors become. Accordingly, the invention relates to novel methods of producing useful polypeptides by culturing growth of such transformed or transferred microbial hosts under the expression of exogenous, vector-borne DNA sequences on a large scale promoting conditions and isolating the desired polypeptides from the growth medium, cell lysates or cell membrane fractions. Isolation and purification of microbially expressed polypeptides of the invention can be accomplished by conventional methods, including preparative chromatographic separations and immunological separations using monoclonal and / or polyclonal antibody preparations.

Presenting the sequence of the amino acid residues of erythropoietin, the present invention contemplates the total and / or partial production of DNA sequences encoding erythropoietin and having such beneficial characteristics as including non-mammalian hosts for "preferred" codons for expression provided that sites are provided for cleavage by restriction endonuclease enzymes, and the further provision of additional initial, terminal or intermediate DNA sequences that facilitate the construction of easily expressible vectors Accordingly, the present invention relates to the preparation (and development by site-specific mutagenesis of cDNA and genomic

DNA) of DNA sequences encoding the microbial expression of polypeptide analogues or derivatives of erythropoietin that differ from naturally occurring forms by the identity or location of the location of one or more amino acid residues (i.e., deletion analogs that are less than all of the EPO-specifying residues and / or substitution analogs wherein one or more of the specified residues is replaced by other residues and / or addition analogs wherein one or more amino acid residues are attached to a terminal or medium portion of the polypeptide); and participate in some or all of the properties of naturally occurring forms.

The novel DNA sequences of the invention contain all sequences necessary for safe expression in procaryotic or eucaryotic host cells of polypeptide products which comprise at least a portion of the primary structural arrangement and one or more of the biological properties of erythropoietin, which include:

(a) the DNA sequences listed in Tables V and VI or their complementary strands; (b) DNA sequences that hybridize (under hybridization conditions, as discussed herein or more stringent conditions) to DNA sequences as defined under (a), or to fragments thereof; and (c) DNA sequences which, however, would hybridize to DNA sequences as defined under (a) and (b) while degenerating the genetic code. Specifically included in part (b) are genomic DNA sequences that encode allelic variant forms of monkey or human erythropoietin and / or decode other mammalian species of erythropoietin. Specifically included in part (c) are prepared DNA sequences encoding EPO, EPO fragments and EPO analogs whose DNA sequences may contain condons that facilitate the translation of messenger RNA into non-vertebrate hosts.

Further assignable to the present invention is the class of polypeptides encoding portions of the DNA complement to the upper strand of the human genomic DNA sequence of Table VI, i. Nucleic Acids Research, 12, pp. 5049-5049 - (1984) Also included within the invention are pharmaceutical compositions containing effective amounts of polypeptide products of the invention, together with suitable diluents , Adjuncts and / or excipients that permit erythropoietin therapy, particularly for the treatment of anemic conditions, and preferably such anemic conditions as occur in chronic renal failure.

The polypeptide products of the present invention can be "labeled" by covalent binding to a detectable marker (e.g., radiolabeled with ¹²⁵ I) to yield reagents used in the detection and quantification of erythropoietin in solid tissue and liquid samples such as blood or blood The DNA products of the invention may also be labeled with detectable markers (such as radiotranslucers and non-isotopic labels such as biotin) and used in DNA hybridization procedures to determine the erythropoietin gene position and / or gene position of any related human gene family They can also be used to detect erythropoietin gene deficiencies based on DNA content and as a gene marker to identify adjacent genes and their discrepancies.

As will be described in detail below, the present invention involves significant improvements in the methods of determination of a particular single-stranded polynucleotide of unknown sequence in a heterogeneous cellular or viral sample containing multiple single-stranded polynucleotides, wherein

(a) preparing a mixture of labeled, single-stranded nucleotide probes having uniformly varying base sequences, each of said probes being potentially specifically complementary to a sequence of bases which is believed to exist only once in the polynucleotide to be determined;

(b) the sample is fixed on a solid substrate,

(c) treating the substrate with the specimen thereon to prevent further binding of polynucleotides thereto except by hybridization to polynucleotides in the specimen;

(d) the treated substrate, with the sample fixed thereon, being transiently contacted with said mixture of labeled probes under conditions which facilitate hybridization only between fully complementary polynucleotides, and

(c) determining the specific polynucleotide by observing the elimination of a hybridization reaction between it and a fully complementary probe within the mixture of labeled probes, as evidenced by the presence of a higher density of labeled material on the substrate at the site of the specific polynucleotide, as compared to a background density on the labeled material resulting from nonspecific binding of labeled probes to the substrate.

The methods are particularly effective in situations requiring the use of 64,128,256,512,124 or more mixed polynucleotide probes 17 to 20 bases in length in DNA / DNA or RNA / RNA or DNA / RNA hybridizations.

As discussed below, the above improved methods have permitted the recognition of cDNA clones encoding erythropoietin originating from monkey species within a library made from monkey kidney anemic cell mRNA. In particular, a mixture of 128 evenly varied 20-mer probes based on amino acid sequence information derived from sequencing fractions of human erythropoietin was used in colony hybridization procedures to add seven "positive" erythropoietin cDNA clones within a total of 200,000 colonies More notably, the practical use of the improved methods of the present invention allows for the rapid isolation of three positive clones by screening 1500,500 phage plaques representing a human gene library This was achieved using the above mixture of 128 20-mer probes along with a second set of 128 17-mer probes based on an amino acid analysis of a different contiguous sequence of human erythropoietin.

The above illustrative methods constitute the first known case of using multiple mixed oligonucleotide probes in DNA / DNA hybridization methods directed to the isolation of mammalian genome clones and the first known case of using a mixture of more than 32 oligonucleotide probes in isolating cDNA -Clone.

Numerous aspects and advantages of the invention will become apparent to those skilled in the art from the following detailed description, in which practical aspects of the invention will be elucidated in its presently preferred embodiments. In accordance with the present invention, DNA sequences encoding some or all of the polypeptide sequence of human and monkey species erythropoietin (hereinafter sometimes referred to as "EPO") have been isolated and further characterized, the monkey or human DNA has become eucaryotic and procaryotic Expression, resulting in isolatable amounts of polypeptides exhibiting biological (eg, immunological) properties of naturally occurring EPO, as well as in vivo and in vitro biological activities of EPO The monkey species DNA was obtained from a cDNA library isolated with a mRNA obtained from monkey kidney tissue in a chemically induced anemic condition, and whose serum, upon immunological determination, contained high levels of EPO compared to normal monkey serum Isolating the desired cDNA clones, the DNA contained encapsulating EPO, one reached under Einsat z of DNA / DNA colony hybridization using a pool of 128 mixed radiolabeled 20-mer oligonucleotide probes involving rapid screening of 200,000 colonies. The pattern of oligonucleotide probes was stationed on amino acid sequence information obtained by enzymatic fragmentation and sequencing of a small human EPO sample.

Human DNA was isolated from a human genomic DNA library. The isolation of clones containing EPO-encoding DNA was achieved by DNA / DNA plaque hybridization using the above-mentioned pool of 128 mixed 20-mer oligonucleotide probes and a second pool of 128 radiolabelled 17-mer soiides whose sequences Amino acid sequence information obtained from a different human enzymatic EPO fragment.

Positive colonies and plaques were checked for accuracy by dideoxy sequencing of clonal DNA using a subset of 16 sequences within the pool of 20-mer probes, and selected clones were subjected to nucleotide sequence analysis, which elucidated the derivation of primary structural identity of the fragments encoded therewith EPO polypeptides allowed. The deduced polypeptide sequences showed a high degree of homology to each other and to a partial sequence generated by amino acid analysis of human EPO fragments.

A selected positive monkey cDNA clone and a selected positive human gene clone were each inserted into a "shuttle" DNA vector amplified in E. coli and used to transfect mammalian cells into culture. Cultured growth-transfected host cells resulted in preparations of the culture medium supernatant containing more than 300 oM EPO per ml of culture fluid.

embodiment

The following examples are illustrative of the invention and are specifically directed to methods previously performed for the recognition of EPO-encoding monkey cDNA clones and human genomic clones, to methods which result in such recognition, and to sequencing, development of expression systems and immunological verification of EPO expression in such systems.

Specifically, Example 1 is directed to the amino acid sequencing of human EPO fragments and the construction of mixtures of radiolabeled probes based on the results of this sequencing. Example 2 is generally directed to methods involving the detection of positive monkey cDNA clones and provides information for treating the animal and preliminary radioimmunoassay (RIA) analysis of animal sera. Example 3 is directed to the preparation of the cDNA library, colony hybridization screening and verification of positive clones for correctness, DNA sequencing of a positive cDNA clone and generation of a primary structure match (amino acid sequence) information of the monkey EPO polypeptide , Example 4 is directed to methods involving the detection of positive human genomic clones leading to information on the origin of the gene library, about plaque hybridization methods, and checking for the correctness of positive clones. Example 5 is directed to the DNA sequencing of a positive genomic clone and the generation of amino acid sequence information of the human EPO polypeptide, including a comparison of these with the monkey EPO sequence information. Example 6 is directed to methods of constructing a vector-containing EPO-encoding DNA derived from a positive monkey cDNA clone, the use of the vector for transfecting COS-1 cells, and cultured growth of the transferred cells. Example 7 is directed to methods of constructing a vector-containing EPO-encoding DNA derived from a positive human genomic clone, the use of the vector to transfect COS-1 cells, and the cultured growth of the transferred cells. Example 8 is directed to immunoassay procedures performed on the media supernatants obtained by culture growth according to Examples 6 and 7 of transferred cells. Example 9 is directed to the in vitro and in vivo biological activity of microbially expressed EPO of Examples 6 and 7.

Example 10 is directed to development of mammalian host expression systems for human monkey species and human genomic DNA EPO cDNA incorporating Chinese hamster ovary ("CHO") cells, as well as the immunological and biological activities of the products of this expression system as well as the art Labeling of Such Products Example 11 is directed to the preparation of genes encoding human EPO and EPO analogs, which include a number of preferential codons for expression in E. coli and yeast host cells, and the expression systems based thereon relates to the immunological and biological activity profiles of the expression products of the system of Example 11.

example 1 A. Human EPO Fragment Amino Acid Sequencing

Human EPO was isolated from urine and subjected to tryptic degradation resulting in the development and isolation of 17 separate fragments in amounts of approximately 100 to 150 picomoles.

The fragments were arbitrarily assigned numbers and analyzed for amino acid sequences by microsequence analysis using a gas phase sequencer (Applied Biosystems) to obtain the sequence information shown in Table I. Single-letter codes were used therein, and "X" denotes a remainder that was not further specified.

TABLEI

Fragment No. Sequence Analysis Result

T4a A-P-P-R

T4b G-K-L-K

T9 A-L-G-A-Q-K

T13 V-L-E-R

T16 A-V-S-G-L-R

T18 · 'L-F-R

T21 K-L-F-R

T25 Y-L-L-E-A-K

T26 a L-I-C-D-S-R

T26d L-Y-T-G-E-A-C-R

T 27 T-I-T-A-D-T-F-R

T28 E-A-I-S-P-P-D-A-A-M-A-A-P-L-R

T30 E-A-E-X-I-T-T-G-X-A-E-H-X-S-L-N-E-X-I-T-V-P

T31 V-Y-S-N-F-L-R

T33 S-L-T-T-L-L-R

T35 V-N-F-Y-A-W-K

T38 G-Q-A-L-L-V-X-S-S-Q-P-W-E-P-L-Q-L-H-V-D-K

B. Pattern and Construction of Oligonucleotide Probe Mixtures

The amino acid sequences listed in Table I were re-examined in connection with the degeneracy of the genetic code as to whether the mixed probe methods can be applied to DNA / DNA hybridization methods in cDNA and / or genomic DNA libraries. This analysis showed that within fragment # T35 there existed a series of 7 amino acid residues (Val-Asn-Phe-Tyr-Ala-Trp-Lys) which can only be characterized by being one of 128 possible DNAs Sequences that spans 20 base pairs is encrypted. A first set of 128 20-mer oligonucleotides was thus synthesized by standard phosphoamidite methods (see, e.g., Beaucage et al., Tetrahedron Letters, 22, pp. 1859-1862 [1981 solid support according to the sequence listed in Table II.

Table II -VaI Asn Phe Tyr Ala Trp Lys rest CAA TTG AAG ATG CGA ACC TT -5 ' 3 ' T A A A T G G C C

Further analysis showed that within the fragment T38 there existed a series of 6 amino acid residues (Gln-Pro-Trp-Glu-Pro-Leu) on the basis of which a pool of 128 mixed oligonucleotide probe was pooled as listed in Table IM below.

Table III Gin Per Trp Glu Per Leu rest GTT GGA ACC CTT GGA GA -5 ' 3 '. C T C T A G G C C

The oligonucleotide probes were labeled at the 5 'end with gamma- ³² P-ATP, 7500-8000 cc / mmole (ICN) using T ₄ polynucleotide kinase (NEN).

Example 2 A. Monkey treatment procedure and RIA analysis

Female cynomolgus monkeys Macacafascicularias (2.5-3 kg, one and a half to two years old) were treated subcutaneously with a pH 7 solution of phenylhydrazine hydrochloride at a dose of 12.5 mg / kg on the first, third and fifth day. Before each injection, the hematocrit value was observed. On the seventh day, or at the time when the hematocrit level fell below 25% of the initial value, serum and kidneys were withdrawn following administration of 25 mg / kg doses of ketamine hydrochloride. The extracted materials were immediately frozen in liquid nitrogen and stored at -70 ⁰ C.

B. RIA for EPO

Radioimmunoassays were performed for the quantitative determination of EPO in samples according to the following procedures: An erythropoietin standard or an unknown sample was incubated together with antiserum for 2 hours at 37 ^° C. ,

After the two hour incubation, the test tubes were cooled in ice, ¹²⁵ L-labeled erythropoietin added and the tubes incubated at ⁰ ° C for at least 15 more hours. Each assay tube contained 500μΙ incubation mixture consisting of diluted immune sera 50μΙ, lOOOOcpm ¹²⁵ l erythropoietin, δμ, Ι Trasylol and 0 to 250μΙ either of the EPO standards or unknown sample comprising 0.1% BSA-containing PBS was achieved the remaining volume , The antiserum used was the second test blood of a rabbit immunized with 1% pure human urinary erythropoietin preparation. The final dilution of antiserum in the assay was adjusted so that the antibody-bound ¹²⁵ I-EPO exceeded 10-20% of the total input counts. In general, this corresponded to a final dilution of the antiserum of 1: 50,000 to 1: 100,000.

The antibody-bound ¹²⁵ l erythropoietin was precipitated by the addition of 150μΙ staph A. After a 40 minute incubation, the samples were centrifuged and the pellets were washed twice with 0.75 ml of 10 mM Tris-HCl pH 8.2 containing 0.15M NaCl, 2 mM EDTA and 0.05% Triton x-100. The washed pellets were counted in a gamma counter to determine the percentage of ¹²⁵ L erythropoietin binding. The counts bound by pre-immune sera were subtracted from all totals to correct nonspecific precipitation. The erythropoietin content of the unknown samples was determined by comparison with the standard curve.

The above method was applied to monkey serum obtained in part A (supra) and also to the untreated monkey serum. Normal serum levels were tested and showed approximately 36mU / ml, while treated monkey serum contained from 1,000 to 1,700mU / ml.

Example 3 A. Construction of the monkey cDNA library

Messenger RNA was isolated from normal and anemic monkey kidneys using the guanidine thiocyanate method of Chirgwin et al., Biochemistry, 18, p. 5294 (1979), and the poly (A) ⁺ mRNA was separated by two passes of oligo (dT). Cellulose column chromatography purified in "Maniatis et al.," Molecular Cloning, A Laboratory Manual, pp. 197-198, Cold Springs Harbor Laboratory, Cold Springs, Harbor, NY, 1982. The cDNA library was constructed according to a modification of the general procedures of Okayama et al., Mol. and Cell. Biol., 2, pp. 161-170 (1982) The key features of presently preferred methods are as follows:

(1) pUC 8 was used as an isolated vector cut with Pst I and then tailed with OligodT from 60 to 80 bases in length; (2) Hinc II digestion was used to remove the oligo dT tail from one end of the vector; (3) the first strand synthesis and tailing of oligo dG was done according to the published procedure; (4) a Barn HI digest was used to remove the oligo dG tail from one end of the vector; and (5) replacing the RNA strand by DNA in the presence of two linkers (GATCTAAAGACCGTCCCCCCCC and ACGGTCTTTA) in a three-fold molar excess over the oligo dG tailed vector.

B. Colony hybridization method for screening the monkey cDNA library

Transformed E. coli was seeded at a density of 9000 colonies per 10 x 10 cm plate on nutrient medium plates containing 50 micrograms / ml ampicillin. GeneScreen filters (New England Nuclear Catalog No. NEF-972) were pre-wetted on a BHI-CAM plate (Bacto brain heart infusion 37g / L, casamino acids 2g / L and agar 15g / L with 500 micrograms / ml chloramphenicol ) and used to lift the colonies off the plate. The colonies were grown in the same medium for 12 hours to amplify the plasmid copy number. The amplified colonies (colony side above) were treated by placing the filters in rows on 2 pieces Whatman 3 MM paper, saturated with the following solutions in each case:

(1) 50 mM glucose-25 mM Tris-HCl (pH 8.0) -1 mM EDTA (pH 8.0) for five minutes;

(2) 0.5M NaOH for ten minutes; and

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

The filters were then air dried in vacuo at 80 ° C. for two hours.

By treatment with a solution containing 50 micrograms / ml of protease enzyme in buffer K [0.1 M Tris-HCl (pH 8, 0) -0.15 NaCl-10 mM EDTA (pH 8.2) -0.2% SDS ] the filters were then subjected to protease K degradation. Specifically, 5ml of the solution was added to each filter, and degradation was allowed to proceed at 55 ° C for 30 minutes. Thereafter, the solution was removed.

The filters were then treated with 4 ml of a pre-hybridization buffer (5 x SSPE - 0.5% SDS -100 micrograms / ml SS E. coli DNA - 5 x BFP). Pre-hybridization treatment was at 55 ° C, generally for 4 hours or longer.

Thereafter, the prehybridization buffer was removed.

The hybridization procedure was performed in a golden manner. To each filter was added 3 ml of hybridization buffer (5 x SSPE - 0.5% SDS -100 micrograms / ml yeast tRNA) containing 0.025 picomoles of each of the 128 probe sequences of Table II (the total mixture was designated as EPV mixture) and the filters were kept at 48 ° C for 20 hours.

This temperature was 2 ⁰ C lower than the lowest of the calculated dissociation temperatures (Td) calculated for any of the probes.

After hybridization, the filters were washed three times for ten minutes on a shaker with 6 x SSC-0.1% SDS at room temperature and washed two to three times with 6 x SSC 1% SDS at derHybridisierungstemperatur (48 ⁰ C). Autoradiography of the filters revealed seven positive clones among 200,000 colonies screened.

The initial sequence analysis of one of the putative monkey cDNA clones (referred to as clone 83) was made for verification for correctness by a modification of the procedure of Wallace et. al., Gene, 16, pp. 21-26 (1981). The plasmid DNA of the monkey cDNA clone 83 was linearized by degradation with Eco Rl and denatured by heating in a boiling water bath. The nucleotide sequence was determined by the dideoxy method of Sanger et al., P.N.A.S. (USA), 74, p.5463-5467 (1997). A subset of the EPV mixture of probes consisting of 16 sequences was used as a primer for the sequencing reactions. ,

C. Sequencing of monkey EPO cDNA

The nucleotide sequence analysis of clone 83 was determined by the method of Messing, Methods in Enzymology, 101, 3.20 ^ 7S

(1983). From Table IV, a preliminary restriction analysis of the approximately 1 600 base pair Eco RI / Hind III-cloned fragment of clone 83 can be seen. The approximate locations of the restriction endonuclease enzyme recognition sites were made 3 'to the Eco RI site at the 5' end of the fragment using the number of bases. Nucleotide sequencing was performed by sequences of the individual restriction fragments with the intention of adapting overlapping fragments, for example, analysis of nucleotides in a restriction fragment determined the overlap of sequence information C113 (Sau 3A at -111 / Sma I at ~ 324), and reverse order sequencing of a fragment C73 (AIu I at ~ 424 / BstE Il at -203.

Approximate location (s)

TABLE IV Appr Restriction enzymes Recognition Site 1 EcoRI 111 Sau3A 180 Smal 203 BstEII 324 Smal 371 KpnI 372 rsal 424 AluI 426 PstI 430 AluI 466 HpaI 546 AluI 601 PstI 604 PvuII 605 AluI 782 AluI 788 AluI 792 rsal 8'07 PstI 841 AluI 927 AluI 946 ncol 1014 Sau3A 1072 AluI 1 115 AluI 1223 AluI

TABLE IV (continued) Approximate location (s)

Restriction enzymes Appr Recognition Site PstI 1301 rsal 1343 AluI 1384 Hind 1449 AluI 1450 Hind 1585

Sequencing approximately 1 342 base pairs (within the Sau 3A site 3 'to the Eco R I site and the Hind Mi-steep spanned region) and analysis of the reading frames allowed the preparation of the DNA and arhiphoid acid sequence information described in U.S. Pat Table V are included. In the table, the probable initial amino acid residue of the amino terminal of the mature EPO (verified by correlation of the above-mentioned sequence analysis of twenty amino terminal residues) is designated by the number +1. The presence of a methionine-specifying ATG codon (designated -27) "upstream" of the initial amino terminal alanine residue as the first residue designated for the amino acid sequence of the mature protein indicates the likelihood that EPO is initially expressed in the cytoplasm of a precursor form was expressed with a 27 amino acid leader region, which is excised before the mature EPO enters the circulation. Potential glycosylation sites within the polypeptide are indicated by asterisks. The estimated molecular mass of the translated region was 21,117 daltons and the molecular mass of the 165 residues of the polypeptide that produced mature monkey EPO was determined to be 18,236 daltons.

TABLE V

Translation of Monkey EPO cDNA

gatcccgcgccccctggacagccgccctctcctccaggccgtggggctggccctgccc cgctgaacttcccgggatgaggactcccggtgtggtcaccgcgcgcctaggtcgctgag

Trp Leu Leu Leu -27 Gly Val His Glu Cys Per -20 Trp Per bad TGG CTT CTC CTG mead GGG GTG CAC GAA TGT CCT Ala TGG CCA AGA -1 + 1 ATG -10 GCC Per Gly Ala Per Leu Val Ser. Leu Per Leu Leu Leu CCG GGC GCC CCA Ser CTC GTG TCG CTC CCT CTG Gly CTC CTG TCT GGC bad Tyr Leu Leu bad Leu me Cys Asp Ser 10 Val mead AGG TAC CTC TTG Per ' CGC CTC ATC TGT GAC AGC bad GTC ATG 30 CCA 20 * CGA Cys Ser Glu Ser Ala Lys Glu Ala Glu Asn Thr Per GGACCCCGGCCAGGCGCGGAG TGT TCC GAA AGC Glu GCC AAG GAG GCC GAG AAT Val ACG CCA Thr Lys Val Asn GAG * GTC Gly Leu ACC AAA GTT AAC Ser Leu Asn Glu Asn He 40 Val GGG CTG 60 Cys AGC TTG crt AAT GAG AAT ATC Thr GTC gin Ala Val Glu TGC Tyr OU Ala Trp Lys bad mead ACC Val Glu Val CAG GCT GTA GAA Phe DID GCC TGG AAG AGG ATG Glu GTC GAA GTC TTC GAG Val Leu bad Gly Trp gin Gly Leu Ala Leu 70 Ser Per Glu GTC CTG CGG GGC Val TGG CAG GGC CTG GCC CTG Leu TCA CCT GAG 90 GTC 80 # CTC Glu Per Leu Gin Ala Val Leu Ala Asn Ser Gin Leu Gly GAG CCC CTG CAG Gin GCC GTG TTG GCC AAC TCT Ser CAG CTT GGC CAG TCC Asp Ser ile Thr Thr His mead Asp Lys Ala me 100 Gly Ala GAC AGC ATC ACC ACT Leu CAC • ATG GAT AAA GCC ATC Ser GGC GCC 120 CTG 110 AGT gin Ser Leu Per Asp Leu bad Ala Leu Gly Ala Glu He CAG TCC CTC CCA GAT Leu CTT CGG GCG CTG GGA GCC. GIn. GAA ATC CTG CAG Ala Ala Asp Thr Phe Ala Ser Ala Ala Per Leu 130 Thr Phe GCT GCT GAC ACT TTC Ala GCC TCG GCT GCT CCA ' CTC bad ACC TTC 150 GCG 140 CGA t Phe bad Gly Lys Leu Lys Leu Phe bad Val Tyr Asn TTC CGG GGA AAG CTG Cys AAA CTC TTC CGA GTC TAC Ser AAT TGC TCC bad Leu Tyr Thr Gly Glu Ala 160 bad CGC Lys CTG TAC ACG GGG GAG GCC Cys AGG AAG TGC He ATC Th r ACT Leu CTC

TABLE V (continued)

165 GIy Asp Arg OP

ggg gac aga tga ccaggtgcgtccagctgggcacatccaccacctccctcaccaaca ctgcctgtgccacaccctccctcaccatcccgaaccccatcgaggggctctcagctaag cgccagcctgtcccatggacactccagtgccagcaatgacatctcaggggccagaggaac tgtccagagcacaactctgagatctaaggatgtcgcagggccaacttgagggcccagagc aggaagcattcagagagcagctttaaactcaggagcagagacaatgcagggaaaacacct gagctcactcggccacctgcaaaatttgatgcaggacacgctttggaggcaatttacctg tttttgcacctaccatcagggacaggatgactggagaacttaggtggcaagctgtcactt ctcaaggcctcacgggcactcccttggtggcaagagcccccttgacactgagagaatatt ttgcaatctgcagcaggaaaaattacggacaggttttggaggttggagggtacttgacag gtgtgtggggaagcagggcggtaggggtggagctgggatgcgagtgagaaccgtgaagac ag g atg ctg g g g g g g g cctctg gttctcgtg gtccaag ctt

Hindi!]

The polypeptide sequence of Table V can be readily checked analytically for the presence of hydrophilic regions and / or secondary matching characteristics that potentially indicate highly immunogenic regions, e.g. By the methods of Hopp et al., P.N. S.S. (USA), 78, pp. 3824-3828 (1981) and Kyte et al., J. Mol. Biol. 157, pp. 105-132 (1982) and / or Chou et al. Biochem., 13, pp. 222-245 (1974) and Advances in Enzymology, 47, pp. 45-47 (1978). Computer-aided analysis according to the method of Hopp et al. is possible via a program called PEP Reference Section 6.7, available from Intelligenetics, Inc. 124 University Avenue, Paolo Alto, California.

Example 4 A. Human Gene Library

A Ch4APhagen-born human fetal liver gene library, prepared according to the methods of Lawn et al., Cell, 18, pp. 533-543 (1979), was obtained and provided for use in a plaque hybridization assay.

B. Plaque hybridization methods for screening a HuMan gene library

Phage particles were lysed and the DNAs fixed on filters (50,000 plaques per filter) according to the procedures of Woo, Methods in Enzymology, 68, pp. 389-395 (1979), except for the use of GeneScreene Plus filters (New England Nuclear Catalog No.NEF-976) and NZYAM plates (NaCl, 5g, MgCl ₂ -6H ₂ O, 2g, NZ-amine A, 10g, yeast extract, 5g, casamino acids, 2g, maltose, 2g, and agar, 15g / l). ,

The air dried filters were heat treated for one hour at 8O ⁰ C and, subsequently degraded with proteinase K as in Example 3, Part B. Pre-hybridization was performed with 1 M NaCl - 1% SDS buffer at 55 ° C for 4 hours or more, after which the buffer was removed. Hybridization and post-hybridization washes were performed as described in Example 3, part B. Both the mixture of 128 20-mer probes designated as EPV and the mixture of 128 17-mer probes from Table III (designated as EPQ mixture) were used. Hybridization was performed at 48 ^° C. using the EPV probe mixture. EPQ probe hybridization was performed at 46 ° C - 4 degrees below the lowest calculated Td for components of the mixture. The hybridization probe for rehybridization was removed by boiling with 1 χ SSC-0.1% SDS for two minutes. Autoradiography of the filters revealed three positive clones (reactive with both probe mixtures) of 150000 screened phage plaques. Verification for the correctness of the positive clones as EPO-encoding was obtained via DNA sequencing and electron micrographic visualization of heteroduplex formation with the monkey cd NA of Example 3. This procedure also revealed multiple introns in the genomic DNA sequence.

Example 5

Nucleotide sequence analysis of one of the positive clones (designated XhEI) was performed. The results obtained are listed in Table VI.

TABLEVI

aagcttctgggcttccagacccagctactttgcggaactcagcaacccaggcatctctgagtctccgccca agaccgggatgccccccaggggaggtgtccgggagcccagcctttcccagatagcacgctccgccagtccc aagggtgcgcaaccggctgcactcccctcccgcgacccagggcccgggagcagcccccatgacccacacgc acgtctgcagcagccccgctcacgccccggcgagcctcaacccaggcgtcctgcccctgctctgaccccgg gtggcccctacccctggcgacccctcacgcacacagcctctcccccacccccacccgcgcacgcacacatg cagataacagccccgacccccggccagagccgxagagtccctgggccaccccggccgctcgcctgccgctg cgccgcaccgcgctgtcctcccggagccggaccggggccaccgcgcccxgctctgctccgacaccgcgccc cttggacagccgccctctcctctaggcccgtggggctggccctgcaccgccgagcttcccgggatgaggxx

-27 -24

Met GIy VaI His

CCCGGTGACCGGCGCGCCCCAAGTCGCTGAGGGACCCCGGCCAAGCGCGGAG ATG GGG GTG CAC G

GTGAGTACTCGCGGGCTGGGCGCTCCCGGCGGCCGGGTTCCTGTTTGAGCGGGGATTTAGCGCCCCGGCT

TABLE VI (cont'd.)

ATTGGCCAAGAGGTGGCTGGGTTCAAGGACCGGCGACTTGTCAAGGACCCCGGAAGGGGGAGGGGGGTGGG GCAGCCTCCACGTGCCGCGGGGACTTGGGGGAGTTCTTGGGGATGGCAAAAACCTGGCCTGTTGAGGGGCA CAGTTTGGGGTTGGGGAGGAGGTTTGGGGTTCTGCTGTGCAGTTGTGTCGTTGTCAGTGTCTCGIl.S.] TTGCACACGCACAGATCAATAAGCCAGAGGCAGCACCTGAGTGCTTGCATGGTTGGGACAGGAAGGACGAG CTGGGGCAGAGACGTGGGGATGAAGGAAGCTGTCCTTCCACAGCCACCCTTCTCCCCCCCCGCCTGACTCT

-23 -20

GIu Cys Pro Ala Trp Leu Trp Leu Leu Leu Ser Leu CAGCCTGGCTATCTGTTCTAG AA TGT CCT GCC TGG CTG TGG CTT CTC CTG TCC CTG

-10 -1 +1

Leu. Leu Pro Leu Lei Pro Lei Lei GIy AIa Pro Pro Arg Leu He Cys CTG TCG CTC CCT CTG GGC CTC CCA GTC CTG GCC CC CCA CCA CGC CTC ATC TGT

'10 20. *

Asp Ser Arg VaI Leu GIu Arg Tyr Leu Leu GIu AIa Lys GIu Ala GIu Asn He GAC AGC CGA GTC CTG GAG AGG TAC CTC TTG GAG GCC AAG GAG GCC GAG AAT ATC 26 Thr

ACG GTGAGACCCCTTCCCCAGCACATTCCACAGAACTCACGCTCAGGGCTTCAGGGAACTCCTCCCAGAT CCAG G AACCTG G CACTTG GTTTGG G GTG GAGTTG G G AAGCTAG ACACTGroughTACATAAG AATAAGTC TG GTG GCCCCAACACATACCTG AAACTAG G CAAG AG CAAAG CCAG CAG ATCCTACG CCTGTG GGCCAGGG

27 30

Thr GIy Cys Ala GIu CCAGAGCCTTCAGGGACCCTTGACTCCCCGGGCTGTGTGCATTTCAG ACG GGC TGT GCT GAA

* 40

His Cys Ser Leu Asn GIu Asn Ne Thr VaI Pro Asp Thr Lys VaI Asn Phe Tyr CAC TGC AGC TTG AAT GAG AAT ATC ACT GTC CCA GAC ACC AAA GTT AAT TTC TAT 50 55

AIa Trp Lys Arg Met GIu

gcc tgg aag agg atg gag gtgagttcci i i i i i i i i i i i il icctttcttttggagaatctcatt tgcgagcctgattttggatgaaagggagaatgatcgggggaaaggtaaaatggagcagcagagatgaggct gcctgggcgcagaggctcacgtctataatcccaggctgagatggccgagatgggagaattgcttgagccct ggagtttcagaccaacctaggcagcatagtgagatcccccatctctacaaacatttaaaaaaattagtcag gtgaagtggtgcatggtggtagtcccagatatttggaaggctgaggcgggaggatcgcttgagcccaggaa tttgaggctgcagtgagctgtgatcacaccactgcactccagcctcagtgacagagtgaggccctgtctca aaaaagaaaagaaaaaagaaaaataatgagggctgtatggaatacattcattattcattcactcactcact CACTCATTCATTCATTCATTCATTCAACAAGTCTTATTG CATACCTTCTGTTTG CTCAG CTTG GTG G CTTG ggctgctgaggggcaggagggagagggtgacatgggtcagctcgactcccagagtccactccctgtag

56 60 70

VaI GIy Gin Gin Ala VaI GIu VaI Trp Gin GIy Leu AIa Leu Leu Ser GIu AIa GTC GGG CAG CAG GCC GTA GAA GTC TGG CAG GGC CTG GCC CTG CTG TCG GAA GCT

80 * 90

VaI Leu Arg GIy Gin AIa Leu Lei VaI Asn Ser Ser GIn Pro Trp GIu Pro Leu GTC CTG CGG GGC CAG GCC CTG TTG GTC AAC TCT TCC CAG CCG TGG GAG CCC CTG

100

GIn Leu His VaI Asp Lys Ala VaI Ser GIy Leu Arg Ser Leu Thr Thr Leu Leu CAG CTG CAT GTG GAT AAA GCC GTC AGT GGC CTT CGC AGC CTC ACC ACT CTG CTT 110 115

Arg AIa Leu GIy Ala GIn

CGG GCT CTG GGA GCC CAG GTGAGTAGGAGCGGACACTTCTGCTTGCCCTTTCTGTAAGAAGGGGA GAAGGGTCTTGCTAAGGAGTACAGGAACTGTCCGTATTCCTTCCCTTTCTGTGGCACTGCAGCGACCTCCT

116 120

Lys GIu Ala He Ser Pro Pro Asp Ala Ala Ser Ala Ala GTTTTCTCCTTGGCAG AAG GAA GCC ATC TCC CCT CCA GAT GCG GCC TCA GCT GCT

130 140

Pro Leu Arg Thr He Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg VaI Tyr Ser CCA CTC CGA ACA ATC ACT GCT GAC ACT TTC CGC AAA CTC TTC CGA GTC TAC TCC

150 160

Asn Phe Leu Arg GIy Lys Leu Lys Leu Tyr Thr GIy GIu AIa Cys Arg Thr GIy AAT TTC CTC CGG GGA AAG CTG AAG CTG TAC ACA GGG GAG GCC TGC AGG ACA GGG

Asp Arg OP GAC AGA TGA CCAGGTGTGTCCACCTGGGCATATCCACCACCTCCCTCACCAACATTGCTTGTGTGCCACA

TABLE VI (cont'd.)

CCCTCCCCCGCCACTCCTGAACCCCGTCGAGGGGCTCTCAGCTCAGCGCCAGCCTGTCCCATGGACACTCC

AGGGCCAACTTGAAGGGCCCAGAGCAGGAAGCATTCAGAGAGCAGCTTTAAACTCAGGGACAGAGCCATGC TGGGAAGACGCCTGAGCTCACTCGGCACCCTGCAAAATTTGATGCCAGGACACGCTTTGGAGGCGATTTAC CTGTTTTCGCACCTACCATCAGGGACAGGATGACCTGGAGAACTTAGGTGGCAAGCTGTGACTTCTCCAGG TCTCACGGGCATGGGCACTCCCTTGGTGGCAAGAGCCCCCTTGACACCGGGGTGGTGGGAACCATGAAGAC AXGATXGGGGCTGGCCTCTGGCTCTCATGGGGTCCAAGTTTTGTGTATTCTCAACCTATTGACAGACTGAA ACACAATATGAC

In Table VI, the initial contiguous DNA sequence designates a 620 base top strand in which an apparently untranslated sequence immediately precedes a translated portion of the human EPO gene. In particular, it appears that the sequence comprises the 5 'end of the gene which directs to a translated DNA region coding for the first four amino acids (-27 to -24) of a leader sequence ("pre-sequence") Base pairs in the sequence that encoded the beginning of the leader sequence were not accurately determined and have therefore been termed "X". This is followed by an intron of approximately 639 base pairs (439 base pairs of which were sequenced and the remaining 200 base pairs of which were termed "IS") and immediately before a codon for glutamine designated as residue -23 of the translated polypeptide Exon sequence is considered to be coding for amino acid residues by an alanine residue (referred to as the + 1 residue of the amino acid sequence of mature ³ human EPO) to the codon specifying threonine at position +26, followed by a second intron consisting of 256 bases This intron is followed by an exon sequence for amino acid residues 27 to 55 and then a third intron containing 612 base pairs begins.

The subsequent exon encodes residues 56 to 115 of Hu-man EPO and then begins a fourth intron of 134 bases as listed. The fourth jntron is followed by an exon coding for residues Nos. 116 to 166 and a stop codon (TGA). Finally, in Table IV, a sequence of 568 base pairs is identified in which an untranslated 3 'region of the human EPO gene appears, two base pairs, of which ("X") has not yet been accurately sequenced.

Table VI thus serves to establish the structural match (amino acid sequence) of mature human EPO containing 166 specific amino acid residues (estimated molar mass = 18,399). In addition, the table shows the DNA sequence encoding a 27 residue leader sequence along with 5 'and 3' DNA sequences which may be significant to promoter / operator functions of the human operon.

Sites for the potential glycosylation of the mature human EPO polypeptide are designated by asterisks in the table. It should be noted that the specific amino acid sequence of Table VI probably forms that of a naturally occurring allelic form of human erythropoietin. This position is supported by the results of ongoing efforts in sequencing urinary isolates of human erythoropoietin, which resulted in finding a significant number of erythropoietin molecules therein having a methionine at residue 126 versus a serine, as shown in the table.

The following Table VII illustrates the degree of polypeptide sequence homology between human and monkey EPO. Single-letter designations were used in the upper continuous row of the table to represent the deduced, translated polypeptide sequences of human EPO starting with the -27 residue, and the lower continuing row shows the. derived polypeptide sequence from monkey EPO starting with the residue designated as number -27. Asterisks indicate the sequence homologies. It should be noted that the deduced human and monkey EPO sequences have an "extra" Lysiη (K) residue at the (human) position 116. Comparison with Table VI shows that this residue is on the brink of a putative In addition, the presence of the lysine residue in the human polypeptide sequence was checked by sequencing a cDNA human sequence clone prepared from mRNA isolated from COS-1 cells linked to the human genomic DNA of Example 7 (see below).

TABLE VII

Comparison of Human and Monkey EPO polypeptides

-20 -10. + 1 10 20 30 40 Human MGVHECPAWLWLLLSLSLSLPLGLPVLGAPPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNENITVPDTK

Monkey MGVHECPAWLWLLLSLVSLPLGLPVPGAPPRLICDSRVLERYLLEAKEAENVTMGCSESCSLNENITVPDTK

50 60 70 80 90 100 110 Human VNFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAQKE

Monkey VNFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQAVLANSSQPFEPLQLHMDKAISGLRSITTLLRALGAQ-E

120 130 140 150 160

Human AISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDR

Monkey AISLPDAASAAPLRT ITADTFCKLFRVYSNFLRGKLKLYTGEACRRGDR

Example 6

The expression system selected for initial experiments in the microwave synthesis of isolatable amounts of the EPO polypeptide material encoded by monkey cDNA therefor, obtained by the procedures of Example 3, was one to which mammalian host cells (i.e., COS-1 cells, ATTC No. CRL-1650). The cells were transfected with a "shuttle" vector capable of autonomous replication in the E. coli host (due to the presence of pBR322-derived DNA) and the mammalian hosts (due to the presence of SV40 virus-derived DN A) ,

Specifically, an expression vector was constructed by the following methods. The plasmid clone 83 obtained in Example 3 was amplified in E. coli and the approximately 1.4 kb monkey ΕPO-encoding DNA was isolated by EcoRI and HindIII digestion. Separately isolated was an approximately 4.0 kb Hind III / Sal I fragment from pBR322. An approximately 3ObP ₁ ECoRIZSaM "left" fragment was obtained from M13mp10RFDNA (PundL Laboratories) .This linker contained - in series - a single-stranded EcoRI end, followed by Sst1, SmaI, BamHI and XbaI recognition sites and a single-stranded Sa11 -The End.

The above three fragments were ligated to give an approximately 54kb intermediate plasmid ("pERS") in which the EPO DNA was flanked on one side by a "bank" of useful restriction endonuclease recognition sites. Then pERS was digested with Hind III and SaIII to give the EPO-DNa and the EcoRI to SaII (M13mp10) linkers. The 1.4kb fragment was ligated into an approximately 4.0kb BamH I / SalI of pBR322 and another M13mp10 Hind HI / BamHl RF fragment linker, which also had about 30bp. The M13 linker fragment was characterized by a single-stranded HindIII end, followed by Pst I, Sail and Xba1 recognition sites and a single stranded BamH1 end. The ligation product was again a useful intermediate plasmid ("pBR-EPO") containing the DNA flanked on both sides by benches of recognition sites.

The vector selected for expression of EPO DNA in Cos-1 cells ("pDSVL 1") was previously designed to allow for selection and self-replication in E. coli, which are due to the origin of replication and the ampicillin resistant Gene DNa sequences present in the region spanned by nucleotides 2448 to 4362 of pBR322 This sequence was structurally modified by the addition of a linker which provided HindIII recognition immediately adjacent to nucleotide 2448 prior to incorporation into the vector. Other useful properties of the selected vector included the ability to self-replicate in COS-1 cells and the presence of a viral promoter sequence function in mammalian cells, as indicated by the origin of the replication DNA sequence and the viral promoter DNA sequence at the " spade gene present in the 342bp sequence containing nucleotides # 5171 spans to 270 of the SV40 genome.

In the vector, a single restriction site (BamH I) was provided and immediately adjacent to the viral promoter sequence by using a commercially available linker sequence (Collaborative Research). Also incorporated into the vector was a 237 base pair sequence (derived as nucleotides Nos. 2553 to 2770 of SV40) containing the viral mRNA polyadenylation signal of the "late gene" (commonly referred to as a transcriptional terminator) correct orientation vis-a-vis the viral promoter of the "late gene" placed over the single BamHI site. Similarly, in the vector, another mammalian gene was present at a site where there was no material for potential transcription of a gene inserted at the single BamH! Site, between the viral promoter and terminator sequences.

The mammalian gene comprised about 2,500 bp mouse dihydrofolate reductase (DHFR) minigene isolated from plasmid pMG-1 as described in Gasser et al., P. N-A. S. (USA), 79, pp. 6522-6526 (1982).

Again, the major operative components of plasmid pDSVLI contained nucleotides 5171 to 270 (342 bp) and 2553 to 2770 (237 bp) of SV40 DNA.

After the following z. From Maniatis et al. (see above), the EPO-encoding DNA was isolated from the plasmid pBR-EPO as a BamH I fragment and ligated into the BamH I cut plasmid pDSVL1. Restriction enzyme analysis was used to confirm the insertion of the EPO gene in the correct orientation in two of the resulting cloned vectors (double vectors H and L) - see Figure 2, which illustrates the plasmid pDSVL-MKE.

Vectors with misaligned EPO genes were repealed for use as negative controls in transfection experiments to determine EPO expression levels in hosts transformed with EPO DNA in the correct orientation vectors.

The vectors H, L, F, X and G were combined with carrier DNA (murine liver and spleen DNA) and used to transfect double 60 mm plates by calcium phosphate microfiltration techniques. The double 60 mm plates were also transferred with carrier DNA as a "mock" negative transformation control.

After 5 days, all culture media were tested for the presence of polypeptides having immunological properties of naturally occurring EPO.

Example 7 A. Initial EPO expression system involving COS-1 cells

The system selected for the initial experiments in microwave synthesis of isolatable amounts of human EPO polypeptide material encoded by the human genomic DNA EPO clone was also expressed in mammalian host cells (ie, COS-1 cells, ATTC # CRL). 1650) included. The human EPO gene was first subcloned into a "shuttle" vector capable of self-replicating both in E. coli hosts (due to the presence of DNA derived from pBR322) and in the mammalian cell line COS-1 (as a result of The shuttle vector containing the EPO gene was then transferred to COS-1 cells and EPO polypeptide material was produced in the transferred cells and secreted into the cell culture media.

Specifically, the expression vector was constructed by the following methods. DNA was isolated from lambda clone \ hE1 containing the human genomic EPO-oen and digested with BamHI and HindIII restriction endonucleases to isolate a 5.6kb DNA fragment which was known to be the entire EPO. Contained gene. This fragment was mixed and ligated with the bacterial plasmid pUC8 (Bethesda Research Laboratories, et al.) Which was degraded in a similar manner to give the intermediate plasmid "PUC8-HUE", thereby obtaining an appropriate source of this restriction fragment.

The vector chosen for the expression of EPO DNA in COS-1 cells (pSV4SEt) was previously constructed. The plasmid pSV4SEt contained DNA sequences that allowed for selection and self-replication in E. coli. These characteristics resulted from the origin of replication and the ampicillin resistance gene DNA sequences present in the region spanning nucleotides 2448 to 4362 of the bacterial plasmid pBR322. This sequence was structurally modified by the addition of a linker which provided a HindIII recognition site immediately adjacent to nucleotide 2448. The plasmid pSV4SEt was also capable of self-replication in COS-1 cells. This tag was revealed by a 342 bp fragment containing the SV40 virus origin of replication (nucleotides # 5171-270). This fragment was modified by the addition of a linker which provided an EcoRIU recognition site adjacent to nucleotide 270 and a linker providing a Sall recognition site adjacent to nucleotide 5171.

A 1061bp fragment of SV40 was also present in this vector (nucleotide numbers 1711 to 2772 plus linker, which provided a Sal I recognition site next to nucleotide number 2772). Within this fragment, there was a single BamHI recognition sequence. Thus, in summary, the plasmid pSV4SEt, containing a single BamHI and HindIiI recognition site, allowed the insertion of the human EPO gene, the sequences allowed replication and selection in E. coli, and the sequences allowed replication in COS-1 cells.

To insert the -EPO gene into pSV4SEt, the plasmid pUC8-HuE was digested with BamHI and HindIII restriction endonucleases and the 5.6 kb EPO-encoding DNA fragment was isolated. pSV4SEt was also digested with BamH I and Hind III and the larger 2513bp fragment isolated (preserves all necessary functions). These fragments were mixed and ligated to form the final vector "pSVgHuEPO" (see Figure 3) .This vector was reproduced in E. coli and the vector DNA isolated Using restriction enzyme analysis, the insertion of the EPO gene was confirmed. The plasmid pSVgHuEPO DNA was used to express human EPO polypeptide material in COS-1 cells More specifically, pSVgHuePO DNA was combined with carrier DNA and transferred to triple 60 mm plates of COS-1 cells Control, carrier DNA alone was also transferred to COS 'cells Samples were taken from the cell culture media five and seven days later and tested for the presence of polypeptides having the immunological properties of naturally occurring human EPO.

B. Second EPO Expression System Including COS-1 Cells

Another system was designed to allow for enhanced production of EPO polypeptide material encoded by the human genomic DNA-EPO clone in COS-i cells (ATCC No. CRL-1650).

In the immediately preceding system, EPO had been experimented in COS-1 cells using its own promoter located within the 5.6 kb BamHI to HindIII restriction fragment. In the following construction, the EPO gene was rearranged to express it using SV40 "late" promoter Specifically, the cloned 5.6kb Bam HI to Hind III genomic human EPO restriction fragment was modified in the following manner. The plasmid pUC8-HuE described above was cut with Bam HI and BstE II restriction endonucleases BstE II cuts within the 5.6kb EPO gene at a position 44 base pairs 5 'after the initiating ATG used for the pre The approximately 4900 base pair fragment was isolated A synthetic linker DNA fragment ending in single-stranded Sail and BstE II ends containing an internal Barn HI recognition site was synthesized and purified, and the two fragments were mixed and ligated with the plasmid pBR322 which had been cut with Sal I and Bam HI to produce the intermediate plasmid pBRgHE The human omega human EPO gene could be isolated therefrom as a 4900 base pair Bam HI digestion fragment carrying the full structural gene with a single ATG 44 base pair 3 'to the Barn HI site adjacent to the amino terminal coding region.

This fragment was isolated and inserted as a Barn HI fragment into the Barn HI cut expression vector plasmid pDSVLI (written in Example 6). The resulting plasmid pDSVLgHuEPO, as illustrated in Figure 4, was used to express EPO polyeptide material from COS-1 cells, described in Examples 6 and 7A.

Example 8

Culture media from the growth of the six transferred COS-1 cultures of Example 6 were analyzed by radioimmunoassay according to the procedures of Example 2, Part B. Each example was assayed at 250, 125, 50 and 25 microliter aliquots. Supernatant from the growth of cells, mock transfected or transferred to Veroren, which had the incorrect gene orientation, was undoubtedly negative for EPO immunoreactivity. For each sample of the two supernatants resulting from the growth of COS-1 cells using vectors (H and L). whose EPO DNA was in the correct orientation transferred the percent inhibition of ¹²⁵ I-EPO binding to antibody from 72 to 88%, bringing all values to the top of the standard curve. The exact concentration of the EPO in the culture supernatant could not be reliably estimated. There was a completely conservative estimate of 300mU / ml, but from the value calculation of the largest aliquot size (250 microliters).

A representative culture fluid according to Example 6 and five and seven day old culture fluids obtained according to Example 7A were tested by an RIA to test the activity of the recombinant monkey and human EPO materials against a standard of naturally occurring human EPO, the results being shown graphically in FIG. Briefly, the results were expected to reveal that the recombinant monkey EPO competed significantly with anti-human EPO antibody, although it was unable to completely inhibit binding under the assay conditions. However, the highest inhibition percentage for recombinant human EPO was close to that of the human EPO standard. The parallel position of the dose response curves indicates immunological identity of the shared sequences (epitopes). Previous estimates of monkey ePO in culture fluids were recalculated at these higher dilution levels and found to be in the range 2.91 to 3.12 U / ml. Estimated human EPO production levels were accordingly set at 392mE / ml for the five-day growth assay and 567mE / ml for the seven-day growth assay. Estimated monkey EPO production levels in the expression system of Example 78 were in the same range or higher.

Example 9

Culture fluids prepared according to Examples 6 and 7 were subjected to an in vitro assay for EPO activity according to the procedure of Goldwasser et al., Endocrinology, 97, 21, pp. 315-323 (1975). Estimated monkey EPO values for culture fluids tested ranged from 3.2 to 4.3 U / ml. Human EPO culture fluids were also active in this in vitro assay and, in addition, this activity could be abrogated by anti-EPO antibodies. The recombinant monkey EPO culture fluids of Example 6 were also assayed for in vivo biological activity according to the general procedures of Cotes et al., Nature, 191, pp. 1065-1067 (1981) and Hammond et al., Ann. N.Y. Acad. Sci., 149, p.516-527 (1986) and activity levels ranged from 0.94 to 1.24 U / ml.

Example 10

In the preceding examples, recombinant monkey or human EPO material was produced from vectors used to transfect COS-1 cells. These vectors replicate in COS-1 cells because of the presence of SV40 antigen in the cell and a SV40 origin of replication on the vectors. Thus, these vectors produce useful levels of EPO in COS-1 cells, expression is only transient (7 to 14 days) due to eventual loss of the vector. In addition, only a small percentage of COS-1 would be productively transferred with the vectors.

The present example describes expression systems that worked with Chinese hamster ovary (CHO) DHFR ~ cells and the selected marker DHFR [for discussion of related expression systems, see US Pat. No. 4,399,216 and European Patent Applications 117058,117,059 and 117060, all on August 29 1984.] "CHO DHFR" cells (DuX-B 11), CHO K1 cells, Urlaub et al., Proc Nat National Acad., (USA), Vol.77, 4461 (1980), lack the enzyme dihydrofolate reductase (DHFR), which must be present in case of mutations in structural genes, and thus the presence of glycine, hypoxanthine and thymidine in the culture medium is required.The plasmids pDSVL-Mke (Example 6) or pDSVL-gHuEPO (Example 7b) were incubated longitudinally with carrier dNA was transferred to CHO DHFR ~ cells grown in a medium containing hypoxanthine, thymidine and glycine in 60mm culture plates The plasmid psVgHuEPO (Example 7A) was mixed with the plasmid pMG 2 containing murine dihydrofolate reductase gene cloned into The bacterial plasmid vector PBR 322 (according to Grasser et al., supra) The plasmid mixture and the carrier DNA were transferred into CHO DHFR ~ cells (cells which take up a plasmid generally also take up a second plasmid.)

After three days, the cells were dispersed by trysinization into various 100 mm culture plates in media lacking hypoxanthine and thymidine. Only those cells stably transformed with the DHFR gene and thereby with the EPO gene survive in this medium. After 7 to 21 days, the colonies of the surviving cells became visible. These transformant colonies, after dispersion by trypsinization, can be continuously reproduced in media lacking hypoxanthine and thymidine to form new cell strains (eg, CHO pDSVL-MkEPO, kcHO pSVgHuEPO, CHO-pDSVL-gHuEPO.

Culture fluids from the above cell strains were tested in an RIA for the presence of recombinant monkey or human EPO. Media for the strains CHO pDSVL-MkEPO contained EPO with immunological properties such as those obtained from COS-1 cells transferred with the plasmid pDSVL-MkEPO. A representative 65-hour culture fluid contained monkey EPO at 0.60 U / ml.

Culture fluids from CHO pSVgHuEPO and CHO pDSVL-gHuEPO contained recombinant human EPO with immunological properties such as that obtained with COS-1 cells transferred with the plasmid pSVgHuEPO or pDSVL-gHuEPO; had been obtained. A representative 3-day culture fluid from CHO pSVgHuEPO contained 2.99 U / ml human EPO and a 5.5-day sample of CHO pDSVL gHuEPO contained E / ml human EPO as measured by RIA. The amount of EPO produced by the cell strains described above can be increased by gene amplification, thereby creating new cell strains of greater productivity. The enzyme dihydrofolate reductase (DHFR), which is the product coding for it by the DHFR gene, can be inhibited by the drug methotrexate (MTX). In particular, in the media lacking hypoxanthine and thymidine, increased cells are inhibited or killed by MTX. Under the appropriate conditions (e.g., minimal concentration of MTX), cells resistant to and capable of growing in MTX can be obtained. It has been found that these cells are resistant to MTX due to amplification of the number of their DHFR genes, resulting in an increase in the production of DHFR enzyme. The surviving cells can be sequentially treated with increased MTX concentrations, resulting in cell strains containing a larger number of DHFR genes. "Passenger genes" (eg, EPO) carried by the expression vector with the DHFR gene or transformed with the DHFR gene, according to recent reports, are also considered to be increasing in their number of gene copies.

 As practical examples of this amplification system, the cell strain CHO pDSVL-MkE was exposed to increasing concentrations of MTX (OnM, 3OnM and 10OnM). Representative 65 hour culture media samples from each amplification step were checked by RIA and determined to contain 0.60, 2.45 and 6.10 E / ml, respectively. The cell strain CHO pDSVL-gHuEPO was exposed to a series of increasing MTX concentrations of 3OnM, 5OnM, 10OnM, 20OnM, 1μΜ and 5μΜ MTX. A representative 3-day culture media sample from the 10OnM MTX step contained 3084 ± 129 U / ml human EPO as determined by RIA.

Representative 48 hour culture medium samples from the 10OnM and 1 μM MTX levels contained respectively human EPO at 466 and 1 352 U / ml as determined by RIA (average of triplicate assays). In these procedures, 1 × 10 ⁶ cells were plated with 5 ml of the medium in 60 mm culture dishes. Twenty-four hours later, the media was removed and replaced with 5 ml of serum-free media (high glucose DMEM supplemented with O ₁ mM non-essential amino acids and L-glutamine). The EPO content was allowed to grow in the serum-free media for 48 hours. The media were collected for RIA and the cells were strypsinated and counted. The average RIA values of 467 U / ml and 1352 U / ml for cells grown at 10OnM and 1μΜ MTX gave yields of 3335 U / plate and 6750 U / plate, which was the average number of cells per plate corresponding to 1.94 χ 10 ⁶ and 3.12 χ 10 ⁶ cells. The effective production rates for these culture conditions were thus correspondingly at 1 264 and 2167 E / 10 ^s cells / 48 hours.

The cells in the cultures described immediately above represent a genetically heterogeneous population. Standard screening methods have been used in attempting to isolate genetically homogeneous clones with the highest production capacity. See Section A, Part 2 of "Points to Consider in the Characterization of Cell Lines Used to Produce Biologies," June 1, 1984, Office of Biology Research Review, Center for Drugs and Biologies, US Food and Drug Administration The proliferation of mammalian cells in culture generally requires the presence of serum in the growth media, and a production method of erythropoietin from CHO cells in media containing no serum greatly facilitates the production of CHO cell lines producing EPO Purification of Erythropoietin from Culture Medium The method described below demonstrates an economical way of producing erythropoietin in serum-free media in large enough quantities for production.

Cells of strain CHO pDSVL-gHuEPO, grown under standard cell culture conditions, were used to inoculate spinner cell culture flasks. The cells were introduced into the spinner cell culture flask as a suspension cell line

Media consisting of a 50-50 mixture of high glucose DMEIVl and Ham's M12 supplemented with 5% fetal calf serum, L-glutamine, penicillin and streptomycin, 0.05mM nonessential amino acids and the corresponding concentration of methotrexate. The suspension cell culture allows to easily expand the EPO-producing CHO cells to large volumes.

Suspended CHO cells were used to inoculate roller bottles at an initial seeding density of 1.5 x 10 ⁷ viable cells per 850 cm ² roller bottles in 200 ml media. The cells were allowed to grow to confluence with a contiguous cell line over a period of 3 days. The media used for this growth phase are the same as for growth in suspension. At the end of the three-day growth period, the serum-containing medium was removed and replaced with 100 ml of serum-free medium:

50:50 mixture of DMEM with high glucose content and Ham's F12, supplemented with 0.05mM non-essential amino acids and L-glutamine, the roller bottles were returned to the roller bottle incubator for 1 to 3 hours, the medium again removed and replaced by 100ml fresh , serum-free medium replaced. The 1 to 3 hour incubation of the serum-free medium reduces the concentration of contaminating serum proteins.

The roller bottles were returned to the incubator for seven days, during which time erythropoietin collected in the serum-free culture medium. At the end of the seven-day production phase, the resulting media was removed and replaced with fresh serum-free media for a second production cycle. As an example of the practical result of this production system, a representative seven-day serum-free sample contained human erythropoietin at 3892 ± 409 U / ml, as determined by RIA. Based on an estimated cell density of 0.9 to 1.8 χ 10 ⁵ cells / cm ² , each 850 cm ³ roll bottle contained 0.75 to 1.5 χ 10 ⁸ cells, resulting in a production rate of EPO in the seven-day 100 ml culture from 750 to 1 470e / 10 ⁶ cells / 48 hours.

Culture fluids of the cell strain CHO pDSVL-MkEPO in 10 nM MTX were subjected to RIA and in vitro and in vivo EPO activity assays. A sample of the resulting medium contained 41.2 ± 1.4E / ml MkEPO, measured by RIA, 41.2 ± 0.064E / ml as measured by in vitro assay of biological activity and 42.5 ± 5E / ml as measured by in vivo assay of biological activity. Amino acid sequencing of the polypeptide products demonstrated the presence of EPO products, a principal species having 3 residues of the "leader" sequence, adjacent to the putative amino terminal alanine, whether this is the result of inadequate membrane function of the polypeptide in CHO cells or a difference in that At the present time, the structure of the amino terminus of the monkey EPO to human EPO is not known Culture fluids of the cell strain CHO pDSVL-gHuEPO were subjected to three assays A five and a half day sample contained recombinant human EPO in the medium of 18.2 E / ml according to RI A assay, 15.8 ± E / ml according to in vitro assay and 16.8 ± 3.0E / ml in vivo assay.

Culture fluid from CHO pDSVL-gHuEPO cells amplified by stepwise 10OnM MTX was subjected to three assays. A 3.0 day sample contained recombinant human EPO at 3089 ± 129E / ml according to RIA, 2589 ± 71.5E / ml according to in vitro assay and 2040 ± 160 U / ml according to in vivo assay. Amino acid sequencing of this product showed an amino terminal corresponding to that of Table VI.

Cell-conditioned medium from CHO cells transferred to the plasmid pDSVL-Mke in 1OnM MTX was collected and the MTX was removed by dialysis for several days to give a medium with an EPO activity of 221 ± 5.1 E / ml (EPO-CCM). To determine the in vivo effect of EPO-CCM on hematocrit levels in BalB / C normal mice, the following experiment was performed.

Cell-mediated medium of untransfected CHO cells (CCM) and EPO-CCM was adjusted with PBS. CCM was used for the control group (3 mice) and two EPO-CCM doses (4 units per injection and 44 units per injection) were used for the experimental group (2 mice per group). Over a period of 3 weeks, the seven mice were injected intraperitoneally three times weekly. After the eighth injection the average hematocrit plants were determined to be 50.4% for the control group, 55.1% for the 4E group and 67.9% for the 44E group. Expression products of mammalian cells can be easily obtained in a substantially purified form from the culture media if HPLC (C ₄₎ is employed using an ethanol gradient, preferably at pH 7. It was made a pre-experiment, the recombinant Glycoprodeinprodukte from the corresponding medium of the COS -1 and CHO to characterize cell expression of the human EPO gene compared to human urine EPO isolates using both Western blot analysis and SDS-PAGE. These studies showed that the CHO-produced EPO material had a slightly higher molecular mass than the COS-1 expression product, which was somewhat larger than that from the collected human urine extract. All products were pretty heterogeneous. Enzyme treatment with neuraminidase to remove sialic acid resulted in COS-1 and CHO recombinant products of approximately equal molecular mass, both of which, nonetheless, were larger than the asioalo-humanurine extract obtained. Treatment of the recombinant CHO product and the urine extract product (for complete removal of carbohydrate from both) with endoglycosidase F enzyme (EC 3.2.1.) Resulted in substantially homogeneous products with mainly identical molecular mass characteristics.

Purified human urine EPO and a recombinant CHO cell-produced EPO according to the invention were subjected to carbohydrate analysis by the method of Ledeen et al., Methods in Enzymology, 83 (part D), 139-191 (1982), modified by the use of the hydrolysis methods by Nesser et al., Anal. Biochem., 142, 58-67 (1984). Experimentally determined carbohydrate formation values (expressed as molar ratios of carbohydrate in the product) were as follows for the urine isolate: hexoses, 1.73; N-acetylglucosamine, 1; N-acetylneuraminic acid, 0.93; Fucose, 0; and N-acetylgalactosamine, 0. Corresponding values for the recombinant product (derived from a 3-day CHO pDSVL-gHuEPO culture medium with 10OnM MTX) were as follows: hexoses, 15.09; N-acetylglucosamine, 1; N-acetylneuraminic acid, 0.998; Fucose, 0; and N-acetylgalactosamine, 0. These found values are consistent with the above Western blot analysis and SDS-PAGE analysis.

Thus, glycoprotein products made by the present invention include products having a primary structural match of sufficient duplication as that of naturally occurring erythropoietin to have one or more of the biological properties thereof and to have an average carbohydrate composition that differs from the naturally occurring erythropietin.

Example 11

The present example relates to the overall preparation by assembling the nucleotide bases of two structural genes encoding the human EPO sequence of Table VI and correspondingly includes "preferred" codons for expression in E. coli and yeast (see cerevisiaej cells Also described is the construction of genes encoding analogs of human EPO. Briefly, the procedure was as described in Alton, et al., supra (WO 83/04053) The genes have been reviewed for an initial compilation designed the component oligonucleotides in duplicate, which were sequentially pooled into three separate sections which were designed for ready amplification and, after removal from the amplification system, could be sequentially or by multiple fragment ligation into an appropriate expression vector The following Tables VIII to XI Figures V illustrate the design and assembly of a prepared gene encoding a human EPO translation product lacking any leader or presequence, but including an initial methionine residue at position -1. In addition, the gene in its essential part includes E. coli prefector codons, and the construction has therefore been termed the "ECEPO" gene.

TABLE VIII

ECEPO SECTION 1 OUGONUCLEOTIDES

1. AATTCTAG AAACCATG AG G GATE AAAATA

2. CCATTATTTTATTACCCTCATGGTTTCTAG

3. ATGGCTCCGCCGCGTCTGATCTGCGAC

4. CTCGAGTCGCAGATCAGACGCGGCGGAG

5. TCGAGAGTTCTGGAACGTTACCTGCTG

6. CTTCCAGCAGGTAACGTTCCAGAACT

7. GAAGCTAAAGAAGCTGAAAACATC

8. GTGGTGATGTTTTCAGCTTCTTTAG

9. ACCACTGGTTGTGCTGAACACTGTTC

10. CAAAGAACAGTGTTCAGCACAACCA

11. TTTGAACGAAAACATTACGGTACCG

12. GATCCGGTACCGTAATGTTTTCGTT

TABLE IX ECEPO SECTION 1

XbaI

Eco RI I ₁ , 3.

AATTCTAG AAACCATGAG GGTAATAAAA TfljATGGCTCC GCCGCGTCTG GATC TTTGGTACTC CCATTATTTT ATTaCC] GAGG CGGCGCAGAC

ATCTGCGAc | CGAG AGTtCT GGAACGTTAC CTGCTG bAAG CTAAAGAAGC TAGACGCTGA GCTCJTCAAGA CCTTGCAATG GACGACCTTc] GATTTCTTCG

TGAAAACATC ACTTTTGTAG 8

CCACTGGTT GTGCTGAACA CTGTTC

11. TTTG AACGAAAACA

GGTGRCCAA CACGACTTGT GACAAGAAACI TTGCTTTTGT 10

Kpn l BamHI TTACGGTACC G AATGCCATGG CCTAG 12

TABLE X

ECEPO SECTION 2 OUGONUCLEOTIDES

1. AATTCGGTACCAGACACCAAGGT

2. GTTAACCTTGGTGTCTGGTACCG

3. TAACTTCTACGCTTGGAAACGTAT 4 ... TTCCATACGTTTCCAAGCGTAGAA

5. GGAAGTTGGTCAACAAGCAGTTGAAGT

6. CCAAACTTCAACTGCTTGTTGACCAAC

7. TTGGCAGGGTCTGGCACTGCTGAGCG

8. GCCTCGCTCAGCAGTGCCAGACCCTG

9. AGGCTGTACTGCGTGGCCAGGCA

10. GCAGTGCCTGGCCACGCAGTACA

11. CTGCTGGTAAACTCCTCTCAGCCGT

12. TTCCCACGGCTGAGAGGAGTTTACCA

13. GGGAACCGCTGCAGCTGCATGTTGAC

14. GCTTTGTCAACATGCAGCTGCAGCGG

15. AAAGCAGTATCTGGCCTGAGATCTG

16. GATCCAGATCTCAGGCCAGATACT TABLE XI

ECEPO SECTION 2.

EcoRI Kpn l 1_.

A ATTCGGTACC AGACACCAAG GT TAACT TCT ACGCTTGGAA ACGTAT PGAA

GCCATGG TCTGTGGTTC caaTtgIaaga tgcgaacctt tgcataccTt]

1 A

5, 7

GTTGGTCAAC AAGCAGTTGA AGT jTTGGC AG GGTCTGGCAC TGCTGAGCGfi

CAACCAGTTG TTCGTCAACT TCAAÄCCJGTC CCAGACCGTG ACGACTCGCT

6. ^Γ

2. 1 il

GGCTGTACTG CGTGGCCAGG CA pTGCT GGT AAACTCCTCT CAGCCGTGGG

CCGJACATGAC GCACCGGTCC GTGÄCGhcCA TTTGAGGAGA GTCGGCACcT

1P_

λΐ ι 15 BgIII BamHI

AACCGCTGCA gctgcatgtt gac baagc ag tatctggcct GAGaTC ^- TG -

TTfcGCGACGT CGACGTACAA ctgtttcgJtc atagaccgga ctctagacctac

  IA '

TABLE XII ECEPO SECTION 3

1. GATCCAGATCTCTGACTACTCTGC

2. ACGCAGCAGAGTAGTCAGAGATCTG

3. TGCGTGCTCTGGGTGCACAGAAAGAGG

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 TCCAGATCTCTG GTCTAGAGAC

¹ ACTACTCTGC

TGATGAGACG 2

GCGT GCTCT GGGTGCACAG AAAGAGG FTA TC TCTCCGCC CGCAtGAGA CCCACGTGTC TTTCTCCGAT AGjAGAGGCGG

GGATGCTGCA TCT CCTACGACGT AGA

- L-

3CTG CAC CGCTGCGTAC CATCACT GCT GAT ACCTTCC

; tg gcgacgcatg gtagtgacga ct ^ jtggaagg

: GAC!

GCAAACTGTT TCG tTGTAT AC TCTAACTTCC TGCGTGGTA CGTTTGACAA AGCACATA (TG AGATTGAAGG ACGCACCAT 10 12

ACTGAAACTG

TGAqTTTGAC

11 sai l

TATACTGGCG AAGC hTGCC G TACTGGTGAC CGCTAATAG ATATGACCGC TTCGTACGOC ATGACCACTG GCGATTATC AGCT 14 16

TABLE XIV ECEPO GENE 3

-11

Xba I - 'MetAla CTAG AAACCATGAG GGTAATAAAA TAATGGCTCC GCCGCGTCTG TTTGGTACTC CCATTATTTT ATTACCGAGG CGGCGCAGAC

ATCTGCGACT CGAGAGTTCT GGAACGTTAC CTGCTGGAAG CTAAAGAAGC TAGACGCTGA GCTCTCAAGA CCTTGCAATG GACGACCTTC GATTTCTTCG

TGAAAACATC ACCACTGGTT GTGCTGAACA CTGTTCTTTG AACGAAAACA ACTTTTGTAG TGGTGACCAA CACGACTTGT GACAAGAAAC TTGCTTTTGT

TTACGGTACC AGACACCAAG GTTAACTTCT ACGCTTGGAA ACGTATGGAA AATGCCATGG TCTGTGGTTC CAATTGAAGA TGCGAACCTT TGCATACCTT

GTTGGTCAAC AAGCAGTTGA AGTTTGGCAG GGTCTGGCAC TGCTGAGCGA CAACCAGTTG TTCGTCAACT TCAAACCGTC CCAGACCGTG ACGACTCGCT

GGCTGTACTG CGTGGCCAGG CACTGCTGGT AAACTCCTCT.CAGCCGTGGG CCGACATGAC GCACCGGTCC GTGACGACCA TTTGAGGAGA GTCGGCACCC

AACCGCTGCA GCTGCATGTT GACAAAGCAG TATCTGGCCT GAGATCTCTG TTGGCGACGT CGACGTACAA CTGTfTCGTC ATAGACCGGA CTCTAGAGAC

ACTACTCTGC TGCGTGCTCT GGGTGCACAG AAAGAGGCTA TCTCTCCGCC TGATGAGACG ACGCACGAGA CCCACGTGTC TTTCTCCGAT AGAGAGGCGG

GGATGCTGCA TCTGCTGCAC CGCTGCGTAC CATCACTGCT GATACCTTCC CCTACGACGT AGACGACGTG GCGACGCATG GTAGTGACGA CTATGGAAGG

GCAAACTGTT TCGTGTATAC TCTAACTTCC TGCGTGGTAA ACTGAAACTG CGTTTGACAA AGCACATATG AGATTGAAGG ACGCACCATT TGACTTTGAC

Sai l

TATACTGGCG AAGCATGCCG TACTGGTGAC CGCTAATAG ATATGACCGC TTCGTACGGC ATGACCACTG GCGATTATCA GCT

In detail, Table VIII shows oligonucleotides used to generate Section 1 of the ECEPO gene, which encodes the amino terminal residues of human polypeptide. Oligonucleotides were combined into doublings (1 and 2, 3 and 4 etc.) and the doublets were then ligated to arrive at ECEPO Section 1 as in Table IX. It should be noted that the unified section includes terminal single-stranded EcoRI and BamHI ends, that "downstream" of the single-stranded EcoRI end is an XbaI restriction enzyme recognition site, and that "upstream" of single-stranded BamHI end is a Kpml recognition site is. Section 1 could be easily amplified using the M13 phage vector used to check the sequence of the section. Some difficulties have been encountered in isolating the section as an XbaI / KpnI fragment from RF DNA generated in E. coli, probably due to methylation of the KpnI recognition site bases within the host. Thus, single-stranded phage DNA was isolated and translated into the double-stranded form in vitro by primer extension, and the desired double-stranded fragment was then readily isolated.

The ECEPO gene sections 2 and 3 (Tables XI and XIII) were constructed similarly from the oligonucleotides of Tables X and XII. Each section was amplified in the M 13 vector, which was used for sequence verification for accuracy, and was isolated from phage DNA.

As can be seen from Table XI, ECEPO Section 2 was constructed with single-stranded EcoRI and BamHI ends and could be isolated as a KpnI / BglII fragment. Similarly, ECEPO Section 3 was prepared with single-stranded BamHI and SaI I ends and could be isolated from phage RFDNAaIs η Bgl II / Sal I fragment. The three sections prepared in this manner could be readily pooled into a contiguous DNA sequence (Table XIV) that encodes the entire human EPO polypeptide with an amino terminal methionine codon (ATG) for E. coli translation initiation. Furthermore, it should be noted that "upstream" of the initial ATG is a series of base pairs that duplicate the ribosomal binding site sequence of the highly expressed E. coli OMP f gene.

To carry the ECEPO, any suitable expression vector can be used. The particular vector chosen for expression of the ECEPO gene as the "temperature-sensitive" plasmid pCFM536 - a derivative of plasmid pCFM414 (ATCC40076) is described in copending U.S. Patent Application No. 636727 filed August 6, 1984 of Charles F.Morris.

In particular, pCFM 536 was degraded with XbaI and HindIII; the large fragment was isolated and used in a two-part ligation with the ECEPO gene. Sections 1 (Xbal / KpnI), 2 (Kpml / Bgl II) and 3 (Bgl II / Sal I) were previously incorrectly combined in M13 and the EPO gene was isolated from it as a single Xba I / Hind III antibody. Fragment. To this fragment belonged a portion of the polylinker from the M13 and mp9 phage spanning the Sal I to Hind I II sites therein. Control of expression in the resulting expression plasmid p536 was by means of a lambda PL promoter, itself controlled by the Cii8S7 repressor gene (as provided by the E. coli strain K12 Δ Htrp).

The prepared ECEPO gene above could be modified in various ways to encode erythropoietin analogs such as [Asn ² , desPro ² to He ⁶ JhEPO and [HiS ⁷ JhEPO as described below.

A. [Asn ² des-Pro ² through lle ^e] hEPO

Plasmid 53b carrying the ECEPO-produced gene of Table XIV as an XbaL to Hind III insert was digested with Hind III and Xho I. The latter endonuclease cleaves the ECEPO gene at a single 6 base pair recognition site spanning the last base of the Asp ⁸ encoding codon to the second base of the Arg ^1c codon.

An Xba I / Xhol "linker" sequence was prepared containing the following sequences:

Xbal +1.2 7 8 9

Met AIa · Asn Cys Asp Xho I

5'-CTAG ATG GCT AAT TGC GAC-3 '

3 '-TAC CGA TTA ACG CTG AGCT-5'

The Xba I / Xho I linker and the Xho I / Hind III ECEPO gene sequence fragment were inserted into the large fragment obtained from the Xba I and Hind III digestion of plasmid pCFM 526 - a derivative of plasmid pCFM414 (ARCC40076). As described in co-pending U.S. Patent Application No. 6,36727, filed August 6, 1984, by Charles F. Morris, to generate a plasmid-born DNA sequence expressing E. coli expression of Met ^. Form of the desired analog coded

For example, [His ⁷² ] hEPO

Plasmid 536 was digested with Hind III and Xho 1 as in Part A above. An Xba I / Xho I linker was prepared having the following sequence: Xba I + 123456789 Xho I

Met AI

5'-CTAG ATG GCT

3 '-TAC CGA

The linker and the Xhol / Hind III ECEPO sequence fragment were then inserted into pCFM 526 to generate a plasmid-born DNA sequence which encodes E. coli expression of the Met ~ ¹ form of the desired analog. The construction of a produced gene ("SCEPO") including yeast preference codons is described in the following Tables XV to XXI As with the ECEPO gene, the formation of three sets of oligonucleotides (Tables XV, XVII and XIX) belongs to the overall construction in duplicates It has been noted that the synthesis was partially facilitated by the use of some suboptimal codons, both in SCEPO and ECEPO construction, ie, oligonucleotides 7 and 7 were pooled into sections (Tables XVI, XVIII, and XX) Section I of both genes were identical, as were the oligonucleotides 1-6 of Section 2 in each gene.

TABLE XV

SCEPO SECTION 1 OLIGOIMUCLEOTIDES

1. · AATTCAAGCTTGGATAAAAGAGCT

2. GTGGAGCTCTTTTATCCAAGCTTG

3. CCACCAAGATTGATCTGTGACTC

4. TCTCGAGTCACAGATCAATCTTG

5. GAGAGTTTTGGAAAGATACTTGTTG

6. CTTCCAACAAGTATCTTTCCAAAC

7. GAAGCTAAAGAAGCTGAAAACATC

8. GTGGTGATGTTTTCAGCTTCTTTAG

9. ACCACTGGTTGTGCTGAACACTGTTC

10. CAAAGAACAGTGTTCAGCACAACCA

11. TTTGAACGAAAACATTACGGTACCG

12. GATCCGGTACCGTAATGTTTTCGTT

2 3 4 5 6 7 8th 9 Per Per bad Leu He His Asp CCG CCA CGT CTG ATC CAT CAC-3 ' GGC GGT GCA GAD DAY GTA CTG AGCT-5 '

TABLE XVI SCEPO SECTION 1

EcoRI HiodIII _1 AATTCA AGCTTGGATA GT TCGAACCTAT 2

AAAGAGCTZCACCAAGATTG ATCTGTGACT CGAGAGTTTT

TTTCTCGAGG TGIGTTCTAAC TAGACACTGA GCTCTJCAAAA

GGAAAGATAC TTGTTG PAAG CTAAAGAAGC TGAAAACATC hCCAC TGGTT CCTTTCTATG AACAACCTTC] GATTTCTTCG ACTTTTGTAG TGGTG & CCAA 6 I

1 ii HEU ¹ BamHI

GTGCTGAACA ctgttc | tttg aacgaaaaca ttacggtacc g

CACGACTTGT GACAAGAÄÄC] TTGCTTTTGT AATGCCATGG CCTAG

TABLEXVII

SCEPO SECTION 2 OLIGONUCLEOTIDES

1. AATTCGGTACCAGACACCAAGGT

2. GTTAACCTTGGTGTCTGGTACCG

3. TAACTTCTACGCTTGGAAACGTAT, 4. TTCCATACGTTTCCAAGCGTAGAA

5. GGAAGTTGGTCAACAAGCAGTTGAAGT

6. CCAAACTTCAACTGCTTGTTGACCAAC

7. TTGGCAAGGTTTGGCCTTGTTATCTG

8. GCTTCAGATAACAAGGCCAAACCTTG

9. AAGCTGTTTTGAGAGGTCAAGCCT

10. AACAAGGCTTGACCTCTCAAAACA

11. TGTTG GTTAACTCTTCTCAACCATG G G

12. TGGTTCCCATGGTTGAGAAGAGTTAACC

13. AACCATTGCAATTGCACGTCGAT

14. CTTTATCGACGTGCAATTGCAA

15. AAAGCCGTCTCTGGTTTGAGATCTG

16. GATCCAGATCTCAAACCAGAGACGG

Kpn l

EcoRI 1

~ ~~~ ÄTTCGGTACC AGACACCAAG GCCATGG TCTGTGGTTC 2

TAACTTCT ACGCTTGGAA ACGTATPGAA GTTGGTCAAC AAGCTGTTGA

CAACCAGTTG TTCGACAACT 6

iTTCnAGA TGCGAACCTT TGCATACCT

AGT | TTGG CA GGTTTGGCCT TGTTATCTG p AGC TGTTTTG AGAGGTCAAG

TCAACFJ3TT CCAAACCGGA ACAATAGACTTCGhCAAAAC TCTCCAGTTC

8 '10

CCT frGTT GGT TAACTCTTCT CAACCATGGG f \ ACCAT TGCA ATTGCACGTC GGAAC ~ ÄÄtCA ATTGAGAAGA GTTGGTACCC TTGGTt \ ACGT TAACGTGCAG

12 '14

11 ML · ai £ L

GAT hAAGC CG TCTCTGGTTT GAGATCTG CTATTTCbGC AGAGACCAAA CTCTAnAPHTA Π

TABLE XVIII SCEPO SECTION 2

TABLE XIX

SCEPO SECTION 3 OLIGONUCLEOTIDES

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

TABLE XX SCEPO SECTION 3

BamHI BgIII 1

GATC CAGATCTTTG ACTACTTTGT T ^ AGAG CTTT GTCTAGAAAC TGATGAAACA ACTCTbGAAA 2

3

GGGTGCTCAA AAGGAAG fcCA TT TCCCCACC AGACGCTGCT TCTGCCGCTC CCCACGAGTT TTCCTTCGGT A) AAGGGGTGG TCTGCGACGA AGÄCGGCGAG

2. 9

CATTGAGAAC CATC PCTGC T GATACCTTCA GAAAGTTJ ^ TT CAGAGTTTAC

GTAACTCTTG GTAGTGÄCfcA CTATGGAAGT CTTTCAATAA GItCTCAAATG

8 10 I!

"'12

TCCAACTTCT [TGAGAGGTAA ATTGAAGTTG TACAC ^ GGT G AAGCCTGTAG

AGGTTGAAGA ACTCfTCCATT TAACTTCAAC ATGTGETTACC TTCGGACATC

¹ 14 ^

AACTGGT GAC AGA TAAGCCC GACTGATAAbAACAGTGTAG TTGACCACTG TCjTATTCGGG CTGACTATTG TTGfTCACATC

Sai l

ATGTAACAAA G TACATTGTTT CAGCT 20

TABLE XXI SCEPOGEN

HLND III AGCTTGGATA ACCTAT

-1 +1 ArgAla AAAGAGCTCC TTTCTCGAGG

ACCAAGATTG TGGTTCTAAC

ATCTGTGACT TAGACACTGA

CGAGAGTTT GCTCTCAAAA

GGAAAGATAC TTGTTGGAAG CTAAAGAAGC TGAAAACATC ACCACTGGTT CCTTTCTATG AACAACCTTC GATTTCTTCG ACTTTTGTAG TGGTGACCAA

GTGCTGAACA CTGTTCTTTG AACGAAAACA TTACGGTACC AGACACCAAG CACGACTTGT GACAAGAAAC TTGCTTTTGT AATGCCATGG TCTGTGGTTC

GTTAACTTCT ACGCTTGGAA ACGTATGGAA GTTGGTCAAC AAGCTGTTGA CAATTGAAGA TGCGAACCTT TGCATACCTT CAACCAGTTG TTCGACAACT

AGTTTGGCAA GGTTTGGCCT TGTTATCTGA AGCTGTTTTG AGAGGTCAAG TCAAACCGTT CCAAACCGGA ACAATAGACT TCGACAAAAC TCTCCAGTTC

CCTTGTTGGT TAACTCTTCT CAACCATGGG AACCATTGCA ATTGCACGTC GGAACAACCA ATTGAGAAGA GTTGGTACCC TTGGTAACGT TAACGTGCAG

GATAAAGCCG TCTCTGGTTT GAGATCTTTG ACTACTTTGT TGAGAGCTTT CTATTTCGGC AGAGACCAAA CTCTAGAAAC TGATGAAACA ACTCTCGAAA

GGGTGCTCAA AAGGAAGCCA TTTCCCCACC AGACGCTGCT TCTGCCGCTC CCCACGAGTT TTCCTTCGGT AAAGGGGTGG TCTGCGACGA AGACGGCGAG

CATTGAGAAC CATCACTGCT GATACCTTCA GAAAGTTATT CAGAGTTTAC GTAACTCTTG GTAGTGACGA CTATGGAAGT CTTTCAATAA GTCTCAAATG

tccaacttct tgagaggtaa attgaagttg tacaccggtg aagcctgtag aggttgaaga actctccatt taacttcaac atgtggccac ttcggacatc

AACTGGTGAC AGATAAGCCC GACTGATAAC AACAGTGTAG TTGACCACTG TCTATTCGGG CTGACTATTG TTGTCACATC

Sai l

ATGTAACAAA G TACATTGTTT CAGCT

The pooled SCEPO sections were sequenced in M13 and sections 1.2 and 3 were isolatable from the phage as HindIII / KpnI, KpnI / BglII and BglI / Sall fragments.

The presently most preferred expression system for SCEPO gene products is a S. Cerevisiae α-factor secretion-based secretion system, as described in copending U.S. Patent Application No. 487753, filed April 22, 1983, by Grant A. Bitter, published October 31, 1984 as European Patent Application 0123294. Briefly, the system includes constructions in which DNA encoding the leader sequence of the yeast a-factor gene product is located immediately 5 'to the coding region of the exogenous gene to be expressed. As a result, the translated gene product contains a leader or signal sequence that is "processed off" by an endogenous yeast enzyme in the course of secretion of the remaining product it was not necessary to include such a codon at the -1 position of the SCEPO gene.

As can be seen from Table XXI, the alanine (+ 1) encrypting sequence is preceded by a linker sequence allowing direct insertion into a plasmid containing the DNA for the first 80 residues of the a-factor leader sequence, which is the a-factor. Promoter follows. The specifically preferred construction for SCEPO gene expression includes a four-part ligation, including the above-mentioned SCEPO section fragment and the large fragment of Hind III / SaI I degradation of the plasmid p C3. From the obtained paC3 / SCEPO, the aa factor promoter and the leader sequence and SCEPO gene were isolated by digestion with Bam HI and ligated into the Barn HI-degraded plasmid pYE to arrive at the expression plasmid pYE / SCEPO.

Example 12

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

Using the expression system intended for use with E. coli host cells, plasmid p536 was transformed into AM 7 E. coli cells previously transformed with a suitable plasmid, pMW1, followed by C ₈ 57 gene was obtained. Cell cultures in LB broth (ampicillin 50 / xg / ml and kanamycin o / zg / ml), preferably with 1OnM mg SO ₄₎ were added at 28 ⁰ C and to the growth of the cells in KuItUr to 0. D. _6O o = 0 , 1 held. EPO expression was evoked by raising the culture temperature to 42 ° C. The cells grew to about 40 OD and resulted in an EPO production (estimated by gel) of about 5 mg / 0.D. Liter.

The cells were harvested, lysed, disrupted with the French press (10,000 psi) and treated with lysozan and NP-40 detergent. The resulting pellet after 24,000xg centrifugation was dissolved with guanidine-HCl and subjected to another one step purification by C ₄ (Vydac) reverse phase HPLC (EtDHm 0-80%, 50 mM NH ₄ Ac, pH 4.5). Protein sequencing gave a product with a purity of greater than 95%, and the products obtained showed two different amino terminals, APPR ... and PPR ... in a relative quantitative ratio of about 3 to 1. This latter observation of hEPO- and [ of AlaHlEPO production showed that amino terminal "work" within the host cells serves to remove the terminal methionine and in some cases the initial alanine.

The radioimmunoassay activity for the isolates was in the range of 150,000 to 160,000 U / mg; the in vitro assay activity is in the range of 30,000 to 62,000 U / mg; and the in vivo assay activity was about 120 to 720 U / mg. (Cf., human urine isolate standard of 70000 U / mg in each assay). The dose-response curve for the recombinant product in the in vivo assay deviated markedly from that of the human urine EPO standard.

The EPO analog plasmids formed in parts A and B of Example 11 were each transformed into pMW1-transformed AM 7 E. coli cells and the cells were cultured as above. Purified isolates were tested both via RIAaIs and in vitro assays. The RIA and in vitro assay values for [Asn ² , des-Pro ² to He ⁶ ] hEPO expression products were approximately 11 000 U / mg and 6000 U / mg protein, while the assay values for [His ⁷ -] hEPO were approximately 4100 E / mg and 14000E / mg protein. This indicated that the analog products were one-quarter to one-tenth as active as the "Eltem" expression products in the assays.

Into the expression product designed for use with S. cerevisiae host cells, the plasmid pYE / SCEPO was transformed into two different strains, YSDP4 (genotype α pep4-3 trpl) and RK81 (genotype α α pep4-3 trpl).

Transformed YSDP4 hosts were grown in SD medium (Methods in Yeast Genetics, Cold Spr. Harb. Lab., Cold Spring Harbor, NY, p. 62 [1983]) supplemented with casamino acids 5%, pH 6.5 at 30 ⁰ C. The medium was harvested as the cells

360. D. and EPO products were obtained in the amount of about 244 U / ml (97 μg / L.D. liter according to RIA).

Transformed RK81 cells grew to either 6.5 O.D. or 60 O.D., yielding media with EPO concentrations of about 80-90 U / ml (34 / j, g / O.D. liter according to RIA). Preliminary analyzes showed significant heterogeneity in the products produced by the expression system, probably because of variation in the glycosylation of expressed proteins and because of the relatively high mannose content of the associated carbohydrate.

The plasmids PaC3 and pYE in HB101 E. coli cells were prepared according to the Implementing Regulation of the US Patent Office on May

Filed September 27, 1984 at the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland under the accession numbers ATCC 39881 and ATCC 39882. The plasmids pCFM526 in AM7 cells, pCFM536 in JM103 cells and pMW1 in JM103 cells were deposited in the same manner on November 21, 1984 as ATCC 33932, 333934 and 33933.

The Saccharomyces cerevisiase strains YSPD4 and RK81 were deposited on November 21, 1984 as ATCC 20734 and 20733.

It should be apparent upon consideration of the examples that numerous, extremely valuable products and procedures have been produced by the present invention in its many aspects.

The polypeptides contemplated by the invention are remarkably useful materials, whether present as microbially expressed products or synthetic products, and their primary, secondary, and tertiary structural arrangement has been first disclosed by the present invention.

As noted previously, the recombinantly produced and synthetic products of the present invention, to varying degrees, share the in vitro biological activity of EPO isolates from natural sources, and accordingly, can be used as substitutes for EPO isolates in culture media used for the growth of erythropoietin cells in culture. To the extent that the polypeptide products of the present invention similarly share the in vivo activity of natural EPO isolates, they are remarkably suitable for use in erythropoietin therapy, as practiced in mammals, including humans, to develop some or all of the effects as attributed in vivo to EPO, e.g. Stimulation of the reticulocyte reaction, development of ferrokinetic effects (such as plasma iron inversion and bone marrow transient effects), erythrocyte mass changes, stimulation of hemoglobin C synthesis (see Eschbach et al., Supra), and as noted in Example, increase hematocrit levels in mammals.

Among the group of people to be treated with the products of the present invention are patients who generally require blood transfusions including traumatic victims, surgical patients, kidney disease patients including dialysis patients and patients with a standard deviation of blood composition affecting diseases such as hemophilia, sickle cell disease , physiological anemia and the like. Limiting the need for transfusion therapy to a minimum by using EPO therapy may reduce the transmission of infectious components. Of the products of the invention can

can be expected to be free from pyrogens, natural inhibiting substances and the like because of production by recombinant methods, and thus they are likely to have an increased overall effect in therapeutic procedures over naturally derived products. Erythropoietin therapy with products of the invention may thus also be useful in increasing the oxygen-carrying capacity of animals suffering from hypoxic environmental conditions, and possibly in the production of cardiovascular effects.

A preferred method of administering polypeptide products of the present invention is by parenteral routes (e.g., i.v., i.m., s.c., or i.p.), and the compositions administered will normally include therapeutically effective amounts of the product in combination with acceptable carriers and / or adjuncts. Preliminary pharmacokinetic studies show a longer in vivo half-life for monkey EPO products when i. m. better than i.v. administration. It is expected that the effective dosage will depend essentially on the conditions to be treated. However, therapeutic doses are currently estimated to be in the range of 0.1 (~ 7E) to 100 (~ 7000E) μg / kg body mass of active material. For pharmaceutical compositions according to the invention is to be expected with standard diluents such as human serum albumin as well as with standard carriers such as saline. Excipients suitable for use in compositions of the present invention include compounds having independently detected erythropoietin-stimulating effects, such as testosterone, progenitor cell stimulants, insulin-like growth factors, prostaglandins, serotonin, cyclic AMP, prolactin and triiodothyronine, as well such as agents commonly used in the treatment of aplastic anemias, such as methenols, stanozolol and nandrolen (see, e.g., Resegotte, et al., Panminerva Medica, 23 243-248 [1981]; McGonigle, et al., Kidney Int , 25 [2], 437-444 [1984], Pavlovic-Kantera, et al., Hematol., 8 [supp. 8], 283-291 [1980], and Kurtt, FEBS Letters, 14a [1 ], 105-108 [1962]). Also suitable as an adjuvant are substances that are reported to increase or synergistically enhance the effects of erythropoietin or asialo-EPO, such as adrenergic agonists, thyroid hormones, androgens and BPA (see Dunn, "Current Concepts in Erythropoieses", John Wiley and Sons [Chichester, England, 1983]; Weiland, et al., Blut, 44 [3], 173-175 [1982]; Kalmanti, Kidney Int., 22, 383-391 [1982]; Shahidi, New. Eng J. Med., 289, 72-80 [1973]; Fisher, et al., Steroids, 30 [6], 833-845 [1977]; Urabe, et al., J. Exp. Med., 149, 1314 -1325 [1979], and Billat, et al., Hpt., Hematol., 10 [1], 133-140 [1980]), as well as classes of compounds termed "hepatic erythropoietic factors" (see Naughton et al. Acta., Haemat., 69, 171-179 [1983] and "Erythrotropins" [described by Congote, et al., In Abstract 364, Proceedings 7th International Congress of Endocrinology Quebec, July 1-7, 1984]; Congote, Biochem., Biophys Res. Comm., 115 [2], 447-483 [1983] and Congo, Anal. Biochem. 140,428-433 [1984] and "erythrogenins" described in Rothman, et al., J. Surg. Oncol., 20 105-108 [1982]).

Preliminary screenings to measure erythropoietic responses of ex-hypoxic polycythemic mice pretreated with either 5-a-dihydrotestosterone or nadrolene and then treated with erythropoietin of the present invention produced dubious results.

The diagnostic use of polypeptides of the invention is similarly extensive and may include use in labeled and unlabeled forms in a variety of immunoassay techniques, such as RIA's, ELISA's and the like, as well as a variety of in vitro and in vivo assays. (see, for example, Dunn, et al., Hematol Ex., 11 [7], 590-600 [1983]; Gibson et al., Pathology, 16, 1555-156 [1984]; Krystal, Hpt., Expt., 11 [7 ], 649-660 [1983]; Saito, et al., Jap. J. Med., 223 [1], 16-21 [1984]; Natahn, et al., New Eng. J. Med., 308 [ 9], 520-522 [1983]) and numerous references relating to assays reported there.

The polypeptides of the invention, including the synthetic peptides, contain sequences of EPO residues reported herein for the first time, also yielding very useful pure materials for the generation of polyclonal antibodies and "banks" of monoclonal antibodies specific for the differentiation of consecutive and discontinuous epitopes of As an example, the preliminary analysis of the amino acid sequences of Table VI in the context of the hydropathicity of Hoppetal., PNAS (USA), 78, pp. 3824-3828 (1981) and of secondary structures by Chou et al., Ann. Rev Biochem., 47: 251 (1978) that the synthetic peptides with duplicated contiguous sequences of residues spanning positions 41 through and including 57,116 through and including 118 and 144 through 166 are likely to result in a highly antigenic reaction and useful monoclonal and generate polyclonal antibodies that bind with both the synth peptide and the entire protein are immunoreactive. It is expected that such antibodies will be useful in the detection and affinity purification of EPO and EPO-related products

 By way of illustration, the following three synthetic peptides were prepared:

(DhEPO 41-57, V-P-D-T-K-V-N-F-Y-A-W-K-R-M-E-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 the above polypeptides have shown a relatively weak positive response to hEPO41-57, a marked response to hEP0116-128, and a strong positive response to hEP0144-166, as measured by the capacity of rabbit serum antibody to immunodeplate ¹²⁵ μl. labeled human urine EPO isolates. Preliminary in vivo activity studies on these three peptides showed no significant activity, either alone or in combination.

While the deduced amino acid residue sequences of mammalian EPO, according to the illustrative examples, essentially define the primary structural organization of the mature EPO, it is believed correct that the specific sequence of 165 amino acid residues of the monkey species EPO is given in Table V and the 166 residues of human EPO. EPO in Table VI does not limit the scope of the useful polypeptides provided by the invention. Included in the present invention are those numerous naturally occurring allelic forms of EPO that the research has previously suggested to exist in biologically active mammalian polypeptides such as human y interferon (See, for example, the human interferon species that are in position with an arginine residue 140, according to EP Application 0077670 and the species having a glutamine residue in position 140 according to Gray et al., Nature, 295, pp. 503-508 [1982]. Both species were characterized as forming "mature" human y interferon sequences Allelic forms of mature EPO polypeptides may differ and from the sequences of Tables V and VI in terms of the length of the sequence and / or deletions, substitutions, insertions or additions of amino acids in the sequence with consistent potential variations in glycosylation capacity

 As previously stated, it is believed that a putative allelic form of human EPO is a methionine residue

at position 126. As expected, natural allelic forms of EPO-encoding DNA genomic and cDNA sequences likely to encode the aforementioned types of allelic polypeptides, or simply using aberrant codons to design the same polypeptides as outlined, are also likely to occur. In addition to naturally occurring allelic forms of mature EPO, the present invention also encompasses other "EPO products" such as polypeptide analogs of EPO and fragments of the "mature" EPO. Using the procedures of the above published application of Alton et al.

(WO / 83/04053) it is easy to design and produce genes which code for the microbial expression of polypeptides having primary arrays which differ from those of mature EPO with respect to the identity or location of one or more residues (e.g. Substitutions, terminal and intermediate additions and deletions).

One by one, modifications and cDNA and genomic EPO genes can be readily achieved by known site-directed mutagenesis techniques and used to generate analogs and derivatives of EPO. Such EPO products would have at least one of the biological properties of EPO but could be different in others. By way of example, intended EPO products of the invention may include those shortened by, for example, US Pat. Deletions [Asn ² , des-Pro ² to He ⁶ JhEPO, [des-Thr ¹⁶⁴ to Arg ¹⁶⁶ ] hEPO and "A27-55hEPO", the latter having residues coding for a deleted whole exon, or the more stable against Or have been modified to delete one or more potential sites for glycosylation (which can lead to higher activities for yeast-produced products); or the one or more have multiple cysteine residues that are deleted or replaced by, for example, histidine or serine residues (as in the analogous [HiS ⁷ IhEPO] and potentially more readily isolatable in active form from microbial systems, or that have one or more tyrosine residues produced by Phenylalanine is replaced as in the analogues [Phe ¹⁵ ] hEPO, [Phe ⁴⁹ ] hEPO and [Phe ¹⁴⁵ ] hEPO and more or less readily EPO receptors can bind to target cells.

Also included are polypeptide fragments that double only a portion of the contiguous amino acid sequence or secondary arrays within the mature EPO whose fragments may have EPO activity (e.g., receptor binding) and no other (eg, erythropoietic activity). Of particular importance in this context are those potential fragments of the EPO that have been elucidated (structurally) using the human genomic DNA sequence of Table VI, e.g. For example, "fragments" of the entire EPO sequence represented by intron sequences and which can form highlighted "domains" of biological activity It is noteworthy that the lack of in vivo activity for any one or more the "EPO products" of the invention do not fully exclude their therapeutic utility (see Weiland et al., supra) or utility in other contexts such as EPO assays or EPO antagonism.

Erythropoietin antagonists may be very useful in the treatment of polycythemia or cases of overproduction of EPO (see Adamson, Hosp. Practice, 18 [12], 49-57 [1983] and Hellman et al., Clin, Lab. Hacmat, 5 , 335-342. [1983]).

According to another aspect of the present invention, the cloned DNA sequences described herein encoding human and monkey EPO polypeptides are remarkably valuable for the information contained therein regarding the amino acid sequence of mammalian erythropoietin that have not been obtained to date except for Sharing analytical procedures of isolates of naturally occurring products. The DNA sequences are also remarkably valuable as products that can be used to extensively describe the microbial synthesis of erythropoietin by a variety of recombinant techniques. On the other hand, the DNA sequences provided by the present invention are useful for the generation of novel and beneficial viral and circular plasmid DNA vectors, novel and beneficial transformed and transferred microbial procaryotic and eucaryotic host cells (including bacterial and yeast cells, and mammalian cells grown in culture), and novel and beneficial methods of cultivated growth of such microbial host cells capable of expressing EPO and EPO products. The DNA sequences of the present invention are also remarkably suitable materials for use as labeled probes in the isolation of EPO and related protein-encoding cDNA and genomic DNA sequences from mammals other than the human and monkey species specifically discussed herein. The content to which the DNA sequences according to the invention are used in the different alternative method for the synthesis of Prokin (eg in insect cells) or in the genetic therapy of humans or other mammals can not yet be calculated. It is expected that the DNA sequences of the present invention may be useful in the development of transgenic mammalian species which may serve as alseucaryotic "hosts" for the production of erythropoietin and erythropoietin products in amounts, see Polmiter et al., Science, 222 (4625), 809 -814 (1983).

From this point of view, it is clearly not intended that the illustrative examples limit the scope of the present invention, and numerous modifications and variations will be apparent to those skilled in the art. As an example, while to the DNA sequences pertaining to the illustrative examples, as this application provides amino acid sequence information essential to the preparation of the DNA sequence, the invention also encompasses those DNA sequences produced as recited in the wake of the art the EPO amino acid sequences can be constructed. These may encode EPO (as in Example 12), as well as EPO fragments and kEPO polypeptide analogues (ie, "EPO products") having one or more of the biological properties of naturally occurring EPO, but not others (or others in different degrees).

The DNA sequences disclosed by the present invention thus include all DNA sequences suitable for safe expression of a polypeptide product in a procaryotic or eucaryotic host cell, which product comprises at least a portion of the primary structural arrangement and one or more of the biological properties of Having erythropoietin selected from:

(a) the DNA sequences listed in Tables V and VI;

(b) DNA sequences which hybridize to those listed under (a) or fragments thereof; and (c) DNA sequences which hybridize to the DNA sequences defined under (a) and (b), but with the genetic code being degenerated. In this regard, it is noteworthy, for example, that existing allelic monkey and human EPO gene sequences and the gene sequences of other mammalian species are expected to hybridize to sequences of Tables V and VI or to fragments thereof. In addition, but with the genetic code degenerating, the SCEPO and ECEPO genes and the produced or mutated cDNA or genomic DNA sequences encoding different EPO

Encrypt fragments and analogs, also hybridize to the above-mentioned DNA sequences. Such hybridizations could easily be performed under the hybridization conditions described herein with regard to the initial isolation of monkey and human EPO-encoding DNA or more severe conditions to reduce background hybridization if desired.

Similarly, a variety of expression systems are within the scope of the invention, while the above examples illustrate the invention of microbial expression of EPO products in the context of mammalian cell expression of DNA inserted into a hybrid vector of bacterial plasmid and viral genomic origin , Particularly encompassed are expression systems that include vectors of homogeneous origin that are applied to a variety of bacterial, yeast and mammalian cells in culture, as well as expression systems that do not contain vectors (such as calcium phosphate transfection of cells). In this regard, the matter is to be understood that the expression of z. B. DNA of monkeys in monkey host cells in culture and human host cells in culture Indeed, cases of "exogenous" DNA expression, as in EPO DNA whose high expression level is sought, would not originate in the genome of the host. In addition, the expression systems of the present invention take into account these habits which lead to the cytoplasmic formation of EPO products and the accumulation of glycosylated and non-glycosylated EPO products in the host cell cytoplasm or membranes (eg accumulation in bacterial periplasmic spaces) or in the supernatant of Culture media, as discussed above, or in more unusual systems, such as P. aeruginosa expression systems (described by Gray et al., In Biotechnology, 2, pp. 161-165 [1984]) Improved Hybridization Methodologies of the Invention Illustrative of the Above DNA / dNA hybridization screenings are also applicable to RNA / RNA and RNA / DNA screening. Mixed probe techniques, as discussed herein, generally consist of a number of improvements in hybridization techniques that allow for faster and more reliable polynucleotide isolation. These many individual process improvements include: improved colony transfer methods and methods; Using nylin-based filters such as GeneScreen and GeneScreenPlus to allow re-probing with the same film and repeated use of the filters, application of new protease treatments (eg, Taub et al., Anal. Biochem., 126, pp. 222-230 [1982]); Use of very low single concentrations (in the range of 0.025 picomoles) of a larger number of mixing probes (eg the numbers above 32); and carrying out hybridization and Nachhybridisierungsschritten under severe temperatures, which is close (di within 4 ° C, and preferably within 2 ⁰ C away from) the lowest calculated dissociation temperature of any of the mixed probes used. The improvements together lead to results that were unexpected from the outset. This is further emphasized by the fact that mixed probing methods with four times the number of probes reported hitherto as successfully employed in the same cDNA screens of messenger RNA species of relatively small amount have succeeded in isolating a single gene in one Gene library were screened from 1 500000 phage piaques. This was achieved substantially contrary to the view published by Anderson et al., Supra, that mixed-probe screening methods ". for the isolation of mammalian protein genes, if the corresponding RNAs are not available. The features disclosed in the foregoing description, in the following claims and / or in the accompanying drawings, both separately and in combination, may be the basis for the realization of the invention in its various forms.

Claims (1)

A method of producing a polypeptide having part or all of the primary structural design and one or more biological functions of the naturally occurring erythropoietin, characterized by (a) culturing - under appropriate conditions - procaryotic or eucaryotic host cells reshaped or transferred with a biologically active , circular plasmid or virus DNA vector, including purified and isolated DNA sequence coding for the procaryotic or eucaryotic host expression of a polypeptide having some or all of the primary structural design and one or more biological properties of the erythropoietin and (b) the Isolation of the desired polypeptide products of the expression of the DNA sequences in said vector. For this 4 pages drawings