AU606585B2 - Human granulocyte-macrophage colony stimulating factor-like polypeptides and processes for producing them in high yields in microbial cells - Google Patents

Description

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AU- A6 5 4 9 3 P CTWORLD INTELLECTUAMO qAN r O!0 INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCI) (51) International Patent Classification 4 International Publication Number: WO 87/ 02060 C12N 15/00, C12P 19/34, 21/02 Al (3 nentoa ulcto ae pi 9.7(90.7 C07K 9/00, A61K 37/02 (3 nentoa ulcto ae pi 97(90.7 (21) International Application Number: PCT/US86/02 106 (22) International Filing Date: (31) Priority Application Number: (32) Priority Date: (33) Priority Country: Parent Application or Grant (63) Related by Continuation us Filed on 3 October 1986 (03.10.86) 783,414 3 October 1985 (03.10.85) us 783,414 (CIP) 3 October 1985 (03.10.85) (72) Inventors; and (75) Inventors/Applicants (for US only) :DeLAMARTER, John [US/CH]; Chemin des Goulettes 13, CH-1256 Troinex ERNST, Joachim, F. [DE/CH]; Chemin de Marais 156, CH-1255 Veyrier (CH).

(74) Agents: HALEY, James, Jr. et al.; Fish Neave, 875 Third Avenue, 29th Floor, New York, NY 10022- 6250 (US).

(81) Designated States: AT (European patent), AU, BE (European patent), CH (European patent), DE (European patent), DK, FR (European patent), GB (European patent), IT (European patent), JP, LU (European patent), NL (European patent), SE (European patent), US.

-Published Withi international search report.

(71) Applicant (for all designated States except US)i-BIO G -cao 1~ I (54)Title: HUMAN GRAN ULOCYTE- MACRO PHAGE COLONY STIM TIDES AND PROCESSES FOR PRODUCING THEM IN HIGH ,0K0 StOUINC AflI I "Is I00~ r00MW To A-S :1r.a r si? C-L LU.

530 50n 5090 M0 SCCC~gOCCCCAACC56M506ALM5CTGCT TUACAgA06u&5ATCTCMM05r56cCCCAACCACC5CCACAGAr 250 270 700 350 Ito ISO 5AWc055~cGCC5CCC0CTCACC3A0CTCAA000CCCCT5ACCAGAACCCC5CMCACAT5CC5CCACCC50UM5 t' 570 590 %to i50 00 470 490 550 550 550 505 Eta 60 650 670 690 Ito St.GCASAGAGAA&ASTASSACGOL ATAAAMSAiS TA T I I ASSSS l IIATATASAI ISASSAI 17KAGI ICATSOSCCAIAISIS0SSCAAGATGI i 750 (57) Abstract Granulocyte-macrophage colony stimulating factor-like polypeptides having a specific activity of at least I x 108 Units/mg and methods of making these products. DNA sequences and recombinant DNA molecules and microbial hosts transformed with them which produce human granulIo cyte- macro phage colony stimulating factor-like polypeptides in high yields, and methods of making these polypeptides. The human granulocyte-macrophage colony stimulating factor-like polypeptides of this invention may be used to treat cancer patients to regenerate leukocytes after radiation or chemotherapy treatment, and to increase white blood cell count to reduce the likelihood of viral, bacterial, fungal, and parasitic infection, especially in immunologically compromised patients such as these suffering from AIDS.

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B72/B72CIP HUMAN GRANULOCYTE-MACROPHAGE COLONY STIMULATING FACTOR-LIKE POLYPEPTIDES AND PROCESSES FOR PRODUCING THEM IN HIGH YIELDS IN MICROBIAL CELLS TECHNICAL FIELD OF THE INVENTION 4 .4 4 4 15 This invention relates to human granulocytemacrophage colony stimulating factor-like polypeptides (hGM-CSF), DNA sequences, recombinant DNA molecules and processes for producing hGM-CSF. More particularly, the invention relates to hGh-CSF-like polypeptides with a high specific activity, and DNA sequences, recombinant DNA molecules and processes that permit the production of hGM-CSF-like polypeptides in high yields in microbial cells.

BACKGROUND OF THE INVENTION Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) is one of four classes of hemopoietic growth factors known as colony stimulating factors GM-CSF is a growth factor which regulates 20 the proliferation and differentiation of multipotential cells R. Stanley and P. T. Jubinsky, "Factors Affecting the Growth and Differentiation of Haemopoietic Cells in Culture," Clinics In Hematoloqy, 13, pp. 329-48 (1984)]. GM-CSF has also been shown to stimulate the formation of clones of neutrophilic granulocytes and mononuclear phagocytic cells from i single bone marrow cells in vitro W. Burgess et al., "Purification and Properties of a Colony Stimulating Factor from Mouse Lung-Conditioned Medium," J. Biol.

Chem., 252, pp. 1998-2003 (1977)]. Accordingly, the GM- CSF's of this invention should be useful in the recovery of white blood cells after irradiation or chemotherapy.

They also should activate white blood cells to combat infections of bacteria, fungi, and parasites and to accelerate the maturation of leukemic cells and thereby stop the regeneration of leukemic cells.

Mouse GM-CSF (also known as mouse CSF-2), a 24- 26,000 molecular weight glycoprotein which contains no Ssubunits, has been purified from endotoxin-injected mouse lung-conditioned medium W. Burgess, supra; N. A. Nicola 15 et al., J. Biol. Chem., 254, pp. 5290-99 (1979)]. It is unable to stimulate the formation of colonies of erythroid, eosinophil, megakaryocyte cells or of T-and B-lymphocytes in suitable culture conditions, which implies that it is highly selective in its proliferative effects on hemopoietic cells W. Burgess and D. Metcalf, "The Nature and Action of Granulocyte-Macrophage Colony Stimulating Factors," Blood, 56, pp. 947-58 (1980)]. The nucleotide sequence of a mouse lung GM-CSF cDNA is known and has been expressed in animal cells at low yield M.

Gough et al., "Molecular Cloning of cDNA Encoding a Murine Haematopoietic Growth Regulator, Granulocyte-Macrophage Colony Stimulating Factor," Nature, 309, pp. 763-67 (1984); N. M. Gough et al., "Structure and Expression of the mRNA for Murine Granulocyte-Macrophage Colony Stimulating Factor," EMBO Journal, 4, pp. 645-53 (1985)].

Human GM-CSF is also a glycoprotein (24-26,000 daltons). It has been cloned and the cDNA sequence reported G. Wong et al., "Human GM- CSF: Molecular Cloning of the Complementary DNA

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1 'niiiiiniiiiiiiiii-iiiiii-iiiiiii i iiiiii 1 WO 87/02060 PCT/US86/02106 -3and Purification of the Natural and Recombinant Proteins," Science, 228, pp. 810-15 (1985)]. Animal cells transfected with this cDNA sequence synthesize glycosylated GM-CSF on the order of Ipg/mi [Wiesbart et al., "Human Granulocyte-Macrophage Colony-Stimulating Factor is a Neutrophil Activator," Nature, 314, pp. 361-63 (1985); Wong, supra]. Therefore, these animal cells have not been able to produce human GM-CSF in sufficient quantities and with the necessary purity for biological and clinical use.

Accordingly, processes enabling the production of biologically active human GM-CSFs of high purity and in clinically useful amounts are needed.

SUMMARY OF THE INVENTION The present invention solves the problems referred to above by providing DNA sequences that code for human GM-CSF-like polypeptides and by expressing those sequences in high yields in appropriate microbial hosts to produce efficiently and economically large quantities of polypeptides displaying a granulocyte and macrophage colony-stimulating activity. According to this invention, it is possible to modify the amino terminal end of a DNA sequence coding for hGM-CSF and thereby to produce hGM-CSF-like polypeptides in high yields in microbial hosts. By virtue of this invention, it is for the first time possible to obtain polypeptides displaying GM-CSF activity in quantities large enough for clinical use.

Furthermore, the present invention provides unglycosylated hGM-CSF-like polypeptides unexpectedly having a specific activity of at least 1 x 108 Units/mg. This specific activity is substantially higher than the specific activity of native hGM-CSF or of hGM-CSF produced in yeast or animal cells in culture.

k I i I The polypeptides of this invention may be used either as produced or after further derivatization or modification, against bacterial, fungal or parasitic infections, in the regeneration of leukocytes after irradiation or chemotherapy, as well as in methods and compositions for the treatment of leukemia. These compounds may also be used to reduce the likelihood of opportunistic infections in immunologically compromised individuals, such as those suffering from AIDS. In this case GM-CSF-like polypeptides are administered to the AIDS patients to increase their white blood cell count so as to prevent opportunistic infections, thus lengthening the life and reducing the expense of treating the AIDS patient.

0* 0r r 00'* 0 0 0 *0 0*0 BRIEF DESCRIPTION OF THE DRAWINGS 0* 0

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S 4 0 0 15 Figure 1 depicts the nucleic acid and deduced amino acid sequence for an hGM-CSF DNA as isolated from the U937 cell line. The cleavage site for the signal peptide is indicated by an arrow. The nucleotide and amino acid differences from Mo-cell derived cDNA are indicated above 20 and below the U937 equivalent. Nucleotide 356 is either T or C in the Mo-cell derived cDNA.

Figure 2 depicts the nucleic acid sequence for a hGM-CSF cDNA as isolated from the 5637 cell line. The cleavage site for the signal peptide is indicated by an 25 arrow.

Figure 3 depicts a restriction endonuclease map of mouse GM-CSF cDNA, wherein both the coding region, and the nick-translated probe which was used to screen the U937 hGM-CSF library are indicated.

Figure 4 depicts the construction of the E.coli expression vector pPLmuGM-CSF (p210*) for high level hGM- CSF production in microbial cells in accordance with this invention.

.I i .rna WO 87/02060 PCT/US86/02106 Figure 5a and 5b depict schematic representation of the E.coli expression vectors (p210* and pCI857, respectively) used to produce hGM-CSF in bacterial cells through a two-plasmid system and their construction through intermediate plasmids.

The synthetic region used to replace the 5' coding sequences of hGM-CSF is indicated by a hatched region.

Figure 6 depicts the nucleotide sequence and deduced amino acid sequence of the synthetic linker used for E.coli expression (NcoI-HgaI).

Figure 7 depicts the construction of the E.coli single plasmid expression vector p241-48.

Figure 8 depicts the yeast alpha mating factor fusion to the hGM-CSF coding region.

Figures 9-10 are a schematic representation of the construction of the yeast expression vector p528/1 for hGM-CSF production.

DETAILED DESCRIPTION OF THE INVENTION In order that the invention herein described may be more fully understood, the following detailed description is set forth.

In the description the following terms are employed: Nucleotide--A monomeric unit of DNA or RNA consisting of a sugar moiety (pentose), a phosphate, and a nitrogenous heterocyclic base. The base is linked to the sugar moiety via the glycosidic carbon carbon of the pentose) and that combination of base and sugar is called a nucleoside. The base characterizes the nucleotide. The four DNA bases are adenine guanine cytosine and thymine The four RNA bases are A, G, C, and uracil DNA Sequence--A linear array of nucleotides connected one to the other by phosphodiester bonds between the 3' and 5' carbons of adjacent pentoses.

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WO 87/02060 PCT/US86/02106 -6- Codon--A DNA sequence of three nucleotides (a triplet) which encodes through mRNA an amino acid, a translation start signal or a translation termination signal. For example, the nucleotide triplets TTA, TTG, CTT, CTC, CTA and CTG encode for the amino acid leucine TAG, TAA and TGA are translation stop signals and ATG is a translation start signal.

Reading Frame--The grouping of codons during the translation of mRNA into amino acid sequences.

During translation the proper reading frame must be maintained. For example, the DNA sequence GCTGGTTGTAAG may be expressed in three reading frames or phases, each of which affords a different amino acid sequence: GCT GGT TGT AAG--Ala-Gly-Cys-Lys G CTG GTT GTA AG--Leu-Val-Val GC TGG TTG TAA G--Trp-Leu-(STOP) Polypeptide--A linear array of amino acids connected one to the other by peptide bonds between the a-amino and carboxy groups of adjacent amino acids.

Genome--The entire DNA of a cell or a virus.

It includes inter alia the structural gene coding for the polypeptides of the substance, as well as operator, promoter and ribosome binding and interaction sequences, including sequences such as the Shine- Dalgarno sequences.

Gene--A DNA sequence which encodes through its template or messenger RNA ("mRNA") a sequence of amino acids characteristic of a specific polypeptide.

Transcription--The process of producing mRNA from a gene or DNA sequence.

Translation--The process of producing a polypeptide from mRNA.

i~ WO 87/02060 PCT/US86/02106 -7- Expression--The process undergone by a gene or DNA sequence to produce a polypeptide. It is a combination of transcription and translation.

Plasmid--A nonchromosomal double-stranded DNA sequence comprising an intact "replicon" such that the plasmid is replicated in a host cell. When the plasmid is placed within a unicellular organism, the characteristics of that organism may be changed or transformed as a result of the DNA of the plasmid.

For example, a plasmid carrying the gene for tetracycline resistance (TET

R

transforms a cell previously sensitive to tetracycline into one which is resistant to it. A cell transformed by a plasmid is called a "transformant".

Phage or Bacteriophage--Bacterial virus, many of which consist of DNA sequences, encapsidated in a protein envelope or coat ("capsid").

Cloning Vehicle--A plasmid, phage DNA, cosmid or other DNA sequence which is able to replicate in a host cell, characterized by one or a small number of endonuclease recognition sites at which such DNA sequences may be cut in a determinable fashion without attendant loss of an essential biological function of the DNA, replication, production of coat proteins or loss of promoter or binding sites, and which contains a marker suitable for use in the identification of transformed cells, tetracycline resistance or ampicillin resistance. A cloning vehicle is often called a vector.

Cloning--The process of obtaining a population of organisms or DNA sequences derived from one such organism or sequence by asexual reproduction.

Recombinant DNA Molecule or Hybrid DNA--A molecule consisting of segments of DNA from different genomes which have been joined end-to-end outside of living cells and able to be maintained in living cells.

i- WO 87/02060 PCT/US86/02106 -8- Expression Control Sequence--A sequence of nucleotides that controls and regulates expression of genes when operatively linked to those genes.

They include the lac system, the p-lactamase system, the trp system, the tac and trc systems, the major operator and promoter regions of phage X, the control region of fd coat protein, the early and late promoters of SV40, promoters derived from polyoma virus and adenovirus, metallothionine promoters, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase, Pho5, the promoters of the yeast a-mating factors, and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells and their viruses or combinations thereof.

GM-CSF-Like Polypeptide--A polypeptide displaying a biological activity of a GM-CSF. This polypeptide may include amino acids in addition to those of a mature GM-CSF or it may not include all of the amino acids of mature GM-CSF. Finally, it may include an N-terminal methionine.

The present invention relates to hGM-CSFlike polypeptides having a specific activity of at 8 least 1 x 10 units/mg, and to processes for the production of those polypeptides. In addition, this invention relates to the production of large amounts of hGM-CSF-like polypeptides in microbial cells. The polypeptides of this invention are clinically useful as described previously; they also permit the production of both polyclonal and monoclonal antisera to human GM-CSF.

I ,1 WO 87/02060 PCT/US86/02106 -9- THE EXPRESSION SYSTEMS OF THIS INVENTION A wide variety of host/expression vehicle combinations may be employed in producing the GM-CSF-like polypeptides this invention in high yields. Furthermore, a wide variety of host/expression vehicle combinations may be employed to produce the high specific activity hGM-CSF-like polypeptides of this invention. For those hosts that glycosylate the produced hGM-CSF, a deglycosylation step is, of course, required to produce the high specific activity hGM-CSF of the present invention.

The selection of an appropriate host is controlled by a number of factors recognized by the art. These include, for example, compatibility with the chosen vector, toxicity of proteins encoded by the hybrid plasmid, ease of recovery of the desired protein, expression characteristics, bio-safety and cost. A balance of these factors must be struck with the understanding that not all host vector combinations may be equally effective for the expression of the particular recombinant DNA molecules of this invention.

Useful expression vectors include, for example, vectors consisting of segments of chromosomal, non-chromosomal and synthetic DNA sequences, such as various known derivatives of SV40, known bacterial plasmids, plasmids from E.coli including col El, pCR., pBR322, pMB9 and their derivatives, wider host range plasmids, RP4, phage DNAs, the numerous derivatives of phage X, NM 989, and other DNA phages, M13 and Filamenteous single stranded DNA phages, yeast plasmids such as the 2p plasmid or derivatives thereof, and vectors derived from combinations of plasmids and phage DNAs, such as plasmids which have been modified to employ phage DNA or other expression control sequences.

WO 87/02060 PCT/US86/02106 In addition, any of a wide variety of expression control sequences sequences that control the expression of a DNA sequence when operatively linked to it may be used in these vectors to express the DNA sequence of this invention. Such useful expression control sequences, include, for example, the early and late promoters of SV40, the lac system, the trp system, the TAC or TRC system, the major operator and promoter regions of phage X, the control regions of fd coat protein, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase, e.g., the promoters of the yeast a-mating factors, and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof.

The preferred expression vectors and control sequences include the PL promoter, the promoter of the yeast a-mating factor, and the yeast actin promoter.

A wide variety of host cells are also useful in producing the hGM-CSF-like polypeptides of this invention. These hosts may include well known eukaryotic and prokaryotic hosts, such as strains of E.coli, Pseudomonas, Bacillus, Streptomyces, fungi such as yeasts, and animal cells, such as CHO cells, African green monkey cells, such as COS1, COS7, BSC1, and BMT10, and human cells and plant cells in tissue culture.

Useful hosts may include strains of E.coli, such as E.coli W3110I E.coli JA221, E.coli C600, E.coli ED8767, E.coli DH1, E.coli LE392, E.coli HB101, E.coli X1776, E.coli X2282, E.coli MRCI, and strains of Pseudomonas, Bacillus, and Streptomyces, yeasts and other fungi, plant cells in culture or other hosts.

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"Purification and Properties of a Colony Stimulating Factor from Mouse Lung-Conditioned Medium," J. Biol.

Chem., 252, pp. 1998-2003 (1977)]. Accordingly, the GM- /2 ~LI 1_ -L rr i 1 WO 87/02060 PCT/US86/02106 The preferred hosts of this invention include E.coli K 12 E.coli strains SG927 [ATCC 39627], SG928 [ATCC 39628], SG935 [ATCC 39623] and SG936 [ATCC 39624] as well as its derivative, A89 [DSM 3869], and yeast strain Saccharomyces cerevisiae BJ1991.

THE hGM-CSF-LIKE POLYPEPTIDES OF THIS INVENTION It should be understood that the hGM-CSF-like polypeptides (prepared in accordance with this invention in those hosts) may include polypeptides in the form of fused proteins linked to a prokaryotic, eukaryotic or combination N-terminal segment to direct excretion, improve stability, improve purification or improve possible cleavage of the N-terminal segment), in the form of a precursor of GM-CSF-like polypeptides starting with all or parts of a GM-CSF-like polypeptide signal sequence or other eukaryotic or prokaryotic signal sequences), in the form of a mature GM-CSF-like polypeptide, or in the form of a met-GM-CSF-like polypeptide.

One particularly useful form of a polypeptide in accordance with this invention, or at least a precursor thereof, is a mature GM-CSF-like polypeptide with an easily cleaved amino acid or series of amino acids attached to the amino terminus. Such construction allows synthesis of the protein in an appropriate host, where a translation start signal that may not be present in the desired GM-CSF is needed, and then cleavage in vivo or in vitro of the extra amino acids to produce the desired GM-CSF-like polypeptides. Such methods exist in the art.

The polypeptides of the invention also include hGM-CSF-like polypeptides that are coded for on expression by DNA sequences characterized by different codons for some or all of the codons of the present DNA sequences. These substituted codons may code for amino acids identical to those coded for by SUBSTITITE

SHEET

i ProThrCysLeuGlnThrArgLeuGluLeuTyrLysGlnGlyLeuArgGly SerLeuThrLysLeuLysGlyProLeuThrMetIleMetAlaSerHisTyr /3 i 1a i

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12 the codons replaced but result in higher yield of the polypeptide. Alternatively, the replacement of one or a combination of codons leading to amino acid replacement or to a longer or shorter GM-CSF-like polypeptide may alter its properties in a useful way increase the stability, increase the solubility or increase the therapeutic activity). It should be understood that these polypeptides are also part of this invention.

Finally, the most preferred hGM-CSFs of this invention are those characterized by a specific activity of at least 1 x 108 Units/mg. These polypeptides may be produced directly in hosts that do not glycosylate their polypeptides, bacterial hosts such as E.coli. They may also be produced by deglycosylating polypeptides 15 produced in hosts that glycosylate their polypeptides, yeasts and animal cells. Such deglycosylation may be accomplished either in vitro or in vivo using conventional methods and compositions well known in the art. It may also be accomplished by inhibiting glycosylation during protein production using conventional agents and methods.

The GM-CSF-like polypeptides of the present invention may be purified by a variety of convention steps *and strategies. These methods are know in the art.

COMPOSITIONS AND METHODS OF USING 25 THE hGM-CSF-LIKE POLYPEPTIDES OF THIS INVENTION While the GM-CSF-like polypeptides of this invention may be administered in compositions and methods of treatment in the form in which they are produced, it should also be understood that they may be formulated using known methods to prepare pharmaceutically useful compositions. Such compositions also will preferably include conventional pharma-

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13 ceutically acceptable carriers and may include other medicinal agents, carriers, adjuvants, excipients, etc., human serum albumin or plasma preparations. See, Remington's Pharmaceutical Sciences W. Martin). The resulting formulations will contain an amount of hGM-CSF-like polypeptides effective in the recipient to stimulate the colony formation of granulocytes and macrophages. Administration of these polypeptides, or pharmaceutically acceptable derivatives thereof, may be via any of the conventional accepted modes of administration of GM-CSF. These include parenteral, subcutaneous, or intravenous administration.

0 •The GM-CSF-like polypeptides of this invention are particularly useful in compositions and methods for increasing the white blood cell count of immunologically- 15 compromised patients so as to reduce the risk of infection in those patients. Immunologically comprised patients are those whose immune systems are not functioning at a normal level.

For example, the compositions and methods of this invention are useful in the therapy of AIDS patients to prevent the occurrence of opportunistic infections, those infections which would normally not occur in persons who are not Co..

immunogloically compromised, which often shorten the life of e the AIDS patient and add to the expense of their treatment.

The dosage and dose rate will depend on a variety

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of factors for example, whether the treatment is given to a cancer patient after radiation therapy or to an AIDS victim to o oprevent opportunistic infection. However, the dosage will likely be between 1 and 10 gg per day or between 10 and 100 Ag per week, if the patient is to be treated steadily over a long period of time.

In order that this invention may be better understood, the following examples are set forth. These examples are for purposes of illustration only and are not to be construed as limiting the scope of the invention.

I II_ transformed with them which produce human granulocyte-macrophage colony stimulating factor-like polypeptides in high yields, and methods of making these polypeptides. The human granulocyte-macrophage colony stimulating factor-like polypeptides of this invention may be used to treat cancer patients to regenerate leukocytes after radiation or chemotherapy treatment, and to increase white blood cell count to reduce the likelihood of viral, bacterial, fungal, and parasitic infection, especially in immunologically compromised patients such as these suffering from AIDS.

-i ZU WO 87/02060 PCT/US86/02106 -14- EXAMPLE 1 CONSTRUCTION AND SCREENING OF A HUMAN cDNA LIBRARY We describe below the construction of a human cDNA library from poly mRNA isolated from human macrophage cell line U937 in Agtl0 and the amplification of that library in E.coli C600 hfl cells.

A. EXTRACTION OF RNA FROM HUMAN U937 CELLS We induced human macrophage U937 cells in -5 culture with dexamethasone (10- M) and phorbol ester -7 7 M) and resuspended the pellets containing 1.2 x 109 cells in 48 ml lysis buffer (0.2 M Tris-HCl (pH 0.1M LiCl, 25 mM EDTA, 1% SDS) plus 5 mM vanadyl complex (Bethesda Research Labs) by vortexing. We lysed the cells by addition of 24 ml phenol and vortexed for 5 min. We added 24 ml chloroform to the lysis mixture which was then shaken for 10 min. We separated the organic and aqueous phases by centrifugation in a clinical centrifuge at room temperature for 10 min. We reextracted the aqueous phase two times with phenol:chloroform then two times with chloroform only. We next ethanol-precipitated the nucleic acids in 0.3 M sodium acetate at -20 0

C

overnight and pelleted the nucleic.acid at 14k rpm in a Sorvall RC2B centrifuge (SS34 rotor) at 4 0 C for min. We resuspended the pellets in 5 ml of 0.3 M sodium acetate buffer, and ethanol-precipitated the nucleic acid again as described above. We resuspended the final pellet in 300 pl H 2 0 and stored it at -20 0

C.

Our RNA preparation was then enriched for poly(A) RNA by passage over an oligo(dT)-cellulose column (PL Biochem).

1 1 WO 87/02060 PCT/US86/02106 B. CONSTRUCTION OF A U937 cDNA-XgtlO LIBRARY 1. cDNA Synthesis We synthesized cDNA from 20 pg poly (A) mRNA isolated as described above. We diluted the poly mRNA to 500 pg/ml in H 2 0, heated it to 0 C for 3 min, quick cooled it in a dry ice-propanol bath and then thawed it. The RNA was then added to a reaction mixture composed of 0.1 M Tris-HCl (pH 8.3) at 42 0 C, 0.01 M MgCl 2 0.01 M DTT, 1 mM dCTP, 1 mM dGTP, 1 mM dTTP, 0.5 mM dATP and 100 pCi e-32P-ATP (3000 Ci/mmole, Amersham or New England Nuclear), pg oligo (dT) 12 18 (PL Biochem), 0.03 M p-mercaptoethanol, 5 mM Vanadyl Ribonucleoside Complex (Bethesda Research Labs), 169 U AMV Reverse Transcriptase (Seikagaku America).' Final volume of the reaction mixture was 200 pi. We incubated this mixture for 2 min at room temperature and 6 h at 44 0 C. We terminated the reaction by addition of 1/10 vol 0.5 M Na 2 EDTA (pH We adjusted the reaction mixture to 0.15 M NaOH and incubated the mixture at 37 0 C for 12 h followed by neutralization with 1/10 vol 1 M Tris-HCl (pH 8.0) and HC1. This was extracted with phenol: chloroform saturated TE buffer (10 mM Tris-HCl (pH and 1 mM Na 2 EDTA). The aqueous phase was chromatographed through a 5 ml sterile plastic pipet containing a 7 x 29 cm bed of Sephadex G150 in 0.01 M (pH 0.4 M NaCl, 0.01 M Na 2 EDTA, 0.05% SDS. We pooled the front peak minus tail and precipitated the cDNA with 2.5 vol 95% ethanol at -20 0 C. This reaction yielded 1 pg of single-stranded cDNA.

2. Double Strand Synthesis We resuspended the single-stranded cDNA in 200 pl (final vol) 0.1 M Hepes (pH 0.01 M MgCl 2 0.0025 M DTT, 0.07 M KC1, 1 mM dXTPs and 75 U Klenow Iwil WO 87/02060 PCT/US86/02106 -16fragment DNA polymerase 1 (Boehringer-Mannheim) and incubated the reaction mixture at 14°C for 21 h.

Reaction was terminated by addition of Na 2 EDTA (pH to 0.0125 M, the mixture extracted with phenol:chloroform, as in the first cDNA step, and the aqueous phase chromatographed on a G150 column in 0.01 M Tris-HC1 (pH 0.1 M NaCI, 0.01 M Na2EDTA, 0.05% SDS. We again pooled the radioactive peak minus the tail and ethanol-precipitated the

DNA.

We then incubated the DNA obtained with 42 U reverse transcriptase in 50 pl 0.1 M Tris-HCl (pH 0.01 M MgCl2, 0.01 M DTT, 0.1 M KC1, 1 mM dXTPS, 0.03 M p-mercaptoethanol for 1 h at 37 0 C to complete double-strand synthesis. The reaction was terminated and processed as described above.

We cleaved the hairpin loop formed during double strand synthesis as follows: We redissolved the pellet in 50 pl 0.03 M sodium acetate (pH 0.3 M NaC1, 0.003 M ZnC12 and treated it with 100 U S 1 nuclease (Sigma) for 30 min at room temperature.

Reaction was terminated by addition of EDTA and processed as described above. The yield after S 1 treatment was 900 ng dsDNA.

To assure blunt ends following S 1 nuclease digestion, we treated the DNA with Klenow in 0.01 M Tris-HCl (pH 0.01 M MgC1 2 1 mM DTT, 0.05 M NaC1, 80 pM dXTP and 12.5 U Klenow in 60 pl for 90 min at 14 0 C, extracted with 50:50 phenol:chloroform, and chromatographed the DNA on a G50 spin column (1 ml syringe) in 0.01 M Tris-HCl (pH 0.1 M NaC1, 0.01 M EDTA, 0.05% SDS.

We next methylated the dsDNA in order to avoid fragmentation under subsequent EcoRI digestion.

We treated the DNA with EcoRI methylase in 30 p1 final vol 0.1 M Tris-HCl (pH 0.01 M Na 2

EDTA,

24 pg BSA, 0.005 M DTT, 30 pM S-adenosylmethionine and 5 U EcoRI Methylase for 20 min at 37 0 C. The r F-7 WO 87/02060 PCT/US86/02106 -17reaction mixture was heated to 70 0 C for 10 min, cooled, extracted with 50:50 phenol: chloroform and chromatographed on a G50 spin column as described above.

We ligated our methylated ds cDNA to phosphorylated EcoRI linkers (New England Biolabs) using the following conditions: 0.05 M Tris-HCl (pH 7.8), 0.01 M MgC1 2 0.02 M DTT, 1 mM ATP, 50 pg/ml BSA, pg linker, 300 U T4 DNA ligase in 7.5 pl final volume for 32 h at 14 0 C. We adjusted the reaction mixture to 0.1 M Tris-HC1 (pH 0.05 M NaCl, mM MgC12, 100 pg/ml BSA and added 125 U EcoRI (New England Biolabs), incubated the mixture for 2 h at 37 0 C, extracted with 50:50 phenol: chloroform and chromatographed the DNA on a G50 spin column as described earlier.

We redissolved the cDNA in 100 pl 0.01 M Tris-HCl (pH 0.1 M NaCl, 1 mM EDTA and chromatographed it on a 1 x 50 cm Biogel A50 (BIORAD) column which had been extensively washed in the same buffer (to remove ligation inhibitors). Aliquots of various fractions were run on a 1% agarose gel in TBE buffer (0.089 M Tris-HCl, 0.089 M boric acid and 2.5 mM Na2EDTA), dried and exposed at -70°C overnight. We pooled all fractions that were larger than 500 base pairs and ethanol-precipitated the DNA in those fractions for cloning into an EcoRI-cut XgtlO cloning vector. The size fractionation column yielded 126 ng of cDNA, average size approximately 1500 bp.

3. Library Construction We incubated 5 pg EcoRI-cut XgtlO with 20 ng cDNA and T4 DNA ligase buffer at 42 0 C for 15 min to anneal cos sites, followed by centrifugation for sec in an Eppendorf centrifuge and addition of ATP to 1 mM and 2400 U T4 DNA ligase (New England Biolabs) in a final vol of 50 pl. [See Huynh, Young and Davis, "Constructing And Screening cDNA Libraries in Xgtl0 i C i I-T ~EN~ F- J PCT/US86/02106 WO 87/02060 -18- And Ngtll", in DNA Cloning: A Practical Approach Glover, IRL Press (Oxford 1984).] The ligation mixture was incubated at 14°C overnight.

We packaged the Xgtl0 cDNA ligation mixture into phage particles using an Amersham packaging mix [Amersham packaging protocol] and diluted with 0.5 ml SM buffer (100 mM NaCl, 10 mM MgSO 50 mM Tris-HCl (pH 7.5) and 0.01% gelatin).

We next infected E.coli C600 hfl cells with these phage particles to form a cDNA library of 1 x 7 independent recombinants [see T. Maniatis et al., Molecular Cloning, p. 235 (Cold Spring Harbor, 1982)].

For plating and amplification of the library, 1 ml of cells plus 250 pl. packaging mix was incubated at room temperature for 15 min, diluted to 50 ml in LB plus MgSO 4 top agarose at 50°C and plated on LB Mg Nunc plates. This represented a plaque density of 2 x 105 plate. The plates were incubated at 37°C for approximately 8 h until plaques were nearly touching.

We flooded the plates with 50 ml of cold SM buffer (0.01 M Tris-HCl (pH 0.01 M MgCl 2 0.1 mM Na 2EDTA) and eluted on a gyro-rotary shaker overnight at 4°C. We pooled the eluants into 250 ml bottles and spun at 6k for 10 min in a Sorvall GSA rotor. We treated the supernatants with an equal volume of cold 20% PEG 4000-2 M NaCl in ice for 3 h and pelleted the phages by centrifugation at 4k for min in an H4000 rotor in an RC-3B Sorvall centrifuge. The phage pellets were thoroughly drained, resuspended in 60 ml SM, and spun at 10,000 rpm in a SS34 rotor to remove debris. The supernatants were adjusted to 3.5 M CsCl by addition of 7 g CsCl to ml supernatant. We obtained phage bands by centrifugation in a 70.1 Beckman rotor at 50,000 rpm for 18 h at 15°C. We pooled the phage bands and stored them at 4°C for library stock. The titer obtained was 2.2 x 1013 PFU/ml.

I

19 4. Screening Of The Human GM-CSF cDNA Library With Murine GM-CSF cDNA We screened the above-described human cDNA library with a fragment of murine GM-CSF cDNA sequence. The basis for our approach was that murine GM-CSF has some amino acid similarity to human GM-CSF M. Gough, et al., Nature and EMBO Journal, supra, p. 2; R. H. Weisbart, et al., Nature, supra, p. Accordingly, we postulated that mouse GM-CSF cDNA might cross hybridize to human GM-CSF cDNA to an extent sufficient to allow selection of a human GM-CSF related cDNA from our human cDNA library.

e* **o aa a a a a., e Preparation of Murine GM-CSF probe We obtained our murine GM-CSF cDNA probe for screening our human cDNA library, as follows: We generated a cDNA library from mRNA isolated from EL-4 mouse thymoma cells [ATCC TIB 39] induced with phorbol-12-myristate-13-acetate (PMA). We then reverse transcribed mRNA into cDNA using the Okayama- Berg protocol Okayama and P. Berg, "High-Efficiency Cloning of Full-Length cDNA," Mol. Cell Biol., 2, pp. 161-70 (1982)]. We inserted the cDNA into the plasmid vector pHG327, a modified pKCR vector O'Hare et al., "Transformation of Mouse Fibroblasts to Methotrexate Resistance by a Recombinant Plasmid Expressing a Prokaryotic Dihydrofolate Reductase," Proc. Natl. Acad. Sci. USA, 78, pp.

25 15, 27-31 (1981)], which has a unique SstI site for cDNA cloning and two flanking BamHI sites to allow convenient excision of the inserted cDNA sequence The resultant library (a gift of W. Boll and C. Weissmann) consisted of approximately 2 X 105 individual cDNA molecules. We screened this mouse cDNA library by hybridization with two oligomer DNA probes synthesized on the basis of the published cDNA sequence for mouse lung cell derived GM-CSF M. Gough et al., "Molecular Cloning of a cDNA encoding a Murine Haematopoietic Growth Regulator Granulocyte-Macrophage Colony L j 1_ WO 87/02060 PCT/US86/02106 Stimulating Factor", Nature, 309, pp. 763-67 (1984)] using the solid-phase phosphotriester method Ito et al., "Solid Phase Synthesis of Polynucleotides: VI. Further Studies on Polystyrene Copolymers .For the Solid Support," Nucl. Acids Res., 10, pp. 1755-69 (1982).

These probes had the following sequences: GMCSF-2 probe CCAACTCCGGAAACGGACTG GMCSF-1 probe CTTAAAACCTTTCTGACTG 0.02% of the 2 x 105 cDNA molecules scored positive.

We determined the sequence of the longest positive insert using conventional methods. For example, either the chain termination method Sanger et al., Proc. Natl. Acad. Sci. USA, 74, pp. 5463-67 (1977); J. Messing and J. Vieira, "A New Pair of M13 Vectors For Selecting Either DNA Strand of Double-Digest Restriction Fragments," Gene, 19, pp. 269-76 (1982)] or by chemical degradation of the DNA chain M. Maxam and W. Gilbert, Methods in Enzymology, 65, pp. 499-560 (1980)] can be used. We found that the DNA sequence was similar to that of GM-CSF from mouse lung cells. Over the coding region there were only three base changes (at positions 186, 213, and 482). However, only the base change at position 482 generates an amino acid substitution, yielding valine instead of glycine. These changes represent one of the two variants reported for different isolates of mGM-CSF M. Gough et al., "Structure and Expression of the mRNA for Murine Granulocyte-Macrophage Colony Stimulating Factor", EMBO 4, pp. 645-53 (1985)].

6. Screening of the Human cDNA library We screened our human cDNA library for human GM-CSF sequences, using a labeled probe which consisted of a 365 base pair NciI-HinfI fragment of our murine GM-CSF (Figure The NciI-Hinf fragment was purified on a 5% polyacrylamide gel and radio-

I

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-~~Jv 86/02106 WO 87/02060 PCT/US86/02106 -21- -69 labled by nick-translation. For screening, we used the plaque hybridization technique of Benton and Davis D. Benton and R. W. Davis, "Screening lambda gt recombinant clones by hybridization to a single 5 plaque in situ", Science, 196, p. 180 f1977)1.

To prepare our library for screening with this DNA probe, we prepared an overnight culture of C600 hfl cells in L broth and 0.2% maltose and pelleted and resuspended the cells in an equal volume of SM buffer. We then pre-adsorbed 0.9 ml of cells with 2 x 105 phage particles at room temperature for min. We diluted the suspension to 50 ml in LB plus 10 mM MgSO 4 and 0.7% agarose at 55'C and plated it on LB Mg Nunc plates. We screened 10 such plates.

We incubated the plates at 37 0 C for approximately 8 h until plaques were nearly touching. We then chilled the plates at 4 0 C for 1 h to allow the agarose to harden. We transferred the XgtlO phage particles from the plaque library plates to nitrocellulose.

We placed the filters onto the plates containing the recombinant plaques for varying times from 30 seconds to 5 minutes depending on the number of different filters used, and 'Chen lifted and incubated the filters with the phage-containing side up on LB 10 m MgSO 4 plates at 37 0 C for 5 h.

These filters were then lysed y placing them onto a pool of 0.5 N NaOH for 5 min, then neutralized on 1 M Tris-HCl (pH submerged into 1 M Tris-HCl (pH 7.0) and then heated at 80 0 C for 2 hours.

We hybridized the filters to the radiolabled 365 base pair NciI-HinfI fragment cDNA probe in [6 x SSC] 0.2% polyvinyl-pyrrolidone 40,000), 0.2% ficoll 40,000), 0.2% bovine serum albumin, 35 0.05 M Tris-HCl (pH 1 M sodium chloride, 0.1% sodium pyrophosphate, 0.1% SDS, 10% dextran sulfate 500,000) and denatured salmon sperm DNA 100 pg/ml) at 65 0 C. They were then washed in a *4 9*9* iE 9.

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'CT/US86/02106 smid I I I -3 I, A Ii -0=W 22 similar buffer (2 x SSC, 0.1% SDS) at 65"C. We detected hybridizing cDNA sequences by autoradiography. By means of this technique, we screened 1 x 106 phage and we picked positive plaques.

These positive cDNAs were further purified and characterized. The largest cloned cDNA was 768 nucleotides in length. We sequenced this cDNA insert using conventional methods. Its sequence is depicted in Figure 1. The longest open reading frame found in this cDNA (nucleotides 7-438) encodes a protein of 144 amino acids which is identical over the coding region to that reported for Mo-cell derived hGM- CSF with the exception of a single base (297). The first 17 residues comprise a series of hydrophobic amino acids consistent with their role as a putative signal sequence for 15 the remaining protein. The cleavage site between serine (the terminal amino acid of the putative signal sequence) and alanine (the first amino acid of the coding sequence) as depicted in Figure 1, is also in agreement with the reported first amino acid of the mature protein (alanine). The A to G substitution at position 297 results in a codon for an isoleucine instead of the reported methionine.

We also constructed a second cDNA library in gtlO from poly(A) RNA of the human bladder carcinoma cell line 5637 (ATCC HTB9) substantially as described previously.

6 25 We screened this library of 10 recombinant phage for hGM-CSF sequences according to the plaque hybridization technique described above using a 240 nucleotide fragment (PstI- ApaI) from the coding region of the hGM-CSF cDNA of Figure 1 as a probe. Positive signals were found at a frequency of one in 500 plaques The largest of these cDNAs was 911 nucleotides in length. We sequenced this DNA insert using conventional methods. Its sequence is depicted in Figure 2. The coding sequence of the 5637-derived cDNA is identical to that reported 1

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23 for the Mo-derived cDNA. The 5637-derived cDNA contains additional non-coding sequences at both the 5' and 3' ends not found in either the U937 or Mo-cell derived cDNA's.

EXAMPLE 2 EXPRESSION OF hGM-CSF A. In E.coli 0 0 1 .4 4 Referring now to Figures 4, 5a and 5b, we have shown therein a schematic outline of one embodiment of a process for preparing a recombinant DNA molecule (pPLmuGM- CSF) characterized in that it produces hGM-CSF in high yield.

It has a U937-derived DNA sequence coding for human GM-CSF, which had been modified at the 5' end of its coding region to minimize any potential disadvantageous RNA secondary structure, fused to a DNA sequence from mu and carrying a Shine Dalgarno sequence from mu, the combined DNA sequence being operatively-linked to a PL promoter derived from bacteriophage To construct expression vector pPLmuGM-CSF, we first prepared a synthetic oligonucleotide DNA sequence or 20 linker to replace the coding sequence between the first alanine codon and a unique Hqa I site at nucleotide 120 of our hGM-CSF cDNA (Figure This NcoI-HqaI linker (Figure 6) was constructed to reduce the potential of mRNA secondary structure which might make the ribosomal binding 25 site inaccessible. In this linker we substituted adenine (A) for the naturally occurring nucleotides wherever the degeneracy of the genetic code allowed the retention of the same amino acid sequence. N. Buell et al., "Optimizing the Expression in E.Coli of a Synthetic Gene Encoding Somatomedin-C Nucl. Acids Res., 13, pp. 1923- 3'8 (1985)]. These base substitutions resulted in a change in the potential free

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6Y Li- 24 energy due to mRNA secondary structure of 15 kcal (dG -28.3 to AG -13.3 kcal) increasing the free energy beyond that predicted to form stable stem and loop structures. We also used our linker to add an ATG start codon directly in front of the N-terminal alanine of our coding sequence.

Referring now to Figure 5a, we depict therein the construction of vector p210* through various intermediates. To carry out the above described DNA sequence modification we subcloned the U937-derived hGM-CSF cDNA of Figure 1 into pUC-8 Viera and J. R. Messing, Gene, 19, pp. 259-268 (1982)] (See Figure The resulting vector, which we designated pUC8 GM-CSF was cut with Hcqal and HindIII. We ligated the resulting small fragment together 15 with our synthetic NcoI-HgaI linker. We then cut our pPLmu vector Remaut et al., Gene, 15, pp. 81-93 (1981); G. Gray et al., Gene, 32, pp. 21-30 (1984)] with NcIo and HindIII and inserted our fragment therein. We then removed the 3' noncoding sequences between the Bal I site and Sma I site. We designated this vector pPLmu:hGM-CSF (p210*). We similarly prepared a second vector which contained the 5637-derived hGM-CSF of Figure 2. This vector was designated (p210*- 5637).

0* *4 S*0 00 00 0 We tested the ability of plasmid p210* to direct the synthesis of hGM-CSF in an in vitro transcriptiontranslation system. For comparison, we assayed the same Sexpression vector carrying the native cDNA sequence deleted to the alanine codon (the initial codon of mature hGM-CSF) by Bal 31 digestion (p208 Bal3l). pPLMU:hGM-CSF (p210*) yielded significantly more hGM-CSF than p208 Bal3l, proving that our synthetic linker and its inhibition of mRNA secondary structure markedly improved the production of hGM-CSF-like polypeptide.

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S 0 *4 0 *0*t 0 4 0 0e 0 0*e S We introduced the expression plasmid p210* into E.coli strain C600 which also carried a second plasmid encoding the thermosensitive respressor (CI 8 5 7 of the PL promoter Remaut et al. Gene, 22, pp. 103-13 (1983)] (Figure 5b). We could have also used strains in which the repressor is part of the chromosome (see description, infra). Transcription was regulated by growing the cells at 28°C, a permissive temperature for repressor activity, or at 42°C, non-permissive for repressor activity. Using this E.coli C600 host strain, we were unable to produce sufficient quantities of hGM-CSF to be visible on SDS-PAGE analysis.

15 Subsequently we tested E.coli strain SG936 [lac trp pho sup C rpsl, mal (am), htpR tsx:TN10, lon R9] [ATCC 39624] which is an htpR lon mutant. This mutant is deficient in its production of lon protease. As a result, this strain, as well as lon mutant strains SG935 [ATCC 39623], SG927 [ATCC 39627], and SG928 [ATCC 39628], exhibits a reduced capacity to degrade foreign proteins upon their accumulation within the cell or at high temperatures. A. Goff et al., "Heat Shock Regulatory Gene htpR Influences Rates of Protein Degradation and Expression of the lon gene in Escherichia coli," Proc.

Natl. Acad. Sci. USA, 81, pp. 6647-6651 (1984).] We transformed E.coli strain SG936 with plasmid 210*, using standard induction protocol. We examined the ability of this strain to produce hGM-CSF under controlled fermentation conditions: we grew the transformed E.coli SG936 at 28°C on L-Broth with 50 mg/ml Ampicillin and 200 mg/ml Kanamycin to an optical density (650) of approximately 30. After r rFI heat shock at 42 0 C, and the addition of extra medium, we grew the cells for three hours and harvested them.

We analyzed total cell aliquots of the fermentation mixture for hGM-CSF production by .SDSpolyacrylamide gel electrophoresis at various intervals: inoculation, 4 hours after inoculation, 1 hour after induction at high temperature (42 0

C)

and 3 hours after induction. A strong protein band appeared 1 hour after induction. The induced protein (with an apparent molecular weight of 14,500) accorded well with the expected 14,574 molecular weight of our expected recombinant hGM-CSF protein. We performed a densitometer scan of a portion of the stained gel which represented the hGM-CSF production of an aliquot taken after harvesting. This scan indicated that 8-9% of the total protein was hGM-CSF.

We also examined other host strains for their ability to produce and accumulate hGM-CSF. We compared wild type E.coli strains C and B with mutant E.coli strain SG936, because the mutant strain grows poorly. We found only B to be positive for hGM-CSF.

This accumulation of hGM-CSF in E.coli B accords with the known low levels of lon protease in that E.coli strain. Thus, our high yields are improved in lon protease mutants which do not break down the recombinant protein once it is produced.

2. Single Plasmid Vector In a more preferred embodiment, we constructed a single plasmid vector for the production of human GM-CSF. This single plasmid was more advantageous then the above-described two-plasmid system for synthesis of hGM-CSF in E.coli for several reasons. First, the two separate plasmids required two different antibiotic selections for their maintenance in the host cell. One plasmid (210*) employed ampicillinase as its resistance marker. Unfortunately, ~1 27 the ampicillin required in its growth medium is an undesirable element in fermentation for a human pharmaceutical product. In addition, coordinate growth of the two plasmids was not certain. Thus, the quantity of repressor encoded by one plasmid (pcI857) could not be certain to match the number of promoter copies present on the second plasmid (210*).

For these and other reasons we constructed a single-plasmid system which would contain all the elements previously found on the two plasmids. Those elements included the leftward transcriptional promoter of phase Lambda (PL) followed by: the ribosome binding site for the ner-1 gene of phage Mu, the altered sequence encoding the mature human GM-CSF protein (preceded by a methionine codon to initiate translation), the transcriptional terminator from 15 phage T4 gene 32, the tetracycline resistance gene from plasmid pBR322, the origin of replication and deletion mutant ofrom plasmid pAT153, phage Lambda cI gene mutant 857 encoding the thermolabile repressor of PL. We combined these elements from previously described plasmids as shown in Figure 7.

In order to construct this single plasmid vector we conducted a three-fragment ligation: the first fragment, *o which contained the c1857 thermolabite repressor, was isolated from plasmid 153-PL-T4-hTNFCA3 cts [DSM 3460] after subjecting it to digestion by EcoRI and SalI; the second S 25 fragment was isolated from plasmid T4 CA5 Dra 6 HinIII; after subjecting it to digestion by SalI and HindIII and isolating the smaller fragment; the final fragment which contained the hGM-CSF cDNA, was isolated from p 210* after EcoRI and HindIII digestion. Single plasmid vector p 241-8 resulted.

Another significant element in our construction of the single plasmid system was the introduction of a transcriptional terminator. Our data indicate "z 'I L;; r that this fragment is useful for stabilizing the single plasmid vector.

The bacterial host strain (SG936) in the high expression system described above was also altered to allow use of the new single plasmid vector. We removed the tetracycline resistance already present in SG936. Selection of the bacteria on fusaric acid for sensitivity to tetracycline yielded a new strain: A89. When the single plasmid vector 241-8 was introduced into strain A89 and production of hGM-CSF was initiated by heat induction, hGM- CSF comprised 8-10% of the total cell protein.

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3. Purification of E.coli Produced hGM-CSF To purify the hGM-CSF we had produced, we lysed the recombinant-protein-bearing E.coli cells using the French press. We centrifuged and then washed the pellet, which included the dense inclusion bodies containing recombinanthGM-CSF, with a solubilizing buffer of 0.75 guanidinium hydrochloride, 1% tween 40, 50mM EDTA, 0.1M tris HC1 (pH to remove soluble contaminants. We extracted the recombinant- hGM-CSF from the pellet using a solution buffer of 5mM KH 2 P0 4 and 6M urea.

We further purified our hGM-CSF from E.coli by gel filtration and traced the eluent of the G-100 Sephadex column by optical density. To do this, we applied the washed pellet to the column. The sample was chromatographed and we monitored the eluent at O.D. 280. Three peaks were observed.

We then analysed our sample containing the recombinant hGM- CSF produced in E.coli by SDS-PAGE, stained by coomassieblue to visualize the results, and found that the last peak yielded a single band of the expected molecular weight.

We also analyzed this last peak using reverse phase HPLC analysis, column: Brownlee RP 4,'

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29 2.1 x 200 mm C8. This further confirmed that the third G- 100 Sephadex column peak showed essentially a single protein peak. The other two peaks produced by the gel filtration chromatography represented contaminants and a dimer molecule, respectively.

4. Biological Activity Assays We assayed samples of lysed E.coli cells which carried the hGM-CSF expression plasmid described above, and were induced for recombinant protein production for biological activity using the bone marrow clonal assay and the CML assay.

SWe first assayed biological activity on human bone marrow cells. Normal human bone marrow was fractionated in Ficoll by density and depleted of macrophages by 15 absorption to plastic Pike and W. A. Robinson, J. Cell.

Physiol, 76, pp. 77-84 (1970)]. We then grew the bone marrow cells (50,000 cells) in serum-free methylcellulose medium or agarose medium in the presence of 10% calf serum F. Eliason and N. Odartchenko, "Colony Formation By 20 Primitive Hemopoietic Progenitor Cells in Serum-Free Medium", Proc. Natl. Acad. Sci. USA, 82, pp. 775-79 (1985)]. We then compared the effect of a reference GM-CSF, a partially purified hGM-CSF supplied by Chugai, and our recombinant hGM- CSF, by growing the bone marrow cells in the presence of the e 25 respective CSFs at different dilutions. For each, duplicate one milliliter aliquots of the cell suspension were plated in mm diameter Petri dishes and incubated with the respective CSFs at 37"C in a fully humidified atmosphere of 5% CO 2 02 and 90% N 2 After 7 days and after 14 days, we counted the colonies. We typed them as granulocyte, macrophage or mixed, based on the size of the cells and the colony morphology. In some cases we also stained with Geimsa to confirm classification of colonies. This assay demonstrated a (4 iA- 0Z) Iand U EcoRI Methylase for 20 min at i WO 87/02060 PCT/US86/02106 concentration dependent stimulation of colony formulation on human bone marrow cells with both the native and recombinant material. Accordingly, the recombinant hGM-CSF is active without glycosylation.

We also performed a CML assay to quantify hGM-CSF activity. This assay measures 3H-thymidine uptake on granulocytes which have been purified from the peripheral blood of patients with chronic myelogenous leukemia (CML). The cell preparation contained 7% blood cells, which proliferated in response to hGM-CSF D. Griffin et al., Blood, 63, pp. 904-11 (1984)]. The cells (10 /ml) were incubated for 48 hours with varying concentrations of GM-CSF and proliferation was measured by a 6 hour incorporation of 3H-thymidine (10p Ci/Ml, 200 Ci/nmole). One unit per ml induces 50% of maximal 3H-thymidine incorporation of CML cells. Such uptake in the presence of GM-CSF is directly proportional to the incubation time and the number of cells.

We assayed the activity of E.coli produced hGM-CSF from: an induced culture of SG936 cells carrying p 210* and pci857; an uninduced culture of SG936 cells carrying p 210* and pcI857; and an induced culture of SG936 cells carrying pPLmu and pcl857. CML cells incubated with samples (2) and incorporated only background levels of 3H-thymidine. CML cells incubated with sample (1) showed a dilution-dependent stimulation of 3 H-thymidine incorporation, confirming the presence of active GM-CSF.

Preferred Method of Purification of E.coli Produced hGM-CSF To reconstitute our host cells, we suspended E.coli cells (100g) in 5 volumes of buffer I (100mM sodium phosphate; 5mM benzamidine-HCl, EDTA; 0.5 mM phenylmethylsulfonyl fluoride (solu- "Constructing And Screening cDNA Libraries in Xgtl0 WO 87/02060 PCT/US86/02106 -31bilized in ethanol, 10% of 25% sucrose; 0.17% (w/w of cells) lysozyme; pH=7.0). After the cells were sonicated for 2 minutes in an ice water bath, we incubated them at 22 0 C for one hour. The mixture was then cooled to 4°C and passed through a french pressure cell twice at 16,000 psi. We then centrigued the crude homogenate at 10,400 xg (GSA rotor, min) and discarded the supernatant. The pellet was sonicated for 90 seconds in buffer II (buffer I, except with 0.75 M guanidium-HCl; 1% Tween and without sucrose and lysozyme). We then centrifuged as above for 30 minutes. The resulting pellet was dissolved in 85 ml of buffer III (l00mM sodium phosphate; 3M gu-HCl; 10mM 2-mercaptoethanol; ImM EDTA; pH=7.0). We centrifuged this solution min) as described above and subjected the supernatant to ultracentrifugation for one hour at 40,000 rpm (Beckman Ti-45 rotor). The supernatant from the ultracentrifugation was applied to a column of Sephacryl S-200 (5.0 x 87 cm) precquilibrated with buffer III. The flow rate of the gel filtration chromotagraphy was maintained at 70 ml/h and we collected 17 ml fractions. We monitored the fractionation of proteins by A 280 nm and SDS gel electrophoresis (12.5% acrylamide, proteins visualized by Coomasie blue staining).

The fractions from the S-200 column that contained hGM-CSF of at least 80% purity were pooled and diluted to a protein concentration of 0.25 mg/ml (708 ml) with buffer IV (15mM sodium phosphate; 3M urea; pH=7.5). We dialyzed this material against the dilution buffer until the concentration of 2-mercaptoethanol was less than 25% of the protein concentration. We also included phenylmethylsulfonyl fluoride (0.5 mM) during the first dialysis to avoid proteolysis.

~~3"a3lr~-~ICPaa~ WO 87/02060 PCT/US86/02106 -32- We monitored the oxidation of protein through an assay for free sulfhydryl groups using DTNB, while continuing dialysis against the same buffer. The half-life of the reduced material under these conditions was found to be about 4 h; therefore, the oxidation was approximately 95% complete after 18 hours. We then dialyzed the material twice against five volumes of a buffer containing 30 mM sodium phospate (pH The resulting solution was centrifuged (30 min, 10,400 x g) and the supernatant concentrated to 230 ml.

We centrifuged the concentrate again, as above, and applied the supernatant to a column of Fast Flow Q (2.6 x 15 cm) which had been equilibrated with 30 mM phosphate buffer (pH The column was washed with the same buffer until the absorbance (280 nm) of the effluent was 0. The column was then developed with a gradient of sodium phosphate (30 to 130 mM, 600 x 600 ml). We analyzed 15 ml fractions containing hGM-CSF for the purest product using SDS gel electrophoresis. Those fractions were pooled and concentrated to 10 ml.

The concentrated material was applied to a column of Ultrogel ACA-54 (LKB, 2.6 x 90 cm) equilibrated with 30 mM sodium phosphate and 130 mM NaCl (pH We pooled and stored 5ml fractions containing pure hGM-CSF at -80° C. Using this purification method we produced in 30 to 60 mg of pure protein, indicating a 12.5% to 25% yield.

B. In Yeast We have also constructed expression vectors for the production in high yield of human GM-CSF in yeast. Figures 9 and 10 show a schematic outline of one embodiment of a process for constructing expression vector p528/1 which, when used to transform WO 87/02060 PCT/US86/02106 appropriate yeast cells, expressed hGM-CSF in high yields.

We made use of the leader or signal peptide of the yeast alpha mating factor to express.

hGM-CSF in yeast. Two genes, denoted MFal and MFa2 have been reported to encode the alpha mating factor of yeast Kurjan and I. Herskowitz, "Structure of a yeast pheromone gene (MFa): a putative factor precursor contains four tandem copies of mature a-factor", Cell, 30, pp. 933-43 (1982); A. Singh et al., "Saccharomyces cerevisiae contains two discrete genes coding for the a-factor pheromone", Nucl. Ac. Res., 11, pp. 4049-63 (1983)]. MFal codes for a precursor of 165 amino acids, containing four copies of alpha factor. The alpha factor repeats are preceded by a secretion leader sequence of 83 amino acids. The junctions between the secretion leader and the first repeat, and between each of the repeats have the following structure: (leader or repeat)-lys-arg-(glu/asp-ala)2_ 3 -(repeat) The optimal cleavage of the secretion leader from the heterologous protein portion of the fusion precursor may be obtained when the first amino acid of the heterologous protein is placed behind the -lys-arg processing site (see Figure Accordingly, we used this alpha mating factor signal sequence hGM-CSF fusion in our vectors.

1. Expression vectors In constructing our expression vector, we used two different genes encoding hGM-CSF. Gene 1, which was present on plasmid p210*, is characterized by a U937-derived DNA sequence coding for hGM-CSF, which was modified at the 5'-end of its coding region by a synthetic oligonucleotide DNA sequence or linker 1 ~I C' 'iiir~liii~i~iiii':: WO 87/02060 PCT/US86/02106 -34- (Figure Gene 2, which was present on plasmid p208 corresponds to unmodified hGM-CSF coding sequence.

In order to place an Nco I site in a position preceding the first hGM-CSF codon in our recombinant molecule, we digested p208 with Pst I, and then treated it with Bal 31. We then restricted the digested sequence with Bam HI and isolated the smaller heterologous fragment. (Pst I is located upstream, and Bam HI downstream, of the hGM-CSF coding sequence.) We next cut p210* with Nco I and filled in with dNTPs. (The Nco I site marks the start of our synthetic linker.) We then restricted our sequence with Bam HI and isolated the large fragment.

We created a group of recombinant plasmids by inserting the heterologous small fragments taken from p208 (containing a DNA sequence coding for hGM-CSF) into the larger fragment taken from p210* (containing the remainder of the expression vector) (see Figure 9), In one selected plasmid, this ligation reconstructed an Nco I site at the start of the hGM-CSF coding sequence.

We cut our two GM-CSF plasmids (p208 Bal 31 and p210*) with Nco I and treated with Sl nuclease (see Figure 10). We next cut the plasmids with Hind III. The smaller fragments, which contained the native hGM-CSF coding sequence (in the case of 208 Bal 31) or the synthetic linker as well as the remainder of the hGM-CSF DNA sequence (in the case of p210*) were isolated.

We next constructed recc.mbinant plasmid p216 using our fragments containing the respective hGM-CSF coding sequences and the larger StuI-HindIII fragment of pMATA 21/51. That fragment contains the promoter and secretion leader of the MATal gene.

To construct that fragment, we first isolated a DNA sequence encoding MFal from a yeast genomic library r -J -I Z- t_ .L.L W CLZ11 U. .L11

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*e *0 0 a a* ova: .0% a *9 s* 4 4 .9.

4904 9 by using an oligonucleotide probe corresponding to amino acids 97 to 102 of MFal precursor GTA CAT TGG TTG G/GCC G/A/TGG which we synthesized according to the published sequence of MFal. A. Nasmyth and S. I. Reed, "Isolation of genes by complementation in yeast: molecular cloning of cell-cycle gene", Proc. Natl. Acad. Sci. USA, 77, 2119-23 (1980).] We subcloned the 1.7 kb EcoRI fragment that we selected with this probe into pUC18. We mutagenized the resulting plasmid (p22 0 3 using primer mutagenesis A. Oostra et al., "Transforming activity of polyoma virum middle-T antigen probed by site-directed mutagenesis", Nature, 304, pp. 456-59 (1983)] to introduce a StuI site at the position corresponding to the -lys-arg cleavage. We also 15 inserted a 500 bp HindIII fragment carrying a synthetic SMC gene starting with a unique NcoI site Buell et al., "Optimizing the expression in E.coli of a synthetic gene encoding somatomedin-C Nucl. Ac. Res., 13, pp.

1923-38 (1985)] into the HindIII site of p220/3. The result of this construction was a plasmid (p216) that had the secretion leader of MFal ligated to the first codon (Ala) of the hGM-CSF coding sequence. We then transferred the MFal/hGM- CSF gene fusion on an EcoRI-HindIII fragment to expression vector p160/1, which carries origins of replication for E.coli and yeast (ori; 2 origins of replication), as well as selectable markers for both organisms (E.coli: p-lactamase; yeast: URA3).

MFal--hGM-CSF AGA GCA CCC MFal/208 Bal 31 lys arg ala pro AGAtGCA CCA MFal/210* KEX2 cleavage j -j WO 87/02060 PCT/US86/02106 -36- Recombinant plasmid p216 was cut with Eco RI and Hind III and we isolated the small fragment, which contained the MFal/hGM-CSF fusion. This fragment was transferred to expression vector which carries an origin of replication for E.coli, the yeast URA 3 gene, the origin of replication of the 2p circle, and the origin of replication of ARS 1 (autonomously replicating sequence, which allows replication in the yeast cell independently of the yeast chromosone), and the upstream region of PYK 1 (PUR) T. Stinchomb et al., "Isolation and Characterization Of A Yeast Chromosomal Replicator," Nature, 282, pp. 39-43 (1979)].

We designated the plasmid that resulted from using the DNA sequence of p208 Bal31 the authentic cDNA sequence) as plasmid 528/1. We designated the plasmid that resulted from using the DNA sequence of p 2 10* the 5' altered sequence) as plasmid 525/2. We also constructed a third expression plasmid, designated p545/1, in which an actin promoter replaced the MFal promoter of pMATA 21/51 and a high-copy number vector (JDB207) was used as a base vector. D. Beggs, "Multiple-copy yeast vectors," Molecular Genetics in Yeast, Alfred Benzon Symposium 16, ed. D. Van Wettstein, J. Fries, M.

Kielland-Brandt Stenderup, Munksgaard, Copenhagen (1981).] The low expression of the LEU 2 gene on this vector requires elevated copy numbers in transformants to allow growth on selective media Erhart and E. P. Hollenberg, J. Bacteriol, '156, pp. 625-35 (1983)].

2. Expression results We transformed the hGM-CSF expression plasmids into Saccharomyces cerevisiae strain BJ1991 (MATa ura3-52 leu2-3,112 trpl prbl-1122 pep4-3) (obtained from E. Jones, Carnegie-Mellon University). We grew WO 87/02060 PCT/US86/02106 -37the transformants in "SD" medium Sherman et al., "Methods in yeast genetics", Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1981)], containing tryptophan and leucine (plasmids p525/2. and p528/1), or tryptophan and uracil (p545/1), to an optical density (600 mm) of 2. These cultures were used to inoculate "production medium", consisting of SD-medium containing 4% casamino acids and tryptophan (inoculum was 10% of final volume of production medium). At appropriate intervals during growth, ml of the cultures were pelleted using a microfuge and then cell pellets were redissolved in 1/10 of the original culture volume by boiling 5 minutes in SDS sample buffer K. Laemmli, "Cleavage of the Structural Proteins During the Assembly of the Head of Bacteriophage T4", Nature, 227, pp. 680-85 (1970)]. Produced proteins were then blotted to nitrocellulose and probed with antibodies against hGM-CSF (Western blotting technique) or with concanavalin A Clegg, "Glycoprotein Detection in Nitrocellulose Transfers of Electrophoretically Separated Protein Mixtures Using Concanavalin A and Peroxidase: Application to Arenavirus and Flavovirus Proteins", Analytical Biochemistry, 127, pp. 389-94 (1982)].

Absolute expression levels realized were as follows (measured at OD 600 nm 10) in mg/litre: plasmid medium cells p525/2 10 p545/1 20 8 p528/1 <0.1 <0.1 The fact that only the two constructions using the modified hGM-CSF gene, but not the construction using the natural cDNA sequence expressed high levels of hGM-CSF indicates that the structure of the mRNA encoding the secretion precursor significantly influences gene expression. Presumably, an unfavorable /11 o 0^ PCT/US86/02106 WO 87/02060 -38structure of the mRNA in p528/1 severely interferes with its translation. This effect is different from the observed effects of mRNA secondary structure in the vicinity of the ATG start codon in bacterial expression [Buell et al., supra] because the altered DNA stretch in the mRNA encoded on plasmids p525/2 and p545/1 is separated from the ATG start codon by 240 base-pairs.

As demonstrated from the above results, 70-80% of the total produced hGM-CSF was secreted into the culture medium. The secreted hGM-CSF occurred in 3 main forms: an unglycosylated form (14.5 kd).

This form comprised approximately 10% of the total secreted hGM-CSF. The size of this molecule corresponded exactly to non-glycosylated native hGM-CSF, indicating that the secretion leader had been correctly processed to generate an hGM-CSF beginning with A.la Pro.

a low-molecular-weight glycosylated form (18 kd). This form comprised up to 5% of the total secreted hGM-CSF. Native hGM-CSF is glycosylated; the hGM-CSF protein sequence contains two potential sites of N-linked glycosylation [Wong et al., Science, 228, pp. 810-15 (1985)]. The size of the 18 kd form was consistent with either the presence of two coreglycosyl side chains attached to each of both potential glycosylation sites, or the presence of an extended core glycosyl chain attached to only one glycosylation site.

a high-molecular-weight glycosylated form (about 43 kd). This form comprised 80-90% of the total secreted hGM-CSF.

Treatment of the above-described glycosylated hGM-CSF forms with endoglycosidase H reduced their molecular weight to approximately 14.5 kd,

I

I

i WO 87/02060 PCT/US86/02106 1 -39indicating that all glycosyl side chains were N-linked to the protein backbone.

Replacement of the MFal promoter by the actin promoter and use of a high-copy-vector (expression plasmid p545/1) improved hGM-CSF expression approximately two-fold. However, while the amounts of the glycosylated forms in the culture medium increased, no increase of the unglycosylated form in the medium was observed. Inr;tead, 2-3 fold more unglycosylated hGM-CSF was cell-associated indicating that solubility may limit the amount of hGM-CSF in the medium.

The supernatants from the cultures of yeast cells producing hGM-CSF were tested for biological activity. In the bone marrow clonal assay, described supra at pp., 23-24, yeast-secreted hGM-CSF stimulated colony-formation in a dose-dependent manner. Similarly, the CML assay, described supra at pp. 24-25, showed a dose dependent response to yeast produced hGM-CSF.

C. Comparison of Glycosylated And Deglycosylated hGM-CSF Natural hGM-CSF is known to be a glycoprotein with a molecular weight of about 22 kd [G.G.

Wong et al., Science, 228, supra]. In the polypeptide chain there are two asparagine residues at positions 27 and 37 which are potential sites for N-linked glycosylation (Asn-X-Thr/Ser). Thus, it was heretofore believed that recombinantly produced hGM-CSF had to be produced in animal cells and glycosylated, in order to be active biologically. If the hGM-CSF was to be produced in bacterial cells, a second glycosylation step was thought to be necessary to confer activity.

Contrary to this supposition, we have discovered that unglycosylated hGM-CSF has an unexpectedly higher specific activity than glycosylated L -I WO 87/02060 PCT/US86/02106 hGM-CSF. Thus such unglycosylated hGM-CSFs are an important part of this invention. As demonstrated in this application, these hGM-CSFs have a specific activity of at least 1 x 10 Units/mg.

Such deglycosylated hGM-CSFs may be produced in several ways. For example, they may be produced in bacterial cells that do not glycosylate the proteins they produce. For example, when the polypeptide chain is produced by E.coli, it contains no attached carbohydrate and possesses a molecular weight of 14.5 kd.

These unglycosylated polypeptides may also be produced by deglycosylating yeast or animal cell produced proteins. For example, we isolated a high molecular weight fraction (MW 50-70 kd) of hGM-CSF, produced in yeast cells, as described above, using ConA-chromatography and gel filtration. We have also produced hGM-CSF in animal cells, by growing transfected Chinese Hamster Ovary (CHO) cells for three days in 10% fetal calf serum containing medium.

We used on CHO-cell clone which was derived from transfection with a vector that had the hGM-CSF gene isolated from U937 cells. The transcription was promoted by a SV40 early and Adenovirus major late j 25 promoter. Gene amplification was carried out with i methotrexate selection. We isolated a high molei cular weight fraction (MW 26-30 kd) by immuno- and lectin chromatography, followed by gel filtration.

We then deglycosylated the isolated fract 30 tion with a mixture of endo-and exoglycosidases.

Deglycosylation of the high molecular weight fractions was established by immunoblotting. The digested fractions showed a reduction of the molecular weight close to that observed with the E.coli produced hGM-CSF 14.5 kd).

We determinated hGM-CSF concentration with a competition Radio Immuno-Assay (RIA), using as i:J,

I

WO 87/02060 PCT/US86/02106 -41tracer 125I labelled E.coli-derived hGM-CSF and antihGM-CSF fixed on Staph. aureus cells. The CML and BMC Marrow assays which we then conducted, are described above.

The results of our deglycosylation assays are summarized in the Table 1: Table 1 Sample A. Mammalian hGM-CSF before digestion after digestion B. yeast hGM-CSF before digestion after digestion C. E.coli hGM-CSF 230 ng/ml 600 ng/ml 0.05 mg/ml 0.20 mg/ml 0.4x10 7 U/mg 3.2x10 7 U/mg 1.2x10 7 U/mg 9.5x10 7 U/mg '2 x 10 8 U/mg Bone Marrow 0.2x108 U/mg 2.3x108 U/mg 0.25x108 U/mg 3.8xl08 U/mg 1.8x109 U/mg Our results demonstrate that the E.coli derived hGM-CSF produced entirely without carbohydrate or deglycosylated yeast or animal cell derived hGM-CSF, are superior to the glycosylated native hGM-CSF for use in clinical applications.

Microorganisms and recombinant DNA molecules prepared by processes of this invention are exemplified by cultures deposited in the Deutsche Sammung von Mikroorganism, Grisebachstrasse 8, D-3400 Gottingen, West Germany, on September 2, 1985 and identified there as B84, B85, B102, and YE464, and on October 4, 1986 and identified there as B1ll (p241-8).

A. E.coli K 12 (p210*) B. E.coli SG 936 (p210*) and (pci857) C. E.coli K 12 (p210*-5637) SUBSTITUTE SHEET

""A

i i PV WO 87/02060 PCT/US86/02106 -42- D. Yeast strain S.cerevisiae BJ1991 (p5 2 5 2 E. E.coli A89 (p241) These cultures were assigned accession numbers DSM 3473, DSM 3474, DSM 3475, DSM 3465, and DSM 3869, respectively.

While we have hereinbefore presented a number of embodiments of this invention, it is apparent that our basic construction can be altered to provide other embodiments which utilize the processes and compositions of this invention. Therefore, it will be appreciated that the scope of this invention is to be defined by the claims appended hereto rather than by the specific embodiments which have been presented hereinbefore by way of example.

~7-c SUBSTITUhTSHEUT

A

Claims (11)

1. A recombinant DNA molecule comprising a DNA sequence encoding a human granulocyte-macrophage colony stimulating factor (hGM-CSF)-like polypeptide as herein before defined, said DNA sequence being characterized by a modification to the 5' terminus, said 5' terminus comprising the nucleotides encoding the first 21 amino acids of mature human granulocyte-macrophage colony stimulating factor, said DNA sequence being operatively linked to an expression control sequence in the recombinant DNA molecule, said modification allowing for the expression of said polypeptide in higher yield than the native DNA sequence coding for human granulocyte-macrophage colony stimulating factor.

2. The recombinant DNA molecule according to 0:. claim 1, wherein the 5' alteration is selected from those of S the formula: ATGGCACCAGCAAGAAGCCCGAGTCCGTCGACACAACCGTGGGAG S* CATGTGAATGCGATCCAGGAG, and GCACCAGCAAGAAGCCCGAGTCCGTCGACAC AACCGTGGGAGCATGTGAATGCGATCCAGGAG.

3. The recombinant DNA molecule according to claim 1 or 2, wherein said expression control sequence is selected from the group consisting of the lac system, the p- lactamase system, the trp system, the tac system, the trc 6. system, major operator and promoter regions of phage X, the control region of fd coat protein, the promoter for 3- phosphoglycerate kinase or other glycolytic enzymes, the 5 0 promoters of acid phosphates, the promoters of the yeast a-mating factors, and other known sequences which control I the expression of genes of prokaryotic or eukaryotic microbial cells and their viruses and combinations thereof. 43 CTS00 L CT/US86/02106 WO 87/0060 PCT/US86/02106 WO 87/02060 4 r Il I I

4. The recombinant DNA molecule according to claim 1, which is selected from the group consisting of p210*, p210*-5-37, p241-8, p525/2, and p545/1, as herein before defined. A microbial host transformed with at least one recombinant DNA molecule according to any of claims 1 to 4.

6. The transformed microbial host according to claim 5, wherein the host organism is selected from the group of microbial hosts consisting of E.coli SG936, E.coli SG935, E.coli SG928, E.coli SG927, E.coli A89, as herein o* before defined, and other E.coli strains which are lon mutants, as herein before defined, and which are charac- terized by production of low levels of lon protease. SeS

7. The transformed microbial host according to claim 5, being Saccharomvces cerevisiae (BJ1991), as herein before defined.

8. A process for producing high yields of human *granulocyte-macrophage colony stimulating factor (hGM-CSF)- like polypeptides comprising the step of culturing a microbial host transformed with a recombinant DNA molecule according to any one of claims 1-4. S

9. The process according to claim 8, wherein said hGM-CSF-like polypeptide is selected from polypeptides having the formulae: MetAlaProAlaArgSerProSerProSerThrGlnProTrpGluHisVal AsnAlaIleGlnGluAlaArgArgLeuLeuAsnLeuSerArgAspThrAla AlaGluMetAsnThrValGluValIleSerGluMetPheAspLeuGlnGlu ProThrCysLeuGlnThrArgLeuGluLeuTyrLysGlnGlyLeuArgGly SerLeuThrLysLeuLysGlyProLeuThrMetIleMetAlaSerHisTyr 44 1 LysGlnHisCysProProThrProGluThrSerCysAlaThrGlnIleIle ThrPheGluSerPheLysGluAsnLeuLysAspPheLeuLeuValIlePro PheAspCysTrpGluProValGlnGlu, AlaProAlaArgSerProSer ProSerThrGlnProTrpGluHisValAsnAlaIleGlnGluAlaArgArg LeuLeuAsnLeuSerArgAspThrAlaAlaGluMetAsnThrValGluVal IleSerGluMetPheAspLeuGlnGluProThrCysLeuGlnThrArgLeu GluLeuTyrLysGlnGlyLeuArgGlySerLeuThrLysLeuLysGlyPro LeuThrMetIleMetAlaSerHisTyrLysGlnHisCysProProThrPro GluThrSerCysAlaThrGlnIleIleThrPheGluSerPheLysGluAsn LeuLysAspPheLeuLeuValIleProPheAspCysTrpGluProValGln Glu and polypeptides coded for by DNA sequences encoding hGM- CSF characterized by a modification of the 5' terminus of said DNA sequence, said 5' terminus comprising the nucleotides encoding the first 21 amino acids of mature hGM- CSF, and said modification allowing the production of said polypeptide in higher yield than the native DNA sequence coding for hGM-CSF. The process according to claim 8, wherein the transformed microbial host is selected from the group consisting of E.coli SG936, E.coli SG935, E.coli SG928, E.coli SG927, E.coli A89, as herein before defined, and other E.coli strains which are lon mutants, as herein before defined, and which are characterized by their production of low levels of lon protease. ao...

11. The process according to claim 8, wherein the transformed microbial host is Saccharomvces cerevisiae (BJ1991), as herein before defined.

12. A pharmaceutical composition comprising a polypeptide produced according to the process of any of claims 8 through 11, is an amount effective to stimulate 1. granulocyte and macrophage formation and a pharmaceutically acceptable carrier.

13. A method of reducing the likelihood of opportunistic infection comprising the step of treating an immunologically compromised human with a pharmaceutical composition as defined in claims 12. DATED this 5th day of November, 1990 BIOGEN, INC. By their Patent Attorneys CULLEN CO. e O 9*44 4 4 e0 4* 0 46 L r **c t 3 cDNA SEQUENCE AND DEDUCED AMINO ACID SEQUENCE FORt k.GtCSi: AS ISOLATED FROM~ THE U937 CELL LINE niETTRPL.EuLNSEREuLELEuLEuGLYTHRVALAL ACV SSER ILESERALAPRoIALAARGERPROSERPROSERTHRGLNPRoTRPGLUH I SVALASNALA ILE6LNGLU t 130 1SO 170 190 210 230 AL AAR GAR 6LEuLEuAsNL.EuSERAR 6AspTHRALAAL AGLJMETAsmGLuTHEVAL6LUVAL I L ESE aGIUIE TPHEAsPLEuGLNGL UPR oTHRCY s E uGL NTH RAR GEuGLLE U 250 270 290 310 330 350 TVRLYsGLNGLYLEIJARGGLYSERLEuTHRLVsL.EuA..sGLYPRo4-EuTHRIIETILEALASERHI sTYRLYsGLNHisCysPRoPRoTHRPRoGLuTHRSERCVsALATHRGLILEILE MIET THu 370 390 410 430 450O 470 ACCTT6GAAAG6ITCAAA6AGMC,.tCTGAAGaA'CTITCTCTT6CATCCCCTACTCGGGACCAGCCAGGAGTAACCGGCCAGA IGAGGCT6GCCAAGCCGGGGA6CT6CTC THRPHEGLUSERPHELYsLuAsLEu&YsAsPP-ELJEUVAL ILEPROPHEASPCVsTRPGLUPROVALGLGLuENID TCTCATGAAACAAGAGCTAGAAACTCAGGATGGTCATCTGGAGGGACCAAGGGGTGGGCCACAGCCAIGGTGGGAGTGGCCTGGACCT6CCC 1GGGCCACAC TGACCCTGATACAGGCA 650 690 FIGI 1~ cDf$A SEQUENCE AND DEDUCED AMlINO ACID SEQUENCE FOR HGMi-CSF AS ISOLATED FROM THE 5637 CELL LINE 30 so .70 90 110 CGA6CTCGAGCGCGGCCGCAAGTCICIGGA66ATGT6GCTGCAGAGCCTGCTGCCTTGGCACTGTGGCC IGCAGCATCTCTGCACCC6CCCGCTCGCCCAGCCCCAGCACGCAGCCC hIErTRPLEuGLNSERLEu1ELEu~fLEuGLYTHRVALALACYSSER ILESERALAPRoALAARGSERPROSERPROSERTHRGLNPRO 130 ISO 170 190 210 230 TRPGLuHitSVALAsNALAI LEGU46LuALAARGARGLEuLEuAsNItEUSERARGAsPTHRALAALAGLU ETASI4GLUTHRVALGLUVAL ILESERGLUME TPHEAsPLEuGLNGLUPRO THRCYsLEU6LwTHRARLuLuLEuTyRLys6LNLYEuAjrGLYSERLEuTRLYsLELYsLYPRot.EuTHRIE-FtiETALASERHI sTYRLYsGLNHI sCysPRoPRoTHRPRO GLuTHRSERCYsALATHRGLN!LEILETHRPHE6LUSERPHELYs6LuAsNL.EuLysAsPPHELEuLEUVAL ILEPR0PHEASPCYSTRPGLUPROVALGLN6LuEND TICATATICCATATTTATTCAAGATGTTTIACCGtAATAATTATTATIAAAAATATGCTICTMMWAAAAAAAAMM-AAGATCAACAGGCTTATTAGAAGAATGAACTAAGGTGTC F16G2 TACCAfIxTTfI ICTAAGCTGGTTG6ITMITAAACAGIACCIGCTCICAAATIGGAAAAAAAAAAAAA L- I I t t I 0 00 0 0 0 HOUSE GHI-CSF cD&M FIG 3 Linear Basin I End 878 PSTI HIMF II I I GGG 35 78 I HIMNF ECU 1W I I mcli I NARI HUll'! I 878 148 333 457 513 643 AAAAAAA CODING REGION I KICK-TRANSLATED PROBE 4k~ LI WO 87/02060 PCT/US86/02106 4/11 FIG 4 Hind DL Nco I Nco I Hga I I I pPLMUSMC Hind MI Hgo h GM -C Hind III 9tJTIT-Tg SHEET I WO 87/02060 PCT/US86/02 106 5/11 A EccR1 ECCR1 HirdIII psti Sall BrnI SEal LcR ECCI Bail Sall pstl Hindzzz EcoI(586) PvuI (27! PitI (2629) AV ai aecama se a competition Radio Immuno-Assay (RIA), using as WO 87/02060 PCT/US86/02 106 6/11 48502/0 F I G 58 177 Hindill 1.*2 kb BgllI-PstI fragnet bringing the C1857 gn BaUto Pstl 3.9 kb fmgffpent r brining Kmr, ori, and part of A C1857 pstI SUBSTITUTE SHEET C -I 'It U) Iv' SYNTHETIC LINKER SE QUE NCE USED FOR E M EXPRESSION fiETALAPRoALAARGSERPROSERPROSERTHR6LNPRoTRPGLUISVALASNALAILE6LN6LU 1 CCATGGCACCAGCAA6AAGCCCGAGTCCGTC6ACACAACCGTGGGAGCAT6T6AAT6CGATCCA66AG 68 C CC CTC6 C C CAGC 6 6 C C AMINO ACID SEQUENCE MATURE PROTEIN COHISTRUCT SYNTHETIC LINKER SEQUENCE NATIVE SEQUENCE DIFFERENCES FIG 6 SUBSTITUJTE SHEET ow WO 87/02060 PCT/US86/02 106 1 8/11 ECR C1.857 FIG 7 r-1857 fragment ECCRI-Sa1I %I 2-csF sail small. fragment SalI-Hirdl= S.LU pstZ Hindl hG4-CSF fr&wmt ZccRl-RJ ndT= Three fragzunt ligati±n EcdZ c1857 Sial SUBTITTESHEET f alpha mating factor fusion aF3 S4 @V4 ci) -4 c: -A rn al) 3: LysArg GluAki AspAloGiu Ala Trp HisTrp Leu Gin Leu Lys Pro Gly Gin Pro Met Tyr SPACEREPTICEMATURE j1-FACTOR I SPAER PETIDEPHEROMONE HYDROPHO8IC POTENTIAL SITES SIGNAL FOR ADDITION4 OF SEQUENCE N -LINKED GLYCOSYL CHAINS -Iys-arg-ala-pro-thr-arg- -Iys-arg-ala-pro-ala-arg- mouse human F/G8 M Fo(l Mo(1 GM-C SF WO 87/02060 10/11 io208 hGM -CSF PCT/US86/02106 Nco I hGM-CSF Barn HI Born HI cut with Pst 1 Bal3l cut with Born H I isolate small fragment cut with Nco I fill -in with dNTPs cut with Born H I isolate large fragment hGM -CSF BSrn HI 208 Bol 31 GCA CCC CC 210 GCA CCA GCA CGC TCG CCG AGC CCC AGC I I III I I III AGA AGC CCC ACT CCC TCC org ser pro ser pro ser ACC ACA CAG CAA alIa pro ala thr gin 208 Bol 31 CCC 210 CCG TGG GAG IGG GAG CAT GTG AAT GC C ATC CAT GTG AAT GCGj ATC a. a. pro trp glu his vol osn ala Hle eti ,aQTIrMYC8_ CLugwOr /1, WO 87/02060 PCT/US86/02 106 11/11 Stut FIG /0 Nco I hGM-CSF ind III SMC HindM cut with NcotI SI cut with Hind III isolate small fragment E co R cut with Stl Hind III. isolate large fragment hGM -CSF Hind II isolate EcoRI Hind II fragment p 160/1 ,,-large EcoRI Hind III fragment MFoc I hGM -CSF H L ARS I PUR 2j 2ori -SUBSTITUTE SHFr INTERNATIONAL SEARCH REPORT International Application No PCT/US86/02106 1 I. CLASSIFICATION OF SUBJECT MATTER (if several classification symbols apply, Indicate all) 3 Accor rnati en cto to bo National Classification and IPr AccrI t a O PI2c .19/34,21/02;C07K90/QQA61K 37/02 U.S. 435/68;514/12;530/351 II. FIELDS SEARCHED Minimum Documentation Searched 4 Classification System Classification Symbols U.S. 435/68,70,91,253,172.3,317;514/2,12;536/27 935/27,38; 530/351, 825 Documentation Searched other than Minimum Documentation to the Extent that such Documents are Included In the Fields Searched 6 CHEMICAL ABSTRACTS DATA BASE (CAS) 1967-1986 BIOLOGICAL ABSTRACTS DATA BASE (BIOSIS) 1969-1986 KEYWORDS: granulocyte-macrophage conlny s imnti1a ing factor gene III. DOCUMENTS CONSIDERED TO BE RELEVANT t* Category Citation of Document, 16 with indication, where appropriate, of the relevant passages 17 Relevant to Claim No. ts Y,E US,A 4,621,050 (SUGIMOTO), 04 November 12-17 1986, see column 1. Y,P THE EMBRO JOURNAL, (Oxford, England), 1-14 Volume 4, issued, October 1985, (MIYATAKE1 ET AL), "Structure of the chromosomal gene for granulocyte-macrophage colony stimulating factor: comparison of the mouse and human genes", see page 2561. Y THE EMBRO JOURNAL, (Oxford, Engalnd), 1-14 Volume 4, issued March, 1985, (GOUGH ET AL), "Structure and expression of mRNA for murine granulocyte-macro- phage colony stiumlating factor", see page 645. Y PROCEEDINGS NATIONAL ACADEMY OF SCIENCES 1-17 (Washington, D.C. USA), Volume 82, issued September 1985, (CANTRELL ET AL), "Cloning, sequence, and expression of a human granulocyte/macrophage colony stimu- lating factor", see page 6250. SSpecial categories of cited documents: 1s later document published after the international filing date or priority date and not in conflict with the application but document defining the general state of the art which is not cited to understand the principle or theory underlying the considered to be of particular relevance invention earlier document but published on or after the international document of particular relevance; the claimed invention filing date cannot be considered novel or cannot be considered to document which may throw doubts on priority claim(s) or involve an Inventive step which is cited to establish the publication date of another document of particular relevance; the claimed Invention citation or other special reason (as specified) cannot be considered to involve an inventive step when the document referring to an oral disclosure, use, exhibition or document Is combined with one or more other such docu- other means ments, such combination being obvious to a person skilled document published prior to the international filing date but in the art. later than the priority date claimed document member of the same patent family IV. CERTIFICATION Date of the Actual Completion of the International Search 5 Date of Mailing of this International Search Report 12 December 1986 0 6 JAN 1987 International Searching Authority I Signaturcq Ahorized lr p j ISA/US St anie Seidman Ph.D. J.D. Form PCT/ISA/210 (second sheet) (October 1981) International Application No. PCT/US/02106 7 III. DOCUMENTS CONSIDERED TO BE RELEVANT (CONTINUED FROM THE SECOND SHEET) Category Citation of Document, 16 with indication, where appropriate, of the relevant passages 17 Relevant to Claim No Is Y Y SPROCEEDINGS NATIONAL ACADEMY OF SCIENCES (Washington D.C. USA), issued June, 1984, (NICOLA ET,AL), "Binding of the differentiation-inducer, granulocyte- colony-stiumlating factor, to responsive but not unresponsive leukemic cell lines", page 3865. BLOOD (New York), Volume 54, issued September, 1979 (NICOLA ET AL), "Separa- tion of Functionally Distinct Human Granulocyte-Macrophage Colony Stimulating Factors", see pages 614-615. S 15-17 12-14 ii j ri Form PCT ISAi210 (extra sheet) (October 1981) PCT/US/02106 International Application No. FURTHER INFORMATION CONTINUED FROM THE SECOND SHEET SCIENCE (Washington D.C. USA), Volume 228 issued May, 1985, (WONG ET AL), "Human GM-CSF:Molecular Cloning of the Complemen- tary DNA and Purification of the Natural and Recominant Proteins", See pages 810 and 815. NATURE (London, England), Volume 309, issued 28 June 1984, (GOUGH ET AL), "Molecular cloning of cDNA encoding a murine haematopoietic growth regulator, granulocyte-macrophage colony stimulating factor", see page 763. 1-17 V.Q OBSERVATIONS WHERE CERTAIN CLAIMS WERE FOUND UNSEARCHABLE to This international search report has not been established in respect of certain claims under Article 17(2) for the following reasons: 1.[j Claim numbers because they relate to subject matter 12 not required to be searched by this Authority, namely: 2.1 Claim numbers because they relate to parts of the international application that do not comply with the prescribed require- ments to such an extent that no meaningful international search can be carried out 13, specifically: VI.Q OBSERVATIONS WHERE UNITY OF INVENTION IS LACKING 11 This International Searching Authority found multiple Inventions in this international application as follows: As all required additional search fees were timely paid by the applicant, this International search report covers all searchable claims of the international application. As only some of the required additional search fees were timely paid by the applicant, this international search report covers only those claims of the international application for which fees were paid, specifically claims: r 3.1 No required additional search fees were timely paid by the applicant. Consequently, this international search report is restricted to the invention first mentioned in the claims; it is covered by claim numbers: 4.F As all searchable claims could be searched without effort justifying an additional fee, the International Searching Authority did not invite payment of any additional fee. Remark on Protest F The additional search fees were accompanied by applicant's protest. No protest accompanied the payment of additional search fees. Form PCT/ISA/210 (supplemental sheet (October 1981)