DK175466B1 - Poly:peptide with granulocyte colony stimulating factor activity - obtd. by recombinant DNA procedures for treating haematopoietic disorders - Google Patents

Abstract

Purified and isolated polypeptide (I) having at least part of the primary structural conformation and a biological property (ies) of naturally occurring pluripotent granulocyte colony stimulating factor (hpG-CSF) is new when the (I) is the prod. of procaryotic or eucaryotic expression of an exogenous DNA sequence. DNA sequence for use in securing expression of (I) in a procaryotic or eucaryotic host cell is new. The DNA sequence is either a defined sequence of its complementary strands, or a sequence that hybridises to such sequences or their fragments, or a sequence that, but for the degeneracy of the genetic code, would hydridise to any of these other sequences.

Description

DK 175466 B1 i

This invention relates to a DNA sequence which, upon expression in a prokaryotic or eukaryotic host cell, encodes a polypeptide product possessing at least a portion of the primary structure and one or more of 5 ae biological properties of naturally occurring pluripotent granulocyte colony stimulating factor. . In this connection, the invention also relates to a biologically functional plasmid or viral DNA vector, a prokaryotic or eukaryotic host cell, a method of producing polypeptide analogs of hpG-CSF in which one or more cysteine residues have been deleted or replaced by alanine cells. or serine residues, a polypeptide analog defined in this way, a pharmaceutical composition containing the polypeptide analog, and finally the use of the polypeptide analog for the preparation of a drug.

The blood-forming (hematopoietic) system in humans destroys a variety of white blood cells (including neutrophils, macrophages and basophils / mast cells), red blood cells (erythrocytes) and clotting-promoting cells (megakaryocytes / platelets). It has been estimated that the hematopoietic system of an average male person produces approx. 4.5 x 1011 granulocytes and erythrocytes each year, which corresponds to a renewal of the entire body weight. Dexter et al., BioEssays, 2, 154-2558 (1985).

It is believed that small amounts of certain hematopoietic growth factors cause a small number of "progenitor cells" to divide into a number of blood cell lines, which propagate to such an extent, and eventually diffuse into fully developed blood cells. As the hematopoietic growth factors are present in immeasurably small quantities, determinations and identification of these factors have rested on a number of experiments, which up to now

DK 175466 B1 I

2 I

only distinguishes the various factors based on I

their stimulatory effect on cell cultures during human I

increase circumstances. As a result, you have

produced a large number of names to denote an I

5 much smaller number of factors. As an example of the I

In the current confusion one can mention that the names I

IL-3, BPA, multi-CSF, HCGF, MCGF and PSF are different I

names which are now believed to all denote the same hematopoietic I

happen growth factor in mice. Metcalf, Science, 229, 16-22 I

10 (1985). See also Burgess et al., J. Biol. Chem. 252, I

1988 (1977), Das, et al., Blood, 58, 600 (1980), Ihle, I

et al., J. Immunol, 129, 2431 (1982), Nicola, et al., J.I.

Biol. Chem., 258, 9017 (1983), Metcalf, et al., Int. J. I

Cancer, 30, 773 (1982), and Burgess, et al., Int. J. I

15 Cancer, 26, 647 (1980), concerning various growth regulations

learning glycoproteins in mice. IN

The use of recombinant genetic engineering has brought about an I

show order in this chaos. For example, you now know you

amino acid and the DNA sequences of human erythropoietin, I

20 which stimulate the production of erythrocytes. (See Lin, I

PCT Application No. 85/02510, published June 20, I

1985). Recombinant methods have also been used

there for isolating cDNA for a human granulocyte I

macrophage colony stimulating factor. See Lee, et al.,

Proc. Natl. Acad. Sci. (USA), 82, 4360-4364 (1985) and I

Wong, et al., Science, 228, 810-814 (1985). See also Yo- I

kota, et al., Proc. Natl. Acad. Sci. (USA), 81, 1070 I

(1984), Fung, et al., Nature, 307, 233 (1984), and Gough, I.

et al., Nature, 309, 763 (1984) on cloning of gel

From mice, as well as Kawasaki, et al., Science, 230, I

291 (1985) on human M-CSF. IN

It has been shown that a human hematopoiesis exists

tical growth factor, called human pluripotent colony I

DK 175466 B1 3 stimulating factor (hpCSF) or pluripoietin in the culture medium of a human bladder carcinoma cell line, called 5637 and deposited under restrictive circumstances at the American Type Culture Collection, Rockville, Maryland as A.T.C.C. No. HTB-9. It has been stated that phCSF, purified from this cell line, will stimulate the propagation and differentiation of pluripotent progenitor cells, thereby producing all the important blood cell types in experiments using human bone marrow output cells. Walte et al., Proc. Natl. Acad.

Sci. In the purification of phCSF, precipitation was used: (NH 4) 2 SO 4; anion exchange chromatography (DEAE cellulose, DE 52); gel filtration (AcA54 column); and C18 reverse phase high capacity liquid chromatography. It was stated that a protein identified as phCSF and eluted in the second of two activity peaks in C18 reverse phase HPLC chromatography should have a molecular weight (MW) of 18,000, determined by sodium dodecyl sulfate (SDS) - polyacrylamide gel electrophoresis (PAGE) using silver staining. Previously, hpCSF was stated to have an isoelectric point of 5.5 [welte, et al., J. Cell. Biochem., Supp. 9A, 116 (1985)] and a highly differentiating activity of the myelomonocytic leukemia cell line WEHI-3B D + from mice [Welte, et al., 25 UCLA Symposia on Molecular and Cellular Biology, Gale, et al., Eds., New Series, 28 (1985)]. Previous studies suggest that the factor identified as phCSF possesses predominantly granolyte colony-stimulating activity for the first seven days of a human CFU-GM trial.

Further, from the human bladder carcinoma cell line 5637, another factor, termed human CSF-β, which is described as a competitor to 125i-labeled granulocyte colony stimulating factor (G-CSF) from mice, has been isolated.

DK 175466 B1 I

for binding to WEHI-3B D + cells in a dose-I

response interaction identical to what you do

finds by unlabeled mouse G-CSF [Nicola, et al., I

Nature, 314, 625-628 (1985)]. You have previously co- I

3 shared that this dose-response relationship should

be unique to unlabeled G-CSF from mice, and not be I

present with such factors as M-CSF, GM-CSF or I

multi-CSF [Nicola, et al., Proc. Natl. Acad. Sci. (USA), I

81, 3765-3769 (1984)]. Among CSFs, CSF-β and I occupy

10 G-CSF the special position that they are both very capable

to induce differentiation in WEHI-3B D + cells. IN

Nicola, et al., Immunology Today, 5, 76-80 (1984). Do you know

high concentrations stimulate G-CSF mixed granulo- I

cyt / macrophage colony forming cells (Nicola, et al., i

15 (1984), see above), which is consistent with previous results

tater suggesting granulocytic occurrence

monocytic and mixed granulocytic / monocytic and I

eosinophilic colonies (CFU-GEMMM) after 14 days of incubation

ring of human bone marrow cultures with phCSF. You have

20 also described CSF-β as a stimulant for the formation of I

neutrophil granulocyte colonies in experiments using I

is the bone marrow cells of mice, a property that one has

used to identify a factor as a G-CSF. IN

On the basis of these similarities, human I has been identified

25 CSF-β with G-CSF (granulocyte colony-stimulating factor). IN

Nicola et al., Nature, 314, 625-628 (1985). IN

It seems, given their common I

properties that human CSF-β according to Nicola, et al., see

above, and the hpCSF of Welte, et al., supra, is I

30 is the same factor which may conveniently be termed an I

human pluripotent granulocyte colony-stimulating factor I

(HpG-CSF). It would be very desirable to be able to buy

background characterize and recombinant produce hpG-CSF

DK 175466 B1 5 of the reported ability of G-CSP from mice to completely suppress in vitro population of WEHI-3B D + leukemia cells at "completely normal concentrations", and considering that uncleaned preparations of G -CSF from mice by injection can suppress existing transplanted myeloid leukemias in mice. Metcalf, Science, 229, 16-22 (1985). See also Sachs, Scientific American, 284 (1), 40-47 (1986).

If hpG-CSP were to prove therapeutically important and it became necessary to be able to produce it in commercial quantities, isolation from the cell culture would hardly be able to provide the necessary amount of material. For example, it should be noted that there appear to be restrictions on commercial utilization of Human Tumor Bank cells, such as human bladder carcinoma cell line 5637 (ATCC HTB9), cited as a source of natural hpCSF -præpara-ter; Welte, et al. (1985), see above).

The DNA sequences of the invention are characterized by encoding a polypeptide analog of hpG-CSF in which one or more cysteine residues are deleted or replaced with alanine or serine residues.

Such sequences may include: incorporated "preferred" codons for expression by selected non-mammalian host cells, providing intersection sites for endoconuclease restriction enzymes; and providing additional DNA sequences placed before, within, or after said sequence, which facilitate the construction of vectors that can be expressed without further ado.

These polypeptide analogs for hpG-CSF differ from naturally occurring forms in the identity or location of one or more amino acid residues (i.e., deletion analogs containing less than the number

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amino acid residues specified for hpG-CSF; or I.

clay substitution analogs, such as [Ser17] hpG-CSF, wherein I

one or more of the prescribed cysteine residues is- I

statted with the other residues mentioned; and they have some electricity

5 clay all properties are common to the natural forms. IN

Such DNA sequences according to the invention I

includes sequences useful for securing expression

in prokaryotic or eukaryotic host cells of I

polypeptide products with at least a portion of the pri- I

10 may have structural conformation and one or more of the I

biological properties of naturally occurring pluri- I

potent granulocyte colony stimulating factor and they can

is prepared by the method of Alton, et al., of I

published PCT application WO 83/04053. IN

The process as described above for preparation

The production of certain hpG-CSF polypeptide analogues is property I

according to the characterizing part of claim 11. IN

The purified and isolated polypeptide products of I

The invention is peculiar to being products of I

20 expression in prokaryotic or especially eukaryotic I

hosts (e.g., bacteria, yeast or cultured cells of I

plants, insects, and mammals) of DNA sequences of I

invention. In this context, it is biologically functional

functional plasmid or the viral DNA vector of op

25 find it peculiar in that it is transformed

or transfected with a DNA sequence of the invention I

in such a way that the host cell can express it- I

current polypeptide product; and a prokaryotic host cell

le according to the invention is peculiar to being trans

propagated with such a DNA vector. "" I

The product from typical yeast cells (e.g. Saccaro-I

myces cerevisiae) or prokaryotic host cells I

[eg. Escherichia coli (E. coli)] is free of associa I

DK 175466 B1 7 with any mammalian protein. The product of microbe expression in vertebrate cells (eg mammals other than humans and birds) is free from association with any human protein. Depending on the host used, the polypeptides of the invention may be glycosylated with mammalian or other eukaryotic carbohydrates or may be non-glycosylated. Polypeptides of the invention may also contain a starting methionine amino acid residue (at scaffold gene -1).

Pharmaceutical compositions of the invention are characterized by comprising a plurality of polypeptide analogues of the invention together with appropriate diluents, adjuvants and / or carriers. Finally, according to the invention, the subject polypeptide analogues are used to prepare drugs for hemapoietic therapy in a mammal.

Polypeptide products of the invention can be labeled as associated with a detectable marker substance 20 (e.g., radiolabelled with 125 I), providing reagents useful in determining the presence and amount of human hpG-CSF in tissue samples and in liquid samples. such as blood or urine. DNA products of the invention can also be labeled with detectable markers (such as radio markers and non-isotope markers such as biotin) and used in DNA hybridization processes to locate the position of the human hpG-CSF gene and / or the position of any related gene family in a chromosome map. They can also be used to identify anomalies related to human hpG-CSF gene at the DNA level and are used as gene markers to identify neighboring genes and their anomalies.

I DK 175466 B1 I

I 8 I

In polypeptide products of the invention, only I

I or in combination with other hematopoietic factors or I

In clay medications are useful in the treatment of hematoma

In poetic disorders, such as aplastic anemia. They can also- I

I 5 so be useful in treating hemapoietic deficiencies · I

In caused by chemotherapy or by radiation therapy. Chan- I

In order for a bone marrow transplant to succeed, I can

For example, if you add hpG-CSF, it is enhanced. Treatment I

I with healing of burns and treatment of bacterial form I

In 10 caused inflammation can also be enhanced by the application of I

In hpG-CSF. In addition, hpG-CSF may be useful in treating I

In leukemia, having been published in I

Able to differentiate leukemia cells. Welte, et I

In al., Proc. Natl. Acad. Sci. (USA), 82, 1526-1530 (1985) I

In 15 and Sachs, see above. IN

In the skilled person, by reading the following detailed I

In a description illustrating the embodiment of the invention, I

In the presently preferred embodiments, you can see

In several aspects and advantages of the invention. IN

I The drawing shows a partial restriction end-I

I ase map of the hpG-CSF gene provided with arrows for illumination I

Creating the Sequencing Strategy Used for I

In obtaining the genomic sequence. IN

In accordance with the invention, one has isolated and characterized

In 25 sequenced DNA sequences encoding the entire polypeptide sequence

In the sequence in hpG-CSF or parts thereof. IN

The following Examples illustrate the invention

In particular, and are directed to procedures performed prior to the identification

In tification of hpG-CSF cDNA and genomic clones, I

For 30 days leading to such identification and to se-

In Quensation, Development of Expression Systems, Based I

On cDNA, genomic and engineered genes and confirmation

I of the expression of hpG-CSF and similar products in so-I

In forming systems. IN

DK 175466 B1 9

More in detail, Example 1 relates to amino acid sequencing of hpG-CSF. Example 2 relates to the preparation of a cDNA library for scanning for colony hydration. Example 3 relates to the preparation of hybridization probes. Example 4 relates to hybridization search, identification of positive clones, DNA sequencing of a positive cDNA clone, and providing information on the primary structural conformation (amino acid sequence) of the polypeptide. Example 5 relates to the identification and sequencing of a genomic clone encoding hpG-CSF. Example 6 relates to the construction of a manufactured gene encoding hpG-CSF using codons belonging to E. coli.

Example 7 relates to methods for constructing an E. coli transformation vector containing DNA encoding hpG-CSF, using this vector for prokaryotic expression of hpG-CSF and analyzing the properties of the recombinant products of the invention. Example 8 relates to methods for preparing 20 inventive analogues of hpG-CSF, wherein cysteine residues are replaced with said other appropriate amino acid residues by mutagenesis performed on DNA encoding hpG-CSF. Example 9 relates to methods for constructing a vector containing DNA encoding an analog for hpG-CSF derived from a positive cDNA clone, using the vector for transfection of COS-1 cells, and culturing the transfected cells. Example 10 relates to the physical and biological properties of the recombinant polypeptide products related to those of the invention.

Example 1 (A) Sequencing performed by literature known

DK 175466 B1 I

methods. IN

One sample (3-4 µg, 85-90% pure SDS, silver staining- I

PAGE) of hpG-CSF was obtained from Sloan Kettering Insti- I

tute, New York, New York, it was isolated and cleaned

5 according to Welte, et al., Proc. Natl. Acad. Sci. (USA), 82, I

1526-1530 (1985). · I

The N-terminal amino acid sequence in this sample of I

hpG-CSP was determined in a first experiment by microse I

sequence analysis using an AB407A gas phase sequence divider (Applied Biosystems, Foster City, California) to obtain the sequence shown in Table I below.

denfor. Tables I-IV use single-letter code I

there, "X" means a residue that could not be determined completely

safe, and residues indicated in parentheses indicate possibility- I

15 there. IN

TABLE I

1 5 10 15 I

K-P-L-G-P-A-S-K-L-K-Q- (G, V, S) -G-L-X-X-X I

20 I

In all parts of the test whose results give I

In Table I, there was a high level of background noise I

present, a sign that the sample contained many conta- I

Mining Ingredients, Probably Chemical Residues I

25 arising from the purification. The sequence is only given by re- I

reference reasons. IN

In trial # 2, another test was used (5-6 l

yg, ca. 95% pure) from Sloan Kettering as in Experiment No. I

1, and the sequencing was performed as in Experiment # 1

30 This sample came from the same material that remained

used to obtain FIG. 4 in Welte, et al., Proc. IN

Natl. Adac. Sci. (USA), 82, 1526-1530 (1985). Result- I

DK 175466 B1 11s are given in Table II.

TABLE II

5 i 5 10 15 20 T-P-L-G-P-A-S- (S) -L-P-Q- (s) -M- <L) -X-K- (R) -X-X- (R) - (L) -X-

Although several residues were identified in Experiment # 2, a sufficiently long safe sequence was not identified, from which a reasonable number of probes could be constructed for searching for hpG-CSF DNA. It was estimated that at least 1536 probes would be required if the sequence of Table II was to isolate cDNA. Again, contamination of the 15 sample was likely to cause this.

Subsequently, a third sample (3-5 vig, about 40% pure) was obtained from Sloan Kettering as above. This sample was partitioned by SDS-PAGE and subjected to the electroplot in a further purification attempt. Sequence analysis of this sample yielded no data.

(B) Sequence breakdown by revised approaches

In order to obtain a sufficient amount of pure material upon which to perform a sufficiently accurate amino acid sequence analysis, cells from Bladder Carcinoma Cell Line 5737 (subclone 1A6) produced by Sloan-Kettering from Dr. E. Platzer. The cells were first grown in Iscove's medium (GIBCO, 30 Grand Island, New York) in small bottles until they collapsed. Then, the culture media were trypsinized and seeded in roller bottles (1½ small bottles / bottle), each containing 25 ml of pre-treated Iscove's medium.

I DK 175466 B1 I

I 12 I

In the 5% CO 2. The cells were grown overnight at 37 ° C

I and 0.3 rpm. IN

In Cytodex-1 Small Spheres (Pharmacia, Uppsala, Sweden) I

I was washed and sterilized by the following procedure. IN

In 5, 8 g of beads were placed in a bottle and 400 I added

In ml of PBS. The pellets were kept in suspension while holding

You shook gently for three hours. The pellets were lowered · I

In the trap, PBS poured off, cleaned the pellets in PBS and added

You put in new PBS. The beads were autoclaved for 15 minutes

10 ter. Before use, wash the balls of Iscove's I

In medium plus 10% fetal calf serum (FCS) before adding I

In fresh medium plus 10% FCS to obtain treated I

In small balls. IN

Culture medium was removed, except 30 ml of I

In 15 from each roll bottle and added 30 ml of fresh medium plus I

In 10% FCS and 40 ml treated beads for the bottles. IN

The bottles were bubbled with 5% CO 2 and sucked

In all bubbles away. The bottles were placed in rollers

I slowed down at 3 rpm ½ hour before slowing down to 1

At 0.3 rpm. After three hours, additional supplies were provided

You made a small bottle of trypsin and the contents were added

You put in all the roll bottles that contained small balls. IN

When the confluence had reached 40-50%, you washed

In the culture in the roll bottles with 50 ml PBS and rolled

For 25 more for 10 minutes before removing PBS. The cells I

You were grown for 48 hours in medium A [iscove's medium with I

Containing 0.2% FCS, 10 “8M hydrocortisone, 2mM glutamine, I

In 100 units / ml penicillin and 100 µg / ml streptomycin]. IN

Then the supernatant from the culture was harvested,

For 30 minutes, spin at 3000 rpm for 15 minutes and store

I at -70 ° C. Medium A I was added to the cultures again

I containing 10% FCS and grown for 48 hours. Cultivated I

The media was discarded, after which cells were washed

DK 175466 B1 13 ne with PDS as above and cultured for 48 hours in medium A.

The supernatant was again harvested and treated as previously described.

Concentrated approx. 30 l of medium containing 1A6 cells to ca. 2 1 on a Millipore Pellicon unit, provided with two cassettes with molecular weight cuts 10,000, filtering 200 ml per ml. minute and withheld approx. 1000 ml per minute. The concentrate was diafiltered to ca. 10 1 50 mM Tris (pH 7.8) with 10 same apparatus and same speeds. The diafiltered concentrate was taken at a rate of 40 ml per ml. per minute on a one liter DE cellulose column, equilibrated with 50 mM Tris (pH 7.8). After application, the column was washed at the same rate with one liter of 50 mM Tris (pH 7.8) and then with two liters of 50 mM Tris (pH 7.8) added with 50 mM NaCl. The column was then sequentially eluted with six 1 liter portions of 50 mM Tris (pH 7.5) containing the following concentrations of NaCl: 75 mM; 100 mM; 125 mM; 150 mM; 200 mM; and 300 mM. Fractions (50 ml) were collected and the active fractions concentrated to 65 ml were collected on an Amicon ultra-filtration stirred cell unit equipped with a YM5 membrane. This concentrate was loaded onto a 2 liter AcA54 gel filtration column. equilibrated with PBS, throughput 25 was 80 ml / h and fractions of 10 ml were collected.

Active fractions were pooled and directly applied to a C4 "high performance liquid chromatography" (HPLC) column.

Samples whose volume was 125-850 ml and contained 1-8 mg of protein, 10% of which was hpG-CSF, were loaded onto the column at a flow rate of 1-4 ml / min.

After application and a first wash with 0.1 M ammonium acetate (pH 6.0-7.0) in 80% 2-propanol with a flow rate

DK 175466 B1 I

rate of lml / minute. One ml of fraction was collected

tions and examined for protein content at 220 nm, 260 I

nm and 280 nm. IN

In this purification, fractions containing

5 hpG-CSF clearly separated (as fractions 72 and 7 3 out of 1

80) from other fractions containing protein. Man I

isolated hpG-CSF (150-300 pg) of a purity of ca. 85 ± I.

5% and with a yield of approx. 50%. From this purified ma- I

9 µg was used in Experiment No. 4, an amino acid

10 sequence analysis, applying the protein sample to an I

TFA-activated fiberglass without polybrene. They performed

sequence analysis with an AB 470A sequence splitter at forward

Hewick et al., J. Biol. Chem., 256, I

7990-7997 (1981) and Lai (Anal. Chem. Acta, 163, 243-248 I

15 (1984). The results of Experiment # 4 are shown in Table I

III. IN

TABLE III

20 in 5 10 I

Thr- Pro - Leu - Gly - Pro - Ala - Ser - Ser - Leu -Pro- I

15 20 I

Gin-Ser - Phe - Leu - Leu - Lys - (Lys) - Leu - (Glu) -Glu- I

25 I

25 30 I

Val-Arg - Lys - Ile - (Gin) - Gly - Val - Gly - Ala -Ala- I

Leu -X - X - I

30 - I

In experiment # 4, after 31 passes I was obtained

(corresponding to residue 31 in Table III) no further sig- I

nificant information about the sequence of the sequence. For achievement I

DK 175466 B1 I

15 I

of a longer distinct sequence in a fifth trial reduce I

14 µg of hpG-CSF were purified from treated culture I

medium with 10 µl of β-mercaptoethanol for one hour at 45 ° C, I

and then dried thoroughly under vacuum. Protein Re- I

Five stones were redissolved in 5% formic acid and applied to a polybrene

treated fiberglass disc. The sequence breakdown analysis was I

performed as in Experiment # 4 above. The results of I

Experiment # 5 is given in Table IV: I

TABLE IV I

15 10 I

Thr- Pro - Leu - Gly - Pro - Ala - Ser - Ser - Leu- Pro- I

15 15 20 I

I Gin-Ser - Phe - Leu - Leu - Lys - Cys - Leu - Glu- Gin- I

I 25 30 I

Val- Arg - Lys - Ile - Gin - Gly - Asp - Gly - Ala- Ala- I

I 20 I 35 40

Leu-Gin - Phe - Lys - Leu - Gly - Ala - Thr - Tyr- Tyr- Lys- I 45 I 25 val- Phe - Ser - Thr - (Arg) - (Phe) - (Met) - X- I The amino acid sequence in Table IV was sufficiently long (44 residues) and accurately determined from which probes could be constructed to obtain hpG-CSF cDNA as described below.

----— - I DK 175466 B1

Example 2

Among standard methods for isolating relevant cDNA sequences are a preparation of plasma-based cDNA "libraries" derived from reverse transcription of mRNA found in donor cells selected on the basis of their expression of a selected gene. Knowing essential portions of the amino acid sequence of a particular polypeptide, labeled single stranded DNA probe sequences can be used that duplicate a sequence which

It is believed to be found in the relevant cDNA by a DNA / DNA

H hybridization procedure performed on cloned copies of cDNA which has been denatured to appear in single strand form. Weissman, et al., U.S. Patent No. 4,394,443; Wallace, et al., Nucleic Acids Res., 6, I 3535-3557 (1979), and Reyes, et al., Proc. Natl. Acad.

In Sci. (USA), 79, 3270-3274 (1982), and Jaye, et al.,

In Nucleic Acids Res., 11, 2325-2335 (1983). See also US

In Patent No. 4,358,535, Falkow, et al., On DNA / DNA

hybridization procedure at diagnosis; and Davis, et al., in 20 "A Manual for Genetic Engineering, Advanced Bacterial in Genetics", Cold Spring Harbor Laboratory, Cold Spring I Harbor, N.Y. (1980) pages 55-58 and 174-176 regarding the colony and plaque hybridization technique.

All RNA was extracted from ca. 1 g of cells from a bladder carcinoma cell line 5637 (1A6) urid the use of 1 guanidinium thiocyanate to quantitatively isolate undamaged RNA. . [surgeon, et al., Biochemistry, 18, I 5294-5299 (1979)].

In the sterile aqueous RNA solution, all I30 contained RNA from the IA6 cells. To provide messenger RNA solely from the solution of total RNA, this I solution was passed on a column containing oligodesoxy-thymidylate [oligo (dT)] (Collaborative Research, Inc.,

DK 175466 B1 I

17 I

Waltham, Massachusetts). Poly-adenylated (poly-A +) I

end pieces characteristic of messenger RNA, I

binds to the column, whereas ribosome RNA elutes. As an I

As a result of this approach, it was possible to isolate approx. 90 I

In 5 µg poly-adenylated messenger RNA (poly-A + mRNA). The ten

In isolated poly-A + messenger RNA was pretreated with I

In methylmercury hydroxide (Alpha Ventron, Danvers, Massa- I

In chusetts) at a final concentration of 4 mM for five minutes

At room temperature before use in a cDNA reaction

In 10 tion. The treatment with methylmercury hydroxide denatu- I

You interact with messenger RNA, both internally

With contaminant molecules that block transla- I

I tion. Payvar, et al., J. Biol. Chem., 258, 7636-7642 I

I (1979). IN

In the Okayama method [Okayama, et al., I

In Molecular & Cellular Biology, 2, 161-170 (1982)]

You set up a cDNA bank, using mRNA, up

In reached from IA6 cells. CDNA was transformed by

In incubated in a host microorganism E. coli K-12 strain I

In 20 HB101 to increase the amount. IN

In Example 3 I

In Hybridization probes, constructed on the basis of the I

In the terminal amino acid sequence of hpG-CSF according to Table IV, I

I 25 consisted of a set of 24 oligonucleotides, each with an I

23 bases in length and containing three inosine residues. The probe oligonucleotides were prepared by the method of Caruthers, et al., Genetic I Engineering, 4, 1-18 (1982) and labeled with γ-32Ρ ATP, id they were kinase with polynucleotide kinase. The probe oligonucleotides corresponding to messenger RNA for residues 23-30 of the sequence in Table IV are illustrated in Table I V: I DK 175466 B1

TABLE V

hpG-CSF without 5 5 'GC IGC ICC * TC ICC TTG GAT TTT3'

The assumption of neutrality for the I bases was based on published works by Takahashi, et al.

Proc. Natl. Acad. Sci. (USA), 82, 1931-1935 (1985) and Ohtsuka, et al., J. Biol. Chem., 260, 2605-2608 (1985).

However, inosine can have a destabilizing effect if it is paired with G or T. In Takahashl, et al., Inosins appear to have a neutral effect because, as a group, they achieve on average neutrality 15 (e.g., if three advantageously pair with C, and two not in favor pair with T).

To test the effect of base pairing between I and G, control experiments were constructed by B using an N-myc gene sequence and clone. The sequence selected from the N-myc gene had the same total content B of G and C at the first two positions in each codon as B was determined for the hpG-CSF probes. The N-myc test probes B were of the same length, contained I in the same relative positions and potentially had the same mean value for Tm 25 (62-66 ° C, with the three or four inosine residues found not being spoken to) as the hpG-CSF probes.

Two groups of N-myc test probes were constructed by the method of Caruthers, et al., Supra.

In Set I, as illustrated in Table VI, I included: 1, a complete adaptation; 2, in which three in third position C have been replaced by I's in the formation of the worst possible case by the addition of I; I and 3, in which four third position Cs have been replaced with I's. The second set of test probes was designed to represent a more random distribution of inosine base pairs, thereby obtaining a base pairing effect that was, on average, neutral. Set II, as illustrated in Table vi, included; 4, containing two I's which will form base pairs with C and I with G; and 5, identical to 4, with an additional I: G base pair added.

TABLE VI

1. 5 'CAC AAC TAT GCC GCC CCC TCC CC3' 2. 5 'CAC AAC TAT GCI GCC CCI YCI CC3' 15 3. 5 'CAI AAC TAT GCI GCC CCI TCI CC3' 4. 5 'AAC GAG CTG TGI GGC AGI CCI GC3 '20 5.5 "AAI GAG CTG TGI GGC AGI CCI GC3"

Five replica filters containing N-myc DNA sequences and chicken growth hormone DNA sequences (as a negative control) were heated for two hours at 25 ° C in a vacuum oven prior to hybridization. All filters were hybridized as described in Example 4 for the hpG-CSF probes, except that the hybridization period was only six hours. Filters were washed three times at room temperature and once at 45 ° C, 10 minutes every 30 times. The filters were checked with a Geiger counter.

The filter, which represented an N-myc probe 3, gave a very weak signal to the other four probes with probes and was not washed further. After

I DK 175466 B1 I

I 20 I

For ten minutes washing at 50 ° C, the Geiger counter gave the following I

In percent signal, with probe 1 set to 100%; probe I.

I 20%; probe 3 (45 ° C), 2%; probe 4 92%; and probe 5 75%. IN

After washing at 55 ° C, the percentages were: probe 2 16%; IN

In 5 probe 4 100%; and probe 5 80%. A final wash at I

At 60 ° C, the following percentages gave: Probe 2 1.6%; probe 4 I

In 90%, and probe 5 70%. IN

Thus, in the presence of I

In three I *, as in probes 2 and 4, you see up to 60 times

In 10 large difference in signal when approaching the theoretical I

In value for Tm (I not included in the calculation) [based I

In the worst case for base pairing with 1 (probe 2)

I and a relatively neutral case with base pairing with I

I (probe 4)]. IN

I 15 The standardization information obtained

I by the N-myc sample hybridizations, were utilized when I

You should wash and control the hpG-CPS hybridization as I did

Below you will assess how much you could have

Trusting the results when using a smaller I

In 20 rigorous washing procedures. IN

Example 4 I

In the method of Hanahan, et al., J.I.

Moth. Biol. 166, 557-580 (1983) spread bacteria, I.

25 containing recombinants with cDNA inserts, I

as prepared in Example 2 of 24 nitrocellulose filters I

(Millipore, Bedford, Massachusetts), placed on agarpla- I

there. The plates were then incubated to obtain I

ca. 150,000 colonies, which were replica-plated in 24 years

30 nitrocellulose filters. Replica filters were incubated

ne until separate colonies were obtained. The bacteria I

on the rattles were lighted on Whatman 3 MM paper I was

nearly saturated with sodium hydroxide (0.5 M) for ten minutes I

21 DK 175466 B1 and then stained with Tris (1M) for two minutes and then stained with Tris (0.5 M) containing NaCl (1.5 M) for ten minutes. When the filters were almost dry, they were allowed to warm for two hours at 80 ° C in a vacuum oven before hybridizing with nucleic acid. [Wahl, et al., Proc. Natl. Acad. Sci. (USA), 76, 3683-3687 (1979)]; and Maniatis, et al., Cell, 81, 163-182 (1976).

The filters were pre-hybridized for two hours at 10 65 ° C in 750 ml of 10 x Denhardt, 0.2% SDS and 6 x SSC. The filters were purified in 6 x SSC and placed four together, then hybridized for 14 hours in 6 x SSC and 10 x Denhardt. There were approx. 15 ml of solution in the hybridization vessel containing 50 x 10 5 cpm of the ³²P-labeled 15 probe (oligonucleotides).

After hybridization, the filters were washed three times in 6 x SSC (1 liter / wash) at room temperature, 10 minutes each time. The filters were washed twice at 45 ° C for 15 minutes, once at 50 ° C for 15 minutes, and 20 times at 55 ° C for 15 minutes, using 1 liter of 6X SSC each time. The filters were autoradiographed at -70 ° C for two hours using a reinforcing screen and Kodak XAR-2 film. On this autoradiograph, there were 40-50 positive signals, including five very powerful signals.

The area containing the strongest five signals and another five positive signals was scraped from the output plates and plated again for a second search using the same probe mix and the same terms. The wash varied, washing at high temperature twice at 55 ° C for 15 minutes, and once at 60 ° C for 15 minutes. Based on the study of the N-myc probe in Example 3, the washing temperature was finally set I DK 175466 B1 I 22

In the second crawl of the weather, combining

Melting point for the 24 23-mer was 60-68eC, as in N-myc I

I sing. Immediately after the second wash at 55 ° C, I allowed

In the filters the autoradiographs in the moist state. And I

5 comparing this autoradiography with another auto- I

In radiography, recorded at the same time after the final wash at I

At 60 ° C, only two of the ten clones tested failed to I

At a significant loss in signal strength when increasing temp

At the rate of 55-60 ° C. It could later be shown that these two I

10 clones were of almost identical length and possessed almost I

In the same pattern for restriction endonucleases. You go out

You selected a clone, designated Ppo2, for sequence splitting. IN

The sequencing of the recombinant I was performed

In hpG-CSF cDNA clone Ppo2, obtained by the above-mentioned assay

In the method of the didesoxy method according to Sanger, et al., I

In Proc. Natl. Acad. Sci. (USA) 74, 5463-5467 (1977). M-13 I

In the single-stranded DNA strand was used as clone I

In vector and provided single stranded DNA templates I

I to the double-stranded cDNA clones. Singer, et al. IN

The procedure gave the sequence shown in Table VTI I

I along with the corresponding amino acid sequence and a com

In the plementary strand of the region encoding polypeptide

In time. IN

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The following characteristics of the sequence in Table I

will should be noted. At the 5 'end of the sequence, bases, I

similar to what one finds in a poly G cDNA together 'I

binds. Then there are approx. five bases (designated "N"), I

5 if sequence could not be determined exactly by Singer,

et al. the method due to the long G sequence, I

that comes immediately before. The sequence that follows, I

exhibits a series of 12 codons encoding part of I

an assumed leader sequence for the polypeptide. On the basis of I

10 correspondence to the terminal amino acid sequence of na-I

hpCSF, isolated in Example 1, is the I

first threonine residue in the noted "finished" form of I

hpG-CSF denoted by +1. Then it is seen that you finished

hpG-CSF comprises 174 amino acid residues, as shown. After I

The "stop" codon (OP codon, TGA) occurs approximately 856 I

bases belonging to an untranslated 3 'sequence and I

a series of A's in the poly A "tail". Unique HgiAi and Apal I

restriction endonuclease sites as well as two Stul ste I

there (discussed below in connection with construction I

20 of a procaryotic and eucaryotic expression system) I

are also shown in Table VII. As no asparagine residues are found in the polypeptide, there are apparently no positions where N-glycosylates can be obtained. They underlined six bases near the end of the 3 · untranslated I sequence representing a possible polyadenylation site.

In One should note that both the two additional cDNA

In clones, identified by the hybridization method I described above out of a total of 450,000 clones, did not include DNA encoding the entire leader sequence from the transcription initiation site onwards. In fact, all three 30 hpG-CSF clones terminated the 5 'region at exactly the same site, a sign that the secondary structure of the transcribed mRNA severely inhibits the formation of the DNA beyond this site. . In practice, therefore, a cDNA expression screening, as described by Okaya-Ima, et al., Mol. and Cell. Biol., 3, 280-289 (1983) and used to isolate GM-CSF by Wong, et al., Science, 228, 810-814 (1985) are not H used to isolate hpCSF DNA. since such isolation systems are generally dependent on the presence of the full-length transcribed cDNA in the clones under investigation.

The above-mentioned sequence is not directly capable of ensuring direct expression of hpG-CSF in an I microcancer. To obtain such an expression, the hpG-CSF coding region must be provided with an initial ATG codon I and the sequence must be placed in a transformation vector in H a position where it is controlled by an appropriate promoter / regulator DNA sequence.

Example 5 Η In this Example, cDNA encoding I20 for hpG-CSF as isolated in the previous Example was used to search a genomic clone. A phage λ human fetal liver genomic library [prepared by the method of Lawn, et al., Cell, 15, 1157-1174 (1978) and obtained from T. Maniatis] was searched using a notch-translated probe. consisting of two hpG-CSF fragments

ter, isolated by digestion with HgiAl and S tul (HgiAI

I to Chair, 649 base pairs; Chair to Chair, 639 base pairs). IN

Total approx. 500,000 phages were plated on 12 (15 cm) triplicate dishes and the stains formed were removed therefrom and 30 probe hybridized by Benton / Davison method [Benton, et al., Science, 196, 180 ( 1977)]. A total of 12 positive clones were observed. Three clones (1-3), which gave the strongest signal by autoradiography in a second scan, were grown in 1 liter cultures and imaged by restriction enzyme digestion and Southern blot using a radiolabeled 24-mer oligonucleotide ( kinase with γ-32p ATP) 5 5'CTGCACTGTCCAGAGTGCACTGTG3 '. The imaging results showed that isolates 1 and 3 were identical, and 2 contained in 2000 additional bases 5 'aligned to the hpG-CSP gene. Therefore, clone 2 was used for further characterization.

Clone 2 DNA was digested with RI to release a 108500 bp fragment containing hpG-CSF, which was then subcloned into pBR322 and further subjected to digestion by restriction endonuclease digestion. Southern Blot, Ml3 subcloning and sequence splitting. The sequence obtained is shown in Table VIII.

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A restriction endonuclease map (approximately 3.4 kb) for genomic DNA containing the hpG-CSF gene is shown in detail in FIG. 1. The restriction endonucleases of FIG. 1 is: Ncol, N; PstI, P; BamHI, B; Apal, A; Xhol, X; and 5 Kpn, K. The arrows below the map show the sequencing strategy used to obtain the genomic sequence. The framed regions are those found in the cDNA clone and the dotted open framing represents a sequence not found in the cDNA clone, but identified only by mRNA probe. Identification of the coding sequences proposed for exon one was performed by Northern blot analysis. A 24 mer I oligonucleotide probe, 5, CAGCAGCTGCAGGGCCATCAGCTT3 'spanning the assumed junctional compounds

In 15 for exons 1 and 2, hybridized with hpG-CSF mRNA

In Northern blot format. The resulting blot shows an H mRNA of the same size (about 1650 bp) as seen with an exon 2 oligonucleotide probe. This result and the ability to direct expression of hpG-CSF from the pSVGM-20 Ppol vector (Example 9) with Met initiation codon, shown in Table VIII, define the coding sequences found in exon 1. Exons 2-5 are defined by the coding sequences obtained in the cDNA clone (Ppo2) of the hpG-1 CSF gene (Table VII).

Example 6 (reference)

The example relates to the preparation of a prepared H gene encoding hpG-CSF and comprising codons to E.

coli.

30x briefly, the same procedure as shown in PCT publication WO83 / 04053, Alton, et al.

The genes were constructed by first assembling component oligonucleotides for multiple duplexes, such as

H

utes were grouped into three separate sections. These sections were designed so that they could be readily amplified and, after removal from the amplification system, assembled sequentially or by ligation of many fragments to an appropriate expression vector.

The construction of sections I, II and III is illustrated in Tables IX-XIV. In the construction of Section I, as illustrated in Tables IX and x, the oligonucleotides 1-14 to 7 duplexes (1 and 8; 2 and H 10 9; 3 and 10; 4 and 11; 5 and 12; 6 and 13; and 7 and 14).

The seven duplexes were then bonded to form Section I as shown in Table X. It should be noted in conjunction with Table X that Section I contains upstream of an XbaI adhesive end and downstream of a BamH1 adhesive end useful for bonding. with multiplication and expression vectors and for section II equation.

37 I

DK 175466 B1 I

TABLE IX

5 I

EChpG-CSFDNA SECTION I I

I CTAGAAAAAACCAAGGAGGTAATAAA 1 I

I TAATG ACTCCATTAGGTCCTGCTTCTTCT 2 I

In 10 CTGCCGCAAAGCTTTCTGCTGAAATGTCTGG 3 In AACAGGTTCGTAAAATCCAGGGTGACGGT 4 In GCTGCACTGCAAGAAAAACTGTGCGCTA 5 In CTTACAAACTGTGCCATCCGGAAGAGC 6 TGGTACTGCTGGGTCATTCTCTTGG 7 In 15 CATTATTTATTACCTCCTTGGTTTTTT 8 GCAGAGAAGAAGCAGGACCTAATGGAGT 9 TGTTCCAGACATTTCAGCAGAAAGCTTTGCG 10 CAGCACCGTCACCCTGGATTTTACGAACC 11 TAAGTAGCGCACAGTTTTTCTTGCAGTG 12 In 20 ACCAGCTCTTCCGGATGGCACAGTTTG 13 GATCCCAAGAGAATGACCCAGCAGT 14 H DK 175466 B1 o o o o o o o o o

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39 DK 175466 B1

As illustrated as in Tables XI and XII in conjunction with Section II construction, oligonucleotides 15-20 were assembled into 8 duplexes (15 and 23; 16 and 24; 17 and 25; 18 and 26; 19 and 27; 20 and 28 ; 21 and 29; 5 and 22 and 30). Then these 8 duplexes were bonded together to obtain Section II, as shown in Table XII. As further shown in Table XII, section II upstream of a BamH1 adhesive end and downstream of an EcoRI adhesive end useful for ligation to a multiplication vector and for ligation to section I. Near the downstream end, section II also includes a downstream Find a site that is useful in any connection of Sections II and III.

I DK 175466 B1 40

TABLE XI

EChpG-CSFDNA SECTION ΙΓ GATCCCGTGGGCTCCGCTGTCTTCT 15 TGTCCATCTCAAGCTCTTCAGCTGGC 16 TGGTTGTCTGTCTCAACTGCATTCTGGT 17 CTGTTCCTGTATCAGGGTCTTCTG 18 CAAGCTCTGGAAGGTATCTCTCCGGA 19 ACTGGGTCCGACTCTGGACACTCTGCA 20 GCTAGATGTAGCTGACTTTGCTACTACT 21 ATTTGGCAACAGATGGAAGAGCTCAAAG 22 GACAAGAAGACAGCGGAGCCCACGG 23 ACCAGCCAGCTGAAGAGCTTGAGATG 24 ACAGACCAGAATGCAGTTGAGACAGACA 25 CTTG C AG AAG ACCCTG AT AC AGG A 26 CAGTTCCGGAGAGATACCTTCCAGAG 27 TAGCTGCAGAGTGTCCAGAGTCGGACC 28 AAATAGTAGTAGCAAAGTCAGCTACATC 29 AATTCTTTG AGCTCTTCC ATCTGTTGCC 30 41 DK 175466 B1

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DK 175466 B1

Finally, section III was constructed as shown in Tables XIII and XIV. For the purpose of this construction, oligonucleotides 31-42 were assembled into 6 duplexes (31 and 37; 32 and 38; 33 and 39; 34 and 40; 35 and 41; 5 and 36 and 42). The 6 duplexes were then combined to form Section III, as shown in Table XIV.

As also shown in Table XIV, section III comprises an upstream BamHI adhesive end and downstream an EcoRl adhesive end, useful for ligation in a multiplication vector H 10 and, at least in the case of EcoRl, in an expression vector. In addition, Section II holds an up-H upstream Sstl site, useful in the eventual ligation of Sections II and III.

I TABLE XIII

ECHGpG-CSFDNA section III

In GATCCAAAGAGCTCGGTATGGCACCAG CTCTGCAACCGACTCAAGGTGCTATGCCG 31 20 32 33 GCATTCGCTTCTGCATTCCAGCGTCGTGC AGGAGGTGTACTGGTTGCTTCTCATCTG 34 CAATCTTTCCTGGAAGTATCTTACCGTGT TCTGCGTCATCTGGCTCAGCCGTAATAG 35 36 25 37 In AGAGCTGGTGCCATACCGAGCTCTTTG ATGCCGGCATAGCACCTTGAGTCGGTTGC 38 TCCTGCACGACGCTGGAATGCAGAAGCGA 39 ATTGCAGATGAGAAGCAACCAGTACACC In CAGAACACGGTAAGATACTTCCAGGAAAG 40 41 I 30 42 AATTCTATTACGGCTGAGCCAGATGACG

DK 175466 B1 I

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The Xbal-BamHI fragment, formed by section I, is ligated to an M13mpl1 phage vector, opened with Xbal and BamH1.

The vector is then reopened by digestion with BamH1 and EcoR1, and then ligated with the BamHI-EcoR1 fragment formed by section II. In this step, sections I and II have been linked together in the correct orientation. Then, another M13mp11 vector is opened by digestion with BamH1 and EcoRl and then ligated with the BamHI-EcoRI fragment formed by section 10

The vector containing sections I and II is allowed to digest with Xbal and Sstl. The vector containing section III is also digested with Sstl and EcoRl. The smallest of the two fragments formed in both H 15 cases by digestion are ligated into a plasmid pCFM1156 previously opened with XbaI and EcoRl. The reaction product is an expression plasmid containing a continuous DNA sequence, as shown in Table XV, which encodes the entire hpG-CSF polypeptide with an amino-terminal methionine codon (ATG) to initiate translation. in E. coli.

45.

X

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47 DK 175466 B1

Although any suitable vector can be used to express this DNA, the expression plasmid pCFMH56 can be readily constructed from a plasmid pCFM836, the construction of which is described in EPO 5 published patent application No. 136 490. First, pCFM836 is intersected with Ndel and by rounding the ends with Poll, so that both existing Ndel locations are destroyed.

Then, the vector is digested with Clal and Sacll, to remove an existing polysaminer before equation to a substitute polysaminer, as illustrated in Table XVI. This substitute can be poly-bonded by the method of Alton, et al., See above. Control of the expression in the expression plasmid pCFMH56 is carried out by means of a lambda PL promoter, which itself may be under the control of a C1857 repressor gene (such as that provided in E. coli strain K12AHtrp).

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Example 7 (Reference)

This Example relates to E. coli expression of a hpG-CSF polypeptide by a DNA sequence encoding [Mef1J]. The sequence used was partially I synthetic and partially derived from cDNA. The synthetic I sequence used codons suitable for E. coli.

In plasmid Ppo2, containing the hpG-1 CSF gene shown in Table III, was digested with HgiAI and Stul to provide a ca. 645 base pair fragment containing the final hpCSF gene (as shown in Table VII) with I 10 seven of the leader sequence codons at the 5 'end and ca. 100 ba-

separ at the non-coding 3 'end. Digestion with HgiAI

You provide a 5 '4 base adhesive end that is identical to what you get with PstI, and digestion with Stul gives a straight end. As a result, the fragment 15 can be inserted into M13 mp8 (Rf) intersected with I PstI, and with the like-forming restriction enzyme.

In Hindi. After multiplication in M13, hpG-CSF DNA was allowed

cut by digestion with Apal and BamHI, which intersect at the Apal site, respectively, containing the codons for 20 residues +3 to +5 in hpCSF and at a BamHI site "downstream" relative to the Hindi site in the Ml3 mp8 restriction. t ions s ammenbinder and. In order to obtain E. coli expression of the hpG-CSF polypeptide, a synthetic fragment was prepared as shown in Table XVII below.

H DK 175466 B1 Η H Table xvii

Η 5 * - C TAG AAA AAA CCA AGG AGG TAA TAA ATA

H 3 '- TTT TTT GGT TCC TCC ATT ATT TAT

H 5 xbai -1 +1

With Thr Pro Leu ATG ACA CCT CTG GGC C - 5 * TAC TGT GGA GAC> 3 '

agal

As can be seen by analysis of Table XVII, the linker is comprised of an Apal adhesive end, codons which

H encodes the first three residues at the amino terminus of hpG-CSF

H 15 (and which "recovers" the codons encoding Thr1,

Pro2 and Leu3, which were deleted by Apal digestion H of M13 DNA, described above, and using H codons, preferably expressed in E. coli), a translation initiating ATG, a sequence of 24 base pairs, providing a ribosome binding site and an Xbai sticky end.

In the Expression Vector used for E.

In coli expression, the one described as pCFM536 in I EPO Patent Application No. 136,490, Morris, was published on April 25, 1985. (See also A.T.C.C. 39934, E. coli JM103 I containing pCFM536). Briefly, plasmid pCFM536 was digested with Xbai and BamHI. The hpG-CSF fragment (Apal / BamH1) and the linker I (Xbai / Apal), described above, were then ligated to form a plasmid designated p536Ppo2.

Plasmid p536Ppo2 was transformed into a phage resistant variant of the E. coli AM7 strain, which had previously been transformed with the plasmid pMW1 51 DK 175466 B1

(A.T.C.C. No. 39933) containing a Cl857 gene. Transformation was monitored by the antibiotic resistance marker gene (amp) carried by the progenitor plasmid pCFM536. Cell cultures in LB herb (ampicillin 5 50 µg 1 ml) were maintained at 28 ° C and after growth of 1 cells in the culture to Ag00 55 0/5, hpCSF was induced.

In expression, raising the temperature of the culture to 42 ° C for three hours. The final O.D. of coal - In the trip Ag00 =! / 2 ·

In 10, the level of expression of hpG-CSF was determined

I of the transformed cells on an SDS-polyacrylamide gel, I stained with blue coomassi staining to be 3-5% of total I cellular protein.

Cells were harvested by centrifugation at 3500 I for 15 g for 10 minutes on a JS-4.2 rotor. Cells were broken into.

In water 25% (w / v), passing the slurry three times through a French pressure cell at 10,000 psi. The disrupted cell suspension was centrifuged at 10,000 g for 15 minutes in a JA-20 rotor. The precipitate I20 was again suspended in water and solubilized with an I content of ca. 5 mg / ml total protein in 1% lauric acid, 50 I mM Tris, pH 8.7. The precipitated solubilized material was centrifuged at 15,000 g for ten minutes and CuSo4 was added to a concentration of 20 mM to the supernatant. * After one hour, this sample was charged with an H C4 HPLC column for purification according to the procedure of Example (B) with corrections for volume and concentration.

H A second purification process was developed for larger amounts of hpG-CSF formulated in a buffer containing inorganic material. This material is suitable for in vivo studies. 150 g of cell paste was suspended for approx. 600 ml 1 mM DTT and let it H DK 175466 B1 Η

pass four times through a Manton Gualin homogenizer at approx. 7000 psi. The crushed cell suspension was centrifuged at 10,000 g for 30 minutes and the precipitate was spent precipitating in 400 ml of deoxycholate (DOC), 5 mM

EDTA, 5 mM DTT, and 50 mM Tris, pH 9. This suspension was admixed for 30 min at room temperature and centrifuged at 10,000 g for 30 min. The precipitate was again suspended for approx. 400 ml of water and centrifuge at 10,000 g for 30 minutes. The precipitate was solubilized in 100 ml of 2% sarcosyl and 50 mM at pH 8. Man H was added CuSO 4 up to 20 µm and kept under stirring for 16 hours at room temperature, after which one-H was trifuged at 20,000 g for 30 minutes. To supernatant was added 300 ml of acetone. This mixture was placed on ice for 20 minutes and then centrifuged at 5000 g for 30 minutes. The precipitate was dissolved in 250 ml of 6 M guanidine and 40 mM sodium acetate at pH 4 and loaded onto a 1200 ml G-25 column, equilibrated and operated with 20 20 mM sodium acetate at pH 5.4. Fractions containing hpG-CSP (about 400 ml) were pooled and loaded onto a 15 ml CM cellulose column, equilibrated with 20 mM sodium acetate at pH 5.4. After application, the column was washed

with 60 ml of 20 mM sodium acetate at pH 5.4 and with 25 mM

25 sodium chloride and then the column was eluted with 200 I ml of 20 mM sodium acetate at pH 5.4 and with 37 mM sodium I chloride. 150 ml of this pass was concentrated I to 10 ml and loaded with one 300 ml G-75 column, equilibrated and operated with 20 mM sodium acetate and 10.0 mM sodium chloride at pH 5.4. Fractions of relevant content I (35 ml) were pooled and filter sterilized. The final concentration of phG-CSF was 1.5 mg / ml, it was more than 95% pure as determined by analysis on a gel and contained less than 0.5 ng of pyrogen per day. 0.5 mg hpG-CSP.

53 DK 175466 B1

The content of the pyrogen was determined using a Limulus Amebocyte Lysate (LAL) test equipment (M. A. Bio-products, Walkersville, Maryland).

Example 8

The example relates to the use of recombinant methods to prepare inventive analogues for hpG-CSP, wherein the cysteine residues found at positions 17, 36, 42, 64 and 74 were individually replaced by a suitable amino acid residue (serine).

Position-directed mutagenesis procedures were performed according to Souza, et al., PCT Application No. W085 / 00817, I

published on February 28, 1985, on DNA from plasmid p536Ppo2 encoding [Met "1;] described below using synthetic oligonucleotides but a length of 20-23 bases, as shown in Table XVIII below.

With oligonucleotide # 1, one could generate a gene encoding [ser173hPG ': CSF' 'ed oligonucleotide # 2, one could generate [sé ^] hpG-CSF and etc.' »· 20

TABLE XVIII

Oligonucleotide_Sequence_ I 25 1 · 51-CTG CTC AAG TCC TTA GAG CAA GT-3 'I 2. 5 * -GAG AAG CTG TCT GCC ACC TACA-3' 3. 51 -TAC AAG CTG TCC CAC CCC GAG-31 4. 5 '-TGA GCA GCT CCC CCA GCC AG-3' 5. 5'-CTG CCA GGC TCC TTG AGC CAA-3 'I 30

The position-directing Cys to Ser j mutagenesis restrictions were performed using M13 mp10 containing a XbaI-BamHI hpG-CSF fragment isolated from p536Ppo2 as a template. DNA from each DK 175466 B1 was processed

H

M13mp10 clone containing a Cys-Ser substitution with XbaI and BamHI. The resulting fragment was cloned into the expression vector pCFM746 and expression products isolated as in Example 7.

Plasmid pCFM746 can be constructed by cleaving a plasmid pCFM736 (the construction of this plasmid from deposited and publicly available material is disclosed in PCT Application No. W085 / 00829, Morris, published February 28, 1985) with Clal and BamHI for the removal of an existing polysaminer and substitution of the following polysaminer.

In TABLE XIX

In Clal

5 'CGATTTGATTCTAGAATTCGTTAACGGTACCATGGAA

3 'TAAACTAAGATCTTAAGCAATTGCCATGGTACCTT

gcttactcgaggatccgcggataaataagtaac3 'I 20 cgaatgagctcctaggcgcctatttattcattgctag5'

Sau 3a by a purification process for Cys to Ser I analogs of the invention was suspended for approx. 10-15 g of cell paste in 40 ml of 1 mM DTT and passed it three times a French pressure cell at 10,000 psi. The broken up I cell suspension was centrifuged at 1000 g for 30 minutes.

nutter. The precipitate was suspended in 1% DOC, 5 mM

In 30 EDTA, 5 mM DTT, 50 mM Tris, pH 9 and blended for 30 minutes at room temperature. The mixture was centrifuged at 10,000 for 30 minutes, suspended in 40 ml H 2 O and centrifuged again at 10,000 g for 30 minutes.

55 DK 175466 B1

The precipitate was dissolved in 10 ml of 2% Sarkosyl, 50 mM DTT? 50 mM Tris, pH 8. After mixing for one hour, the mixture was centrifuged at 20,000 g for 30 minutes, then applied to a 300 ml G-75 column, equilibrated and operated with 1% Sarkosyl, 50 mM Tris, pH 8. Fractions containing the analog compound were pooled and oxidized with air by standing with air access for at least one day. The final concentrations were 0.5-5 mg / ml.

10

Example 9 (Reference) In this Example, a mammalian cell expression system was used to determine whether an active polypeptide product from phG-CSF DNA could be expressed in and secreted by mammalian cells (COS-1, A.T.C.C. CRL-1650). The system was designed to provide secretion of a polypeptide analogous to phGCSF by expression and secretion of a partially synthetic, partially cDNA-derived construct encoding [Ala1] hpG-CSF, where a leader was positioned in front. polypeptide having a sequence of amino acid residues attributed to human G-I-CSF according to Wong, et al., Sciense, 228, 810-815 (1985) I and Lee, et al., Proc. Natl. Acad * Sci. (USA), 82, 4360- 4343 (1985).

The expression vector used for preliminary studies on the expression of polypeptide products of the invention was a "shuttle" vector incorporating both pBR322 and SV40 DNA and which had been engineered for autonomous replication at both E. coli and in 30 mammalian cells, the expression in mammalian cells of inserted exogenous DNA being under the control of a viral promoter / regulator DNA sequence. This vector, designated pSVDM-19 in E. coli Hn 101, was deposited August 23, H DK 175466 B1 Η 1985 at American Type Culture Collection, 12301 Par-claw DRive, Rockville, Maryland, with No. A.T.C.C.

53,241th

In constructing the expression vector, the following specific manipulations were performed. A DNA sequence encoding leader was synthesized, as mentioned in H Table 20 below.

TABLE XX

In Hindlix With Trp

H 5 '- A GCT TCC AAC ACC ATG TGG

3 '- AGG TTG TGG TAC ACC

-10

Leu Gin Ser Leu Leu Leu Leu Gly Thr Val

I CTG CAG AGC CTG CTG CTC TTG GGC ACT GTG

20 GAC GTC TCG GAC GAC GAG AAC CCG TG A CAC

In Ala Cys Ser Ile Ser Ala Pro Leu In GCC TGC AGC ATC TCT GCA CCC CTG GGC G -3 '25 CGG ACG TCG TAG AG A CGT GGG GAC -5 * In Apal 1 30 As seen in Table XX, the sequence comprises Hin - dlll and Apal adhesive ends and codons for the 17 amino acid residues that are believed to belong to the "leader" of human GM-CSF. Then, codons for an alanine residue, a proline residue and a leucine residue are followed. The proline and leucine residues duplicate the amino acids found at positions +2 and +3 in hpG-CSF, whereas the alanine residue duplicates the first amino acid residue (+1) in GM-CSF rather than the first in hpG-CSF.

The exchange of threonine with alanine had been designed, thereby facilitating the removal of the GM-CSF leader peptide by cellular mechanisms, usually associated with secretion of I-10 GM-CSF, by the current host cell.

In Man, plasmid pSVDM-19 was digested with KpnI and filled to even ends with Klenow enzyme. Then DNA is digested with HindIII. The resulting large I fragment was combined and ligated with the HindIII / Pvull I fragment shown in Table VII (isolated from plasmid mid Ppo2 as the second largest fragment after a HindIII I digestion and a partial digestion with Pvull) during formation. plasmid pSV-Ppol. Then it was ligated

I produced GM-CSF leader sequence fragment in Table VIII

I 20 in pSV-Ppol (after cleavage with HindIII and Apal) I to obtain plasmid pSVGM-Ppol.

Calcium phosphate precipitates I (1-5 pg) of plasmid pSVGM-Pol DNA were transformed into duplicate 60 mm plates of COS-1 cells, essentially as described in Wigler, et al., Cell, 14, 725 731 (1978). As a control, plasmid pSVDM-19 1 COS-1 cells were also transformed. The supernatant was harvested over the tissue culture

five days after transfection and analyzed for hpG-CSF

In activity. The yield of [Ala1] hpG-CSF from the culture supernatant was on the order of 1-2.5 µg / ml.

Following the positive expression of the plasmid I pSVGM-Ppol encoding [Alaa ^ hpG-CSF in COS-1 cells, another vector was constructed which included the human GM-CSF leader sequence but had a codon for a threonine residue (naturally occurring at position 1 of hpG-CSF) replacing the codon for alanine at this R position. Briefly, an oligonucleotide 5 (s'cAGCATCTCTACACCTCTGGG) was synthesized for position-directed mutagenesis H (SDM). The HindIII - Bam HI hpG-CSF fragment was ligated into the R13 pSVGM-Ppol in M13mp10 for SDM. The newly synthesized Rp-hpG-CSF gene containing a Thr codon in PoHitlon 1 was isolated by cleavage with HindIII and 11 R Eco RI. Then, the fragment was cloned into pSVDM-19, H prepared by cleavage with the same two restriction H endonucleases. The resulting vector pSVGM-Ppo (Thr)

H was transformed into COS cells and the yield of hpG-CSF

H measured in the supernatant from the culture was in the range of 1-5 to 15 µg / ml.

Finally, the genomic sequence, whose isolation is described in Example 5, was used to generate an expression vector for expression of hpG-CSF in mammalian cells. More detailed, pSVDM-19 was digested with Kpnl 20 and HindIII and used the large fragment in a quadruple alloy with a synthetic linker with HindIII and NcoI adhesive ends, as shown in Table XXI. An NcoI - I BamHl fragment containing exon 1 isolated from pBR322 I (8500 hpG-CSF), a genomic subclone, and a BamH1 - Kpbl 25 fragment containing exons 2-5 isolated from plasmid pBR322 (8599 hpG -CSF genomic subclone). The resultant expression vector for mammalian cells, in pSV / ghG-CSF, produced 1-2.5 µg / ml hpG-CSF from trans-propagated COS cells.

DK 175466 B1 I 59

In TABLE XXI

In Hindlll

I 5'AGCTTCCAACAC

EXAMPLE 10 In the example, physical and biological properties of I 10 relate to recombinant polypeptide products of the invention and related recombinant polypeptide products.

In Molecular Weight I Recombinant hpG-CSF products from E. coli ex-priming as in Example 7 had an apparent molecular weight of 18.8 kD, as determined by reducing SDS-PAGE (which one would expect from the indicated amino acids in Table III), whereas natural isolates purified I as in Example 1 have an apparent molecular weight of 20 19.6 kD. A theory of the presence of N-glycans, associated with the natural isolates, may be rejected as lacking asparagine residues in the primary sequence of hpG-CSF in H Table will, and as a result, a method of determining, whether O-glycans were responsible H 25 for differences in molecular weight between the natural isolates H and the non-glycosylated recombinant products. Man H treated approx. 5 µg of natural isolate with neuraminidase (Calbiochem, LaJolla, CA), a 0.5 µg sample was taken and the residue incubated with 4 mU of O-glycanase (endo-xn-acetylgalactose aminidase, Genzyme, Boston, Massachusetts) at 37 ° C. Equal portions were taken after thawing, 2 and 4 hours of incubation. These samples were subjected to SDS-PAGE side by side with the recombinant H DK 175466 B1 Η H material, derived from E. coli. After treatment with H neuraminidase, the apparent molecular weight of the H isolate changed from 19.6 kD to 19.2 kD, which could be due to H removing a sailic acid residue. After two 5 hours treatment with O-glycanase, the molecular weight H changed to 18.8 kD - this value is identical to the apparent molecular weight of the material derived from E. coli. Due to the sensitivity of the carbohydrate structure to neuronal H minidases and O-glycanase, the following structure can be proposed for the carbohydrate component: N-acetyl-neuramin-H acid-α (2-6) (galactose β (1-3) N- acetylgalactose-amine-R, wherein R is serine or threonine.

2. Uptake of 3H-Thymidine 13 Induction of growth by cell division I1 for human bone marrow cells was investigated, based on growing incorporation of 3H-thymidine. Human bone marrow from healthy donors were subdivided with respect to this site by Ficoll-Hypaque (1.077 g / ml, Pharmacie) and low density suspended cells in H Xscove's medium (GIBCO). containing 10% fetal calf-H serum and glutamine pen-strep. Then, 2x10 4 human bone marrow cells were incubated with either control medium or the recombinant E. coli material of Example 7 H 25 in 96-well flat bottom plates at 37 ° C with 5% CO 2 in the air for two days. Double assays I were performed and the concentration varied by a factor of 10,000.

The cultures were then kept under pulse for four I hours with 0.5 μ Ci / well 3H-thymidine (New England 30 Nuclear, Boston, Massachusetts). The uptake I of 3 H-thymidine was measured as described in Ventua, et al., Blood,

61, 781 (1983). In this experiment, human hpG-CSF

In isolates, incorporation of 3 H-thymidine into human 61 marrow cells induces bone marrow cells in a ca. 4-1 times higher than when using control supernatants. The hpG-CSF material, derived from E. coli, of Example 6, had similar properties.

Another study on cell division of human bone marrow cells was used, using a culture medium from transfected COS-1 cells of Example 9, and obtained the same results, indicating that the encoded polypeptide product was in fact acting as tive material was excreted in the culture medium.

I 3 · Induction of Differentiation in WEHI-3B D * In The ability of recombinant material derived from E. coli to induce differentiation in the myelomonocyte leukemia cell line WEHI-3B D + was investigated in mice. a semi-solid agar medium, as described in Metcalf,

Int. J. Cancer, 25, 255 (1980). The recombinant hpG-CSF product and a control medium were incubated with ca.

60 WEHI-3B D + cells / well at 37 ° C with 5% CO 2 in the air for seven days. The samples were incubated in 24-well flat bottom plates and the concentration varied by a factor of 2000. The colonies were divided as non-differentiated, partially differentiated, or completely differentiated, and cells were counted in the colonies under a microscope. It was found that the recombinant material from E. coli induced differentiation.

4. Analyzes for CFU-GM, BFU-E and CFU-GEMM

It was found that natural isolates of pluripotent human G-CSF (hpG-CSF) and the recombinant pluripotent human G-CSF (rhpG-CSF) could induce human bone marrow cells to undergo cell division and differentiate. These activities were measured by CFU-GM [Broxmeyer, I DK 175466 B1 Η

H et al., Exp. Hematol., 5, 87, (1971)] BFU-E and CFU-GEMM

assays [Lu, et al., Blood, 61, 250 (1983)] using noncontiguous low-density bone marrow cells from healthy volunteers. A comparison of CFU-GM, BFU-E and CFU-GEMM biological activities H using either 500 units of hpG-CSF or ^ R rhpG-CSF is shown in Table XXII below.

^ R All colony experiments were performed with non-conjugate ^ R low-density bone marrow cells. Under 10 human bone marrow cells were discarded with a density distribution

Ficoll-Hypague (density 1.077 g / cm 3; Pharmacia). Low-density cells were suspended in Iscove's MO-H, Dulbecco's medium containing calf fetal-H serum was detected and placed on Falcon tissue culture dishes (no.

H 15 3003, Becton Dickenson, Cockeysville, MD.) For 1½ hours at 37 ° C.

I TABLE XXII (Reference)

20 __CFU-GM BFU-E CPR-GEMM

R Control medium 0 ± 0 26 il 0 ± 0

Natural hpG-CSF 83 ± 5.4 83 ± 6.7 4 ± 0 rhpG-CSF 87 ± 5 81 ± 0.1 6 ± 2 The control medium consisted of Iscove's modified Dulbecco medium plus 10% FCS, 0.2 mM hemin and 1 unit of recombinant erythropoietin.

In a CFU-GM assay, the I 30 cells were plated in a number of 1 x 10 5 in 1 ml of 0.3% agar culture medium which included supplemented McCoy's 5A medium and 10% heat-inactivated fetal serum. The cultures were searched for colonies (more than 40 cells per cell).

63 DK 175466 B1 clustering) and examined the morphology of the seventh day of culture. The number of colonies is shown as mean I ± standard deviation, determined from quadruple trials.

In BFU-E and CFU-GEMM assays, cells (1 L x 108) were added to a 1 ml mixture of Iscove's modified Dul-I becco medium (Gibeo), 0.8% methylcellulose, 30% calf-fetal serum, 0.05 nM 2-mercaptoethanol, 0.2 mM hemin and 1 unit of recombinant erythropoietin. The dishes were incubated in a humid atmosphere containing 5% CO 2 and I 5% O 2. A low oxygen pressure was obtained by an oxygen reducer from Reming Bioinstruments (Syracuse, N.Y.).

In Man colonies spoke after 14 days of incubation. The number of colonies is shown as mean ± standard deviation, H 15 determined by duplicate.

All colonies formed in the CFU-GM assay were found to be positive for chloroacetate esterase and negative for non-specific esterase (α-naphthylacetate esterase), consistent with the colonies of the granulocyte type. It was found that both natural hpG-CSF and rhpG-CSF had a specific activity of approx. 1 x 108 U / mg of pure protein when examined by growth.

send dilution in a CFU-GM assay. BFU-E and CFU-GEMM

data in Table XXII are representative of three separate extents and the results are similar to data previously reported for natural hpG-CSF. It is important to note that rhpG-CSF is exceedingly pure and free of other possible mammalian growth factors due to its production in E. coli. Thus, rhpG-CSF is capable of supporting mixed colony formation (CFU-GEMM) and BFU-E when added in the presence of recombinant erythropoietin.

DK 175466 B1 5. Cell Blend Testing It has been previously announced that WEHI-3B (D +) H cells and human leukemia cells from freshly diagnosed leukemia will bind 12i5I-labeled G-CSF from mice and that 5 may be competitive. this binding by the addition H of unlabeled G-CSF or human CSF-β. H tested the ability of natural hpG-CSF and rhpG-CSF to compete H for binding of 125l-hpG-CSF to human and mouse leukemia cells. High purified natural hpG-CSF 10 (> 95% pure, 1 µg) was iodinated [Trejedor, et al., Anal. Biochem., 127, 143 (1982)] and it was separated from the reactants by gel filtration and ion exchange chromatography. The specific activity of the natural 125 I-hpG-CSF was about γCi / γg protein. WEHI-3B (D +) 15 was tested from mold and two preparations of per if all human myeloid leu- H germ cells (ANLL, one classified as Η M4, another as M5B) for their ability to bind 125I-hpG- CSF.

The mouse leukemia cells and freshly collected human H20 peripheral blood myeloid leukemia cells were washed three times with PBS / 1% BSA. WEHI-3B (D +) cells (5 x 106) or fresh leukemia cells (3 x 106) were incubated in duplicate in PBS / 1% BSA (100 µl) in the absence or in the presence of different concentrations (volume: 10 µl). labeled hpG-CSF, rhpG-CSF or GM-CSF and in the presence of 125 I-hpG-CSF (about 100,000 cpm or 1 ng) at 0 ° C, for 90 minutes. (Total volume; 120 yl). The cells were resuspended and placed as a layer I over 200 µl of ice cold FCS in 350 µl plastic centrifuge tubes and 30 centrifuged (1000 g, 1 min). The precipitate was collected by cutting off the end of the tube and speaking the precipitate and supernatant separately in a gamma counter (Packard).

65 DK 175466 B1

Specific binding (cpm) was determined as total binding in the absence of a competitor (average of two trials) minus the binding kid (cpm) in the presence of a 100-fold excess of unlabeled hpG-CSF (non-specific binding). The non-specific binding was a maximum of 2503 cpm for WEHI-3B (D +) cells, 1072 cpm for ANLL (M4) cells, and 1125 cpm for ANLL (M5B) cells. The first and second experiments were performed on separate days with the same preparation of 125 I-hpG-CSF, and the experiments I 10 exhibited intrinsic consistency with respect to the percent value of inhibition obtained for 2000 units of hpG-CSF. Data I is given in Table XXIII below.

H DK 175466 B1 Η χ: ι ο ο η · η S Μ> 5 _ «νο ο ^ · <Ε (Μ Γ" Οι Η (ΐ) Η <υ χι Η £ Λ r- η Ή * "χ C θ 'C0 <U ^ · Η «χ w' χ '<*> Η ^ · Η Ο Η Η 5 QC Tf ν

Η η ** · r-4 <rj Q

8 Ε Μ Γ4

Dj I l · * Η f * .c ιο ^ -Φοοιιη ο οο ο + CO 'tx \ 0 ο C0 φ Γχ Q Ή rt Η χ ^ η «ρ Η m Η Η Κ CO IT) fNJ rt Ο in Ο Ο 00 ιΗ Η_ Ο 00 O 'CO CD fo rH (Ν Η * Ε \ ονΰνοο —im θ' κο ro H 0 <· C) KO Ή c \} r <IN fv f »7 ** <· oooooooooo · -} oooooo oo

e O C M O O csj O O

Χχ '· ·. .

D> O N O N fKJ ΓΜ x "O r-i c-1 4J ·· ..

C Db) .. bj Φ O '03 tbi O' 10 ^ fi O 03 ΉΟ ·· U W <- || Cu

3 C U O I CUOM

* · «3 O * O * V 3 Tue O

c »σ» + J .c ° <«O '+ J.c i 2 ri £% f *« ¥ c <e χ £ 1 W H C u WWI2 C5 67 DK 175466 B1

As indicated in Table XXIII, 125 I-hpG-CSF showed binding to the WEHI-3B (D +) leukemia cells. The binding was dose dependent inhibited by unlabeled natural hpG-CSF

I or rhpG-CSF, but not of GM-CSF. In addition, binding of native hpG-CSF to human myeloid monocytic leukemia cells (ANLL, M4) was observed. The binding to I these cells has a parallel in the response of liquid cultures to natural hpG-CSF, differentiating into fully developed macrophages, judged by morphology.

I 10 The failure to bind natural 125 I-hpG-CSF to monoclonal leukemia cells from another patient (ANLL, MSB) I may be due; that certain leukemia cells may differentiate differently or lack receptors for hpG-CSF. The ability I of rhpG-CSF to compete with natural hpG-CSF for I binding to natural 125l-hpG-CSF is an indication that the receptors recognize both forms equally.

These experiments demonstrating the binding of natural "Si-labeled hpG-CSF to leukemia cells have a parallel in the ability of natural hpG-CSF to induce 20 granulocytic and monocytic differentiation of low-density bone marrow cells obtained from a patient. with acute promyelocytic leukemia (M3) and another patient with acute myeloblastic leukemia (M2). Cells from the two patients were cultured for four days either in culture medium 25 alone or in the presence of 1 x 10 5 units of rhpG-CSF. Cells from the M3 control medium incubated alone H here were still of promyelocyte type; whereas cells grown in the presence of rhpG-CSF exhibited fully developed cells of the myeloid type, including a metamyelocyte, a giant band form, and segmented meutro-H philis and monocyte. For this patient, the division of 100 cells into the control group was 100% promyelocytes, and of rhpG-CSF treated cells 22% blast cells plus promyeles, 7% myelocytes, 35% metamyelocytes, 20% band-H forms plus segmented neutrophils, 14% monocytes and 2% macrophages. An interesting fact is that one of the polymorphonuclear granulocytes still contained an H 5 dominant auer body, indicating that at least this cell represented a differentiated cell that cured to the leukemia clone. Cells from the other patient with myeloblastic leukemia (M2) were also cultured for four days in the absence or presence of rhpG-CSF.

By looking at M2 cells grown in the medium alone, large "blast-like" cells could be observed, some of which had nuclei. Some of the M2 cells differe-H, when treated with rhpG-CSP, to H fully developed segmented neutrophils with residues of Au-H cell bodies in the center of the neutrophil, a sign that H had been differentiated in a cell. that belonged to the leukemia clone. A breakdown of 100 cells that had been examined morphologically showed that the cells from the control group consisted of 100% blast cells. The cells treated with rhpG-CSP consisted of 43% blast cells, 1% myelocytes, 15% metamyelocytes, 28% band forms plus segmented neutrophils, 2% promonocytes and 11% monocytes. Leukemia cells were also tested for differentiation at four other concentrations of 25 rhpG-CSF (5 x 10 3, 1 x 10 4, 2.5 x 10 4 and 5 x 10 4 U / ml, data not shown). Even at the lowest tested concentrations of rhpG-CSF (5 x 10 3 U / ml), there was clear differentiation (cells differentiated longer than I to myelocytes) of M3 (50%) and M2 (37%) leukemia cells. .

DK 175466 B1 69 6. Immunoassay

For the preparation of polyclonal antibodies for use in immunoassay, as antigenic pluripotent G-CSF, purified from human bladder carcinoma cell line 5637 I 5 (1A6), as prepared in Example 1 (B), was used, based on I silver nitrate staining of polyacrylamide gels. to be 85% pure. Six 1-week-old Balb / C mice were immunized with many subcutaneous injections of the I10 antigen. The antigen was suspended in PBs and emulsified with equal volumes of Freund's complete I adjuvant.

The dose was 5-7 µg antigen per ml. mouse per injection.

18 days later, immunization was enhanced by the same amount of antigen emulsified with an equal amount of space in Freund's incomplete adjuvant. Four days later, mouse serum was taken for testing for the content of the I antibody specific to human pluripotent G-I CSF.

Dynatech Immulon II Removawell strips were coated into holders (Dynateck Lab., Inc., Alexandria, Virginia) with hpG-CSF 5 µg / ml in 50 mM carbonate-bicarbonate buffer, pH 9.2. Plates in sheets were coated with 0.25 µg in a volume of 50 µl. The antigen-coated plates were incubated for two hours at room temperature overnight at 4 ° C. The solution was decanted off and incubated.

Prepare the plates for 30 minutes with PBS containing 5% BSA

to block the reactive surface. This solution was decanted off and either diluted pre-H immune serum or serum was added to assay for the wells and incubated for two hours at room temperature. Sera were diluted with PBS, pH 7.0, containing 1% BSA. Man H discarded the serum solution and washed the plates three times with Wash Solution (KPL, Gaithersburg, Maryland).

H DK 175466 B1 Η

Added approx. 200,000 cpm iodized rabbit anti-mouse igG (NEN,

Boston, Massachusetts) in 50 µl PBS, pH 7.0, containing 1% BSA for each well. After incubation for 1½ H at room temperature, this solution was decanted off and the plates washed five times with Wash solution.

The recesses were removed from the holder and counted in H a Beckman 5500 gamma counter. Highly active mouse serum showed more than 12 times greater reactivity than the corresponding pre-immune serum at a dilution of 1: 100.

The immunological properties of E were determined.

coli-derived hpG-CSF upon reactivity to highly active mouse serum specific to mammalian cell-derived hpG-CSF. Immulon IX Removawells were coated with 0.25 µg 90% pure E. coli derived protein in a volume of H 15 50 µl and the mouse serum analyzed as described above.

High-activity mouse sera exhibited a 24-fold higher re-H activity against E. coli material than the corresponding pre-immune sera at a dilution of 1: 100.

H 7. Serial analog bioassays according to the invention

[Ser17] hpG-CSF, [ser36] hpG-CSF, [Ser42] hpG-CSF, [Ser64] hpG-CSF, and [ser74] hpG-CSF products prepared according to Example 9 for hpG-CSF activity were investigated. 25 by recording 3H-thymidine, CFU-GM and WEHI 3B D + assays. In all assays, the [Ser17] analog possessed an activity comparable to the activity of the recombinant molecule with its natural structure. The other analogs possessed approx. 100 times less activity in the 3H thymidine uptake experiment, 250 times less activity in the CFU-GM assay, and 500 times less activity in the WEHI-3B D + assay. These data support the theory that cysteine residues are required at positions 36, 42, 64 and DK to achieve full biological activity.

In 8. In vivo bioassay Alzet osmotic pumps (Alzet Corp., I $ Palo Alto, CA; Model 2001) were connected to permanent catheters in the right carotid artery and implanted them under the skin of seven acidic gold hamsters. Four of the pumps contained a buffer I [20 mM sodium acetate (pH 5.4) and 37 mM sodium chloride] I and 1.5 mg / ml E. coli-derived hpG-CSF, whereas 3 contained only 10 buffer. The pump speed was set to be 1 μΐ / hour for up to seven days. Three days after the application of the pumps, the mean number of granulocytes in the four treated hamsters was six times higher than that of the three control hamsters (buffer), and at the same time the increased number of granulocytes was a quadruple in the number. of total lymphocytes. The number of H erythrocytes was not altered during treatment. These results indicate that the recombinant material produces a specific enhancement of the production of 20 and / or the release of granulocytes in a mammal.

The inventive DNA sequences encoding hpG-CSF polypeptide analogs may be useful in that regard. It provides information on the amino acid sequence of mammalian proteins that has previously been unavailable, despite assay attempts on isolates of naturally occurring products. DNA sequences of the invention may be suitable material for use as labeled probes in isolating hpG-CSF and related protein encoded by human genomic DMA, as well as cDNA and genomic DNA sequences from other mammalian species. The DNA sequences may also be useful in various other methods of protein synthesis (e.g. in insect cells) or in genetic therapy in humans and other mammals. It is expected that DNA sequences of the invention are useful in the development of transgenic mammalian species which can serve as eukaryotic hosts in producing hpG-CSF and hpG-CSF products in large quantities. 5, Conn. Palmiter, et al., Science, 222 (4625), 809-814 (1983).

Of relevance to the hpG-CSF fragments and the polypept-H time analogs of the invention may be mentioned reports of the immunological activity of synthetic peptides which essentially duplicate amino acid sequences found in naturally occurring proteins, glycoproteins and nucleoproteins. In more detail, it has been shown that relatively low molecular weight polypeptides are involved in immune-H reactions that, in terms of duration and distribution, are similar to the immune responses of physiologically significant proteins such as viral antigens, polypeptide hormones, and the like. Among the immune responses to such polypeptides are the provocation of formation of specific antibodies in immunologically active animals. See, e.g.

Lerner, et al., Cell, 23, 309-310 (1981); Ross, et al., In Mature, 294, 654-656 (1981); Walter, et al., Proc.

In Natl. Acad. Sci. (USA), 77, 5197-5200 (1980); Lerner, et al., Proc. Natl. Acad. Sci. (USA), 78, 3403-3407 (1981); Walter, et al., Proc. Natl. Acad. Sci. (USA), 78, 4882-4886 (1981); Wong, et al., Proc. Natl. Acad.

Sci. (USA), 78, 7412-7416 (1981); Green, et al., Cell,. 28, 477-487 (1982); Nigg, et al., Proc. Natl. Acad.

Sci. (USA), 79, 5322-5326 (1982); Baron, et al., Cell, 28, 395-404 (1982); Dreesman, et al., Nature, 295, 185-160 (1982); and Lerner, et al., Scientific American, 248, No. 2, 66-74 (1983). See also Kaiser, et al.,

Science, 223, 249-255 (1984) as regards biological and immunological activities of synthetic peptides, such as DK 175466 B1 73

with approximation, the secondary structure shares with peptide I

hormones but may not have primary structural con- I

formation is shared with them. IN

Claims (13)

DNA sequence according to claim 1, characterized by encoding [Ser17] hpG-CSF. The cDNA sequence of claim 1. The genomic DNA sequence of claim 1. DNA sequence according to claim 1, characterized in that it is covalently associated with a detectable marker substance, in particular a radioactive marker. The single stranded DNA sequence of claim 5. A biologically functional plasmid or viral I DNA vector, characterized in that it includes a DNA sequence according to claim 1. A prokaryotic or eukaryotic host cell, B characterized in that it is transformed or transfected with a DNA sequence according to claim 1 in such a way that the host cell can express the polypeptide product in question. A prokaryotic or eukaryotic host cell, characterized in that it is stably transformed or transfected with a DNA vector according to claim 7. Host cell according to claim 8 or 9, characterized in that the host is E. coli. DK 175466 B1 I 75 I Process for the preparation of polypeptide analogues of hpG-CSF in which one or more cysteine residues are deleted or replaced with alanine I or serine residues, characterized in that prokaryotic I or I are cultured under appropriate nutritional conditions. in particular, eukaryotic host cells transformed or transfected with a DNA sequence according to claim 1 in such a way that they can express said polypeptide and isolate the desired polypeptide product from the expression of DNA sequence. IN 12. Non-naturally occurring polypeptide analogue of hpG-CSF, in which one or more cysteine residues are deleted or replaced by alanine or I serine residues, characterized in that it is a product of expression in a prokaryotic or especially I eukaryotic host cell of DNA sequence according to claim 1. Pharmaceutical composition, characterized in that it comprises an effective amount of the polypeptide analogue of claim 12 and / or prepared in accordance with claim 11, and a pharmaceutically acceptable diluent, adjuvant or carrier. Pharmaceutical composition according to claim 13, further characterized by being free from association with any human protein. Use of the polypeptide analog of claim 12 for the manufacture of a medicament for providing hematopoietic therapy to a mammal.